Advances in Marine Biology, Volume 29

Advances in

MARINE BIOLOGY VOLUME 29

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Advances in

MARINE BIOLOGY VOLUME 29

Edited by

J. H. S. BLAXTER Dunstaffnage Marine Research Laboratory, Oban, Scotland and

A. J. SOUTHWARD

The Laboratory, Citadel Hill,Plymouth, England

Academic Press Harcourt Brace & Company, Publishers London San Diego New York Boston Sydney Tokyo Toronto

ACADEMIC PRESS LIMITED 24/28 Oval Road London NW17DX United States Edition published by ACADEMIC PRESS INC. San Diego, CA 92101 Copyright 01993 by ACADEMIC PRESS LIMITED ‘The Bristol Channel Sole (Solea solea (L.)): A Fisheries Case Study’ by J. Horwood - Crown Copyright 01993. All rights of reproduction in any form reserved No part of this book may be reprinted in any form by photostat, microfilm, or any other means, without written permission from the publishers A catalogue record for this book is available from the British Library.

ISBN 0-12-026129-4 ISSN 0065-2881

Filmset by Keyset Composition, Colchester Printed and Bound in Great Britain by Hartnolls Limited, Bodrnin, Cornwall.

CONTRIBUTORS TO VOLUME 29 J. HORWOOD, Ministry of Agriculture, Fisheries and Food, Directorate of

Fisheries Research, Fisheries Laboratory, Lowestoft, Suflolk NR33 OHT, UK.

T. KIORBOE,Danish Institute f o r Fisheries and Marine Research, Charlottenlund Castle, DK-2920 Charlottenlund. Denmark. H. KUOSA,Finnish Institute of Marine Research, PO Box 33, SF-00931 Helsinki, Finland.

J. KUPARINEN, Finnish Institute of Marine Research, PO BOX 33, SF-00931 Helsinki, Finland,

T. SUBKAMONIAM, Department of Zoology, University of Madras, Guindy Campus, Madras 600 025, India.

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CONTENTS CONTRIBUTORS TO VOLUME 29

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v

Turbulence, Phytoplankton Cell Size, and the Structure of Pelagic Food Webs T. KI@RBOE

.. .. .. .. .. .. Introduction .. .. Turbulence, Water Column Structure and Phytoplankton Cell .. .. .. .. .. .. Size .. .. .. A. Empirical evidence . . .. .. .. .. .. .. B. Cell size and sinking .. .. .. .. .. C. Cell size and nutrient uptake kinetics: the case of diffusion .. .. .. .. .. .. limitation .. .. D. Effect of cell motility and sinking on nutrient uptake .. E . Effect of turbulence on nutrient uptake .. .. .. F. Photosynthesis, phytoplankton cell size and turbulence . . G. Predation and phytoplankton cell size .. .. .. 111. Implications of Phytoplankton Cell Size and Turbulence for the Fate of Pelagic Primary Production . . .. .. .. A . Grazing, phytoplankton cell size and turbulence . . .. B. Excretion of DOM, phytoplankton cell size and productivity of pelagic bacteria . . .. .. .. .. .. C. Sedimentation: Turbulence, cell size and the formation of phytoplankton aggregates .. .. .. .. .. IV Vertical Mixing and the Structure of Pelagic Food Webs . . A. Seasonal events .. .. .. .. .. .. .. .. .. .. .. B. Wind events .. .. .. .. .. .. .. .. C. Fronts .. .. . . . . . . Summary and Conclusions . . . . V. Acknowledgements . . . . . . . . . . . . . . VI . References . . . . . . . . . . . . . . . . VII.

I.

2

11.

vii

4 4 8 10 12 14 15 18 22 22 29

35 41 42 47 50 60 61 61

... Vlll

CONTENTS

Autotrophic and Heterotrophic Picoplankton in the Baltic Sea J. KUPARENEN A N D H. KLJOSA

Preface .. .. .. .. .. .. .. .. .. I. Introduction .. .. .. .. .. .. .. A. The Baltic Sea .. .. .. .. .. B. Picoplanktonic algae .. .. .. .. .. .. 11. Methods.. .. .. .. .. .. .. .. .. A . Autotrophic picoplankton .. .. .. .. .. B. Bacterioplankton . . . . .. .. .. .. .. .. .. .. 111. Phytoplankton Succession in the Baltic Sea .. .. .. IV. Autotrophic Picoplankton in the Baltic Sea .. .. .. A . Areal and vertical distribution .. B. Seasonal variation. . . . .. .. .. .. .. V. Bacterioplankton in the Baltic Sea .. .. .. .. A. Annual and seasonal variation of bacterioplankton pro.. .. .. .. .. .. .. .. duction B. Distribution of bacterioplankton .. .. .. .. VI. Factors Controlling Autotrophic Picoplankton . . . . .. A . Nutrients and temperature .. .. .. .. .. .. .. .. .. B. Grazing .. .. .. VII. Factors Controlling Bacterioplankton .. .. .. .. A. Nutrient- and carbon-limited bacterioplankton growth . . B. Predation control of bacterioplankton .. .. .. VIII. Bacteria in the Pelagic Food W e b . . . . .. .. .. IX. Acknowledgements . . .. .. .. .. .. .. .. .. .. .. X. References .. .. .. .. t

.

I

.

73 75 75 77 81 81

85 87 87 87 90 92 92 97 101 101 104 105 10s 111 1IS 119 119

Spermatophores and Sperm Transfer in Marine Crustaceans T. SUBRAMONIAM

I. 11.

Introduction . . .. .. .. .. .. .. Sperrnatophore Morphology, Composition and Transfer A. Decapoda . . . . .. .. .. .. .. B. Copepoda . . . . .. .. .. .. .. C. Euphausiids .. .. .. .. .. ..

.. ,

.

. .. .. .

129 133 133 174 183

CONTENTS

111. IV . V. VI . VII

1X

D. Stomatopoda .. .. .. .. .. .. . . 184 E. Mysidacea and other spermatophore-producing marine crustaceans .. .. .. .. .. .. . . 184 Spermatophore Hardening .. .. .. .. . . 186 Cryopreservation of Spermatophores .. .. .. . . 187 Spermatophores and Artificial Insemination .. .. . . 189 .. .. .. .. . . 193 Spermatophore Pathology . .

Comparison with Other Spermatophore-producing Marine .. .. .. .. .. Invertebrates . . .. .. .. .. .. .. .. A . Polychaeta . . . . .. .. B. Pogonophora .. .. .. .. .. .. .. .. .. .. C. Chaetognatha .. .. .. .. .. .. .. .. D , Mollusca . . .. .. VIII. Conclusion .. .. .. .. .. .. .. .. .. .. IX . Acknowledgements . . .. .. .. .. .. .. References .. .. X. I

.

195 195 196 196 196 197 200 201

The Bristol Channel Sole (Solea solea (L.1): A Fisheries Case Study J. HORWOOD

I.

11.

111

IV

Introduction . . .. .. .. .. .. A. Classification and identification .. .. .. .. B. Description and related genera Distribution and Movements .. .. .. .. A. Physical characteristics of the region . . B. Eggs and larvae . . .. .. .. .. C. Juveniles . . .. .. .. .. .. .. .. .. .. D . Adults .. .. E. The Bristol Channel “stock” . . .. .. Feeding, Size and Growth . . . . .. .. A . Feeding .. .. .. .. .. .. .. .. B. Size and growth: general aspects C. Length at age .. .. .. .. .. D . Weight at age .. .. .. .. .. .. .. .. .. Reproduction . . .. A . Spawning behaviour .. .. .. .. B. Seasonaldevelopment and time of spawning

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216 219 220 221 221 229 237 246 249 251 251 254 255 262 266 267 268

X

CONTENTS

C. Distribution, size and age with maturity D. Fecundity .. .. .. V. Natural Mortality Rates .. .. .. A. Eggs and larvae . . .. .. .. .. .. .. B. Juveniles . . .. .. .. .. C. Adults .. .. D. Comments . . .. .. .. .. VI. Harvesting Options . . .. .. .. A. Yield per recruit . . .. .. B. Absolute yields . . .. .. .. C. Spawning stock biomass per recruit . . D. The stock and recruitment relationship E. Bioeconomics and dynamics . . .. F. Appropriate fishery targets .. .. VII. Exploitation of the Bristol Channel Sole A . Early fisheries .. .. .. .. B. Early trawl fisheries .. .. .. C. Early quantitative information.. .. D . Catches from 1903 .. .. .. E. Evolution to the modern fishery .. VIII. Status of the Stock . . .. .. .. A. ICES assessments .. .. .. B. Egg-production based assessments . . C. Comparison of assessment methods . . D. Mark-recapture estimates .. .. E. Simulation of population trajectories. . F. Concluding remarks .. .. .. IX. Some Final Comments .. .. .. X. Acknowledgements . . .. .. .. .. .. .. XI. References .. .. .

Taxonomic Index .. .. .. Subject Index .. Cumulative Index of Titles Cumulative Index of Authors

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. . 29 1 . . 294 . . 295 . . 298 . . 299 . . 300 . . 311 . . 315 . . 316 . . 319 . . 32 1 . . 322 . . 323 . . 324 . . 326 . . 327 . . 330 . . 334 . . 335 . . 336 . . 339 . . 342 . . 343 . . 347 . . 348 . . 352 . . 352

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. . 369 . . 373 . . 389

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. . 27 5 . . 279 . . 290

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Turbulence, Phytoplankton Cell Size, and the Structure of Pelagic Food Webs T. Kiarboe Danish Institute for Fisheries and Marine Research, Charlottenlund Castle, DK-2920 Churlottenlund, Denmark

.. .. .. .. .. .. .. Introduction .. .. .. Turbulence, Water Column Structure and Phytoplankton Cell Size . . A . Empirical evidence . . . . .. .. .. .. .. .. B. Cell size and sinking .. .. .. .. .. .. .. C . Cell size and nutrient uptake kinetics: the case of diffusion limitation .. D. Effect of cell motility and sinking on nutrient-uptake .. .. .. E. Effect of turbulence on nutrient uptake .. .. .. .. .. F. Photosynthesis, phytoplankton cell size and turbulence .. .. .. G. Predation and phytoplankton cell size .. .. .. .. .. 111. implications of Phytoplankton Cell Size and Turbulence for the Fate of Pelagic .. .. .. .. Primary Production .. .. .. .. A . Grazing, phytoplankton cell size and turbulence .. .. .. .. B. Excretion of DOM, phytoplankton cell size and productivity of pelagic bacteria ., .. .. .. .. .. .. .. .. C. Sedimentation: Turbulence, cell size and the formation of phytoplankton aggregates . , .. .. .. .. .. .. .. .. .. .. IV. Vertical Mixing and the Structure of Pelagic Food Webs A. Seasonal events .. .. .. .. .. .. .. .. B. Wind e v e n t s . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. C . Fronts .. .. Summary and Conclusions .. .. .. .. .. .. .. V. VI. Acknowledgements VII. References .. .. ..

1. 11.

ADVANCES IN MAKINE BIOLOGY VOLUME 29 ISBN 0-12-02612Y-4

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2 4 4 8 10 12

14 1s 18

22 22

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35 41 42 47

so 60

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T. KIORBOE

1. Introduction Until a few decades ago it was generally accepted that the majority of the phytoplankton production in the oceans was consumed by mesozooplankton, first of all copepods, and that these, in turn, were eaten by planktivorous fish. According to this classical description, the pelagic food chain is mainly linear and short, and there is a relatively close coupling between the primary production and the production of (pelagic) fish in the oceans (see Steele, 1974). It was further assumed that input of organic material to the sea floor is first of all made up of sedimenting copepod faecal pellets which thus provide the ultimate fuel for benthic heterotrophic processes. During the seventies and eighties it was realized that pico- and nano-sized phytoplankton (e.g. cyanobacteria) and heterotrophic microorganisms (heterotrophic bacteria, heterotrophic nanoflagellates and ciliates) play a much larger quantitative role in production and mineralization, respectively, of the phytoplankton than formerly believed. A new concept of pelagic food webs, known as the microbial loop, was developed (Azam et al., 1983). Since the discovery of the microbial loop the study of microbial processes in the pelagic realm has become increasingly popular. This popularity was, among other things, based on budgetary scaling arguments, showing that the biological activity in the water column is primarily due to microbial processes. Because the biomass of pelagic organisms is approximately constant in logarithmic size groups (Sheldon et ul., 1972), and because specific metabolic rates depend o n the body mass raised to an exponent of c. -%, it follows that the contribution to overall community metabolism decreases with the size of the organisms. In fact, with these assumptions more than 90% of the community metabolism is due to organisms smaller than 100 pm. According to such arguments, microorganisms account for the majority of the biological activity in the water column and “huge” organisms like fish in the centimetre to metre size range are absolutely uninteresting. However, there are still fish in the ocean, and from a fisheries point of view it is still interesting to know what fraction - however small it may be - of the primary production is channelled to fish; and, particularly, what the mechanisms are that determine the magnitude of this fraction. More recently the alternative views of microbial versus classical food webs have been combined into an emerging concept, according to which strongly stratified, oligotrophic environments are dominated by smallsized phytoplankters and a microbial loop type of food web, whereas weakly stratified or mixed, turbulent environments are dominated by large-sized phytoplankters and a classical type of food chain (Legendre and Le Fevre, 1989; Legendre, 1990; Cushing, 1989; Kierrboe ef uf.,

IUKUULENCE, PHYTOPLANKTON CELL SIZE A N D I’FI AGlC F001> WEUS

3

1090a). The production of fish in the sea is related primarily t o t h e latter type of environment where, potentially, a relatively large fraction o f the primary production is channelled to higher trophic levels. I t is the purpose of this paper to summarize t h e theoretical and empirical basis of this concept and in particular to investigate the significance of occanic turbulence and water column structure in determining the relative significance of microbial versus classical food chains in varying environments and, thus, the magnitude of fish production in the sea. Turbulence in the ocean is generated by winds, waves, currents and tides. Turbulence modifies the physical and chemical environment of planktonic organisms in several ways. First, turbulence will tend to erode vertical density structure of the water column and, although turbulence and vertical density structure are not unambiguously related, we shall here generally assume that stratified water columns are less turbulent than mixed water columns. This simplification is made among other things because it is relatively easy to measure the vertical density structure of a water column but difficult to quantify the turbulence. A related implication of turbulence is that it generally increases the availability of inorganic nutrients in the euphotic zone due to enhanced vertical mixing or entrainment of deep water into the surface layer. Thus, turbulent, mixed water columns are typically rich in inorganic nutrients, while inorganic nutrients are most often exhausted by the phytoplankton in t h e euphotic zone of stratified waters. Finally, turbulence modifies the light climate experienced by the phytoplankton. Phytoplankton cells suspended in a vertically mixed water column generally experience a more variable and, on average, lower light intensity than phytoplankton cells occupying the photic zone of a stratified water column. Turbulence also has direct effects on planktonic organisms, first of all by moving the organisms around and increasing the contact rate between suspended plankters. In the following sections we shall discuss how these several direct and indirect effects of turbulence and water motion influence the size distribution of the phytoplankton and affect the structure of pelagic food webs and the fate of pelagic primary production. We shall first (Section 11) consider the adaptive significance of phytoplankton cell size to the contrasting environments of stratified (stagnant) and mixed (turbulent) waters (nutrient uptake, light harvesting, predation). Subscquently (Section 111) we shall discuss the implications of phytoplankton cell size and turbulence to t h e fate of pelagic primary production (grazing, exudation of solute organics, aggregate formation and sedimentation). And finally (Section 1V) we shall synthesize all the individual processes considered in the preceding sections and compare pelagic food web structure in stratified versus verticallv mixed waters.

4

T. KI0RBOE

II. Turbulence, Water Column Structure and Phytoplankton Cell Size A.

Empirical Evidence

It is generally believed that small (e.g. <10pm) and often motile phytoplankters (pico- and nanoplankton, e.g. flagellates, cyanobacteria) characterize vertically stratified, stagnant, oligotrophic waters, where they occur in relatively low biomasses, while larger cells (net-plankton, e.g. diatoms) dominate eutrophic, turbulent and partially mixed water columns, where they occur in relatively high biomasses (e.g. Margalef, 1978; Malone, 1980; Legendre, 1981; Cushing, 1989). In deep (say > lo o m ) entirely mixed water columns, where light is limiting, phytoplankton biomass is low, and it appears that small cells also dominate in this type of environment. Thus, net-phytoplankton is typically restricted to, and blooms at, spatio-temporal transitions between mixed and stratified water columns (Legendre et al., 1986). These empirical relations between water column structure and phytoplankton cell size have been schematically outlined in Fig. 1. Classical examples of such spatio-temporal successions in phytoplankton composition and biomasses in relation to water column structure include the vernal temperature stratification in temperate waters and the associated spring bloom of diatoms and subsequent development of a nanophytoplankton community (e.g. Hallegraeff and Reid, 1986; Sournia et al., 1987; see also Figs 2 and 10). Such successions can also be found on much smaller spatio-temporal scales, such as in association with tidal and other types of fronts (e.g. Le Fevre, 1986; Richardson et al., 1986; Kiorboe ef al., 1988b; see also Figs 3 and 4), wind-mixing events (e.g. Hitchcock et al., 1987; Tanaka et al., 1988; Marra et al., 1990; Kiorboe and Nielsen, 1990; see also Fig. 21), spatial or temporal variation in water column structure due to tides (e.g. Demers e f al., 1986; Kiorboe et al., 1990a) as well as mesoscale upwelling events (e.g. Hanson et al., 1986; Peterson et al., 1988). Yet another example of the idea outlined schematically in Fig. 1 is given in Fig. 5. Thus, small and large phytoplankters appear to characterize different physical environments. Another characteristic difference between the occurrence of small and large cells in the oceans is their different degree of variability in abundance. Much of the variability in phytoplankton biomass in the sea is due to pulses in abundance of net-phytoplankton, while the concentration of pico- and nano-sized phytoplankton is much less variable (e.g. Malone, 1980; Furuya and Marumo, 1983). As a consequence chlorophyll concentrations are typically positively correlated to t h e mean cell size of the

TURBULENCE, P€IYTOPLANKTON CELL SIZE A N D PELAGIC FOOD WEBS

Vertical stability Small flagellates

5

Biomass

Large diatoms

Small flagellates

Large diatoms

Small flagellates

Time (days-week-season) S p a c e (km-oceanwide)

FIG.I , Schcmatic outline of the relation between spatio-temporal variation in water column structure and phytoplankton biomass, size and species composition. Modified after idea of W . T. Peterson.

Percent nanoplankton

'I/ 0 F

'

M

'

A

'

M

'

J

'

J

I

A

l

S

l

O

'

N

'

D

'

1985

FIG.2. Seasonal variation in the relative proportion of nanoplankton (i.e. cells < 2 0 p m ) in Long Island Sound, USA. The black bar represents the period when the water column is strongly stratified (i.e. when the vertical density gradient is >0.05 sigma-t u n i t s h ) . After Peterson and Bellantoni (1987).

6

T. K I 0 R B O E

Ternperature."C

20 40

60

80

40t

Chlorophyll, pg/l

60

56"45'N 01 "50'W

90 nautical miles

56'08" OO"40'W

FIG.3. Variation in phytoplankton biomass across a tidal front in northeastern North Sea in October. (a) Temperature distribution. (b) Distribution of chlorophyll. Data from Kimboe er crl. (1988b).

phytoplankton (Harris et al., 1987). The relative constancy of pico- and nanoplankton abundance appears also to characterize other small suspended cells; e.g. bacterioplankton that typically occur at strikingly constant concentrations. Thus, Fogg (1986) in his picoplankton review found that, independent of temperature, salinity or nutrient status of the water, the concentration of bacteria and picophytoplankton in the ocean tends to be around 10' and lo4 cellsiml, respectively. In the following sections (1I.B-I1.G) we shall consider the adaptive significance of phytoplankton cell size to physical (turbulence, vertical mixing), chemical (nutrient concentration) and biological (predation) ~~

FIG.4. Horizontal variation in water column structure (as illustrated by the distribution of sigma-t, panel b) across the Skagerrak (panel a), and associated variation in the volume-ratio of large (>S p m ) to small ( 4p m ) suspended phytoplankton (panel c) in May. Note the striking correspondence hetwen the isopycnals and volume ratio iscilines. The lower panel (d) shows for the same data the relation between the volume ratio and the depth of the upper mixed layer, as represented by the depth o f the 26 sigma-t isopycnal. Redrawn from Kiorboe ef a / . (1990a).

TIJRBULENCE, PIIYTOPLANKTON CELL SIZE A N D PELAGIC FOOD WEBS

7

a

58

Depth, 0

DENMARK 1 2 3 4 5

6

7

8

9

10

NORWAY 11 12 Station No

rn

24

10

b

20

24 25 26

30

27

Sigma-t

40 Depth, rn

0

1 2

3 4 5

6

7

8

9

10

11

12 Station No.

10

20

2

C

30 5

40 Volume ratio of >8 prn to <8 pm particles 10.0

5.0

d 1 .o 0.5

1111/11111111111 10 20 30 Depth of upper mixed layer, rn

Volume ratio, >8 p m i < 8 prn

8

T. KIORBOE

Particle concentration, ppm 0'30

F

0-24 m 24-60 m

) .

........

6

n 1.4

r

I

I

I

I

l

l

,

[

I

I

.

:I 0.6

I

d ;M

I

I

I

O

I

I

P....

-

0.4 0.2 -

........

.....

..... .....

'...

''W

Fic;. 5. Particle (phytoplankton) size distributions (equivalent spherical diameter) at a stratified (upper panel) and a partially mixed (lower panel) station in the English Channel in July. Note the difference in size distribution and total particle volume between the two stations. The phytoplankton at the stratified station was dominated by small, naked flagellates. while diatoms (mainly Rhizosolenia stolrerfothii) characterized the partially mixed station. Modified from Holligan et al. (1984).

properties of the environment, and its implications, in an attempt to explain t h e characteristic differences in occurrence and distributional patterns related to cell size as outlined above.

B.

Cell Size and Sinking

Sinking of organic particles out of the photic zone represents a major loss of organic matter to the sea floor. A prerequisite for a given phytoplankton species to occur in a particular environment is, of course, that it is able to remain suspended. According to Stokes' law the sinking rate (v) of a spherical particle is proportional to the square of its radius (2) and to t h e differential density between the fluid and the particle ( p - p ' ) , i.e.: v

= 0.222p-'?(p

-p'),

TURBUL-ENCE, P H Y T O P L A N K T O N CELL SIZE A N D PELAGIC FOOD WEBS

9

where g is the gravitational acceleration (982 cm/s2) and 7) is the viscosity of the fluid (approx. lo-' cm2/s for sea water). Flagellated forms, of course, have the potential ability to regulate their vertical position in the water column, but immobile forms, such as most diatoms, are at the mercy of Stokes' law. Several species have been shown to be able to exert some degree of buoyancy control by regulating their chemical composition (Eppley et al., 1967). Thus. generally, exponentially growing populations of diatoms have a lower differential density than nutrient-limited populations (e.g. Eppley et al., 1967; Smayda, 1970) and the latter, therefore, sink faster. The emphasis of this section, however, is the effect of cell size. A typical value of differential density, 0.05 gicm', is therefore inserted in eq. 1 to generate Table 1. Sinking rates calculated on the assumption of a size-independent, constant cell density tend to overestimate the sinking rate of large cells and underestimate sinking rates of small cells, because the specific carbon content and, hence, cell density tends to decline with cell size (Mullin et al., 1966). Taking the empirical carbon content vs. phytoplankton cell size of Mullin et al. into consideration (Jackson, 1989) yields more realistic estimates of sinking rate (Table 1). However, irrespective of the analyses performed it is evident from Table 1 that sinking rates of cells smaller than say 10 pm in diameter are insignificant, while cells 3 1 0 0 p m tend to fall rapidly out of the water column. Therefore, large cells depend on water motion to remain suspended. While this does not explain the dominance of large cells in turbulent environments it shows that turbulence is a necessary prerequisite for large cells to remain suspended.

TABLE1. SFTTLINO VELOCTT~ES FOR SPHERICAL PHYTOPLANKTON CELLSOF DIFFERENTSrzb CALCULATED FROM STOKES'LAW (EQ. 1) ~

Cell diameter (Pn)

Settling velocity (miday)

1 10 100 1000

2.36 X 2.36 x lo-' 2.36 X 10' 2.36 x 10'

1.99 x 2.94 x 4.35 x 6.44 x

lo-* lo-' 10" 10'

In ( I ) a constant differential cell density of 0.0Sgicm' has been assumed; i.e. v (cm/s) = 1091 x ?cm/s. In (2) the declining cell density with cell size has been taken into

account;

1)

=

2.48 x rl "emis (Jackson. 1989).

10

T. Kl@RBOE

C . Celt Size and Nutrient Uptake Kinetics: the Case of Diffusion Limitation

In the following sections nutrient uptake in single cells from fluid dynamical considerations will be considered. The classical work on this subject is that of Munk and Riley (1952), but much has been done since. The entire literature will not be reviewed here but an attempt will be made to give a fairly simple overview of the most recent results of these efforts in the context of cell size effects, the perspective of this section. It is frequently assumed that nutrient uptake rate in phytoplankters depends on the cell surface area and that nutrient uptake, therefore, is most efficient in small cells due to their higher specific surface area (e.g. Smetacek, 1985; Legendre and Le Fkvre, 1989). However, this is true only in the relatively uninteresting case of a high environmental concentration of inorganic nutrients. If the saturated nutrient uptake rate (I/) is proportional to the cell surface area ( A ) :

then the specific uptake rate in spherical cells is inversely related to cell radius:

where V is the cell volume. This is not a serious constraint to large cells because all vital rates (respiration, growth, etc.) depend on cell size in approximately the same manner (see also Section II.G, however). At low nutrient concentrations diffusion rate of molecules towards the cell surface may limit the nutrient supply to the cell. If the potential uptake rate exceeds the diffusion rate, a nutrient-depleted region around the cell will be established (Fig. 6) and the uptake rate becomes diffusion-limited. In this situation the uptake rate is independent of the cell surface area (there is always a sufficient number of uptake “sites”). Consider the extreme situation where the substrate concentration is zero at the cell surface. The substrate concentration (C) will then increase monotonically with increasing distance from the cell surface and eventually reach C’ far away. This can be described by (Berg and Purcell, 1977):

where R is the distance from the centre of the cell. Consider now a

TURBULENCE,

PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

11

Substrate concentration, C

I

,

I;oncenrric sneiis withR>r

'\/ \

\

R,

R,

R,

etc.

R

FIG.6. Estimating diffusion-limited nutrient uptake in phytoplankton cells. After idea of T. Fenchel. See text for further explanation.

number of concentric, imaginary shells around the cell (Fig. 6). The flux per surface area ( J ) through each shell will be (Fick's first law):

J =

-

DdCldR.

The total flux, integrated over the entire surface area, through each of these shells will be the same and equal the uptake rate (U) of the cell: U

=

4JnR2 = - 4nR2 DdCldR,

(4)

where D is the coefficient of diffusion. Differentiating eq. 3 and combining with eq 4 yields (e.g. Fenchel, 1987; Lazier and Mann, 1989):

U

=

(5)

4rrDC',

and the specific uptake rate (UIV) is, therefore:

UIV = 4nrDC' (4/3rrr3)-'

=

3DC' r-2.

(6)

Since potential vital rates are proportional to the inverse of the cell radius and the diffusion-limited nutrient uptake rate is proportional to the inverse of the squared cell radius, small size is a major competitive

12

T. K1Q)KBOE

advantage at low nutrient concentrations. The above considerations explain why large (immobile) phytoplankters cannot exist in oligotrophic environments, although it does not explain the dominance of large cells in turbulent environments.

D. Effect of Cell Motility and Sinking on Nutrient Uptake If the phytoplankton cell is sinking or is able to move, the microzone of nutrient-depleted water surrounding the cell will be replaced faster than if the cell remains motionless; in effect the steepness of the nutrient gradient and. hence, the nutrient uptake rate will increase due to this advective transport of nutrients towards the cell surface. The contribution of advective transport to nutrient uptake can be described by a slight modification of eq 5 (Logan and Hunt, 1987):

U = 4rrrDShC'.

(7)

where Sh, the Sherwood number, is the dimensionless ratio of rate of mass transport by advection and rate of mass transport by diffusion. If the advective transport is zero, Sh = 1, and eq. 7 reduces to eq. 5 , which describes mass transport by diffusion alone. The problem is to determine the Sherwood number. Logan and Alldredge (1989) analysed the experimental data of Canelli and Fuchs (1976) on nutrient uptake in diatoms (Tha/lasiosira weisflogi) fixed in a laminar flow field. They found that the empirical relation:

where Re is the familiar Reynolds number (dimensionless), provided a good description of the experimental data. The Reynolds number is given by:

If we first consider the case of a settling algae we can combine eqs. 8 and 9 with the settling velocities given in Table 1 to yield the Sherwood numbers for differently sized algae given in Table 2. The Sherwood numbers indicate the relative increase in nutrient uptake due to advective mass transport due to sinking. I t is evident that the advective contribution is insignificant for cells smaller than say 10 p m . For larger cells, however, the increase in nutrient uptake is significant or even dramatic. Thus. the

TURBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

13

TABLE2. REYNOLDS(Re) AND SHERWOOD (Sh) NUMBERSCALCULATED FOR SETTLING PHYTOPLANKTERS OF VARIOUS SIZE

1 10 100 1000

2.73 x lo-' 2.73 x 2.73 X 2.73 X 10'

2.30 x lop7 3.40 x lop6 5.03 X 7.45 X lop2

1.002 1.113 6.78 297

1.007 1.034 1.116 11.24

The Sherwood numbers indicate the relative increase in nutrient uptake due to sinking. The settling velocities given in Table 1 have been used. (1) and (2) refer to settling velocities calculated assuming size-independent and size-dependent differential cell densities, respectively (see footnote to Table 1).

diffusion-limited nutrient uptake in large cells may be compensated by increased advective transport to the cells due to a high settling velocity. Of course this has the fatal disadvantage that the cells will sediment rapidly out of the photic zone in a stagnant water column and it is, therefore, probably not of much help to these cells. The pattern emerging from this analysis is, therefore, consistent with the lack of large, immobile cells in stratified waters. The effect of cell motility on nutrient uptake can be analysed along very much the same lines of reasoning. Swimming velocities of small (say 10prn) flagellated forms approximate to 10 body lengths/s. For larger forms (say 100 pm), such as dinoflagellates, relative swimming velocities are lower, c. 1 body lengthis (Levandowsky and Kaneta, 1987). Sommer (1988), analysing data compiled by Throndsen (1973) and Sournia (1982), found the general relation for marine flagellates between swimming velocity ( v , cm/s) and size (equivalent spherical diameter, ESD, pm) to be: v = 9.3 x 10-2ESD".24.In Table 3 Sherwood numbers for swimming algae of different sizes have been calculated from this relation and eqs. 8 and 9. Not unexpectedly, the effect of swimming on nutrient uptake rate is insignificant for small cells. However, nutrient uptake in large cells is significantly enhanced by swimming. This is consistent with the observation that stagnant waters are characterized either by very small cells (e.g. cyanobacteria <1 pm) or flagellated forms. However, diffusion-limited nutrient uptake in large cells is only partially compensated by swimming; for example, an increase in cell diameter from 1 to 100 p m causes a factor of 104 decrease in specific diffusive mass transport to the cell surface (cf. eq 6) but less than a factor of 10 increase in specific advective mass transport (Table 3). Therefore, the occurrence of large, swimming

14

T. K I ~ R B O E

TABLE3. REYNOLDS(Re) A N D SHERWOOD(Sh) N U M H ~ R CA SL C l l l A TED SWIMMING PHYTOPLANKTERS OF VARIOUS Sizts

Cell diameter (Pm) 1

10 100

Swimming velocity (cmis)

Re

Sh

9.3 x 10-3 1.6 X 2.8 x lo-'

9.3 x lo-' 1.6 X 2.8 x lop2

1.23 5.26 6.86

I-OK

Swimming velocity (v, cm/s) = 9.3 x 10 ESD"24,where ESD is the equivalent spherical diameter of the alga (Sommer. 1988). The Sherwood numbers indicate the relative increase in nutrient uptake due to swimming.

dinoflagellates in oligotrophic waters must be facilitated by other mechanisms; e.g. die1 migration into the nutrient-rich water below the nutricline to collect nutrients during the night (Dortch and Maske, 1982; Raven and Richardson, 1984; Levandowsky and Kaneta, 1987).

E. Effect of Turbulence on Nutrient Uptake Like cell motility, turbulence may increase the advective transport of nutrients to the cell surface and, hence, increase the nutrient uptake rate. Turbulence, generated by winds or currents, first appears as large-scaled eddies that dissipate into subsequently smaller and smaller sized eddies. There is a minimum size of eddies, related to the Kolmogorov length scale, below which all turbulent energy dissipates as heat while water motion can be characterized as laminar shear (e.g. Lazier and Mann, 1989). Since the minimum size of eddies is in the order of 1 mm or more for realistic values of turbulent energy in the oceans (e.g. Lazier and Mann, 1989), turbulence can be quantified as equivalent shear for phytoplankters smaller than c. 1 mm. The Sherwood number for a sphere in a laminar flow field is (Frankel and Acrivos, 1968; cf. Logan and Alldredge, 1989): Sh

=

+

1 0.26Pe"-5,

(10)

where Pe, the dimensionless Peclet number, is given by (Logan and Alldredge, 1989):

TURBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

15

4. PECLLT (Pe) A N D S t I F R W O O D (Sh) NUMBERSCALCULATED FOR D I H - F RI ~ N SIZES OF SPHERICAI PHYTOPLANKTERS AT TURBULENT SHEAR RATESOF (1) 0.1is AND (2) l.O/s

'rABl,E

1 10

100 1000

2.5 x 2.5 x 2.5 x 2.5 x

10-5 lo-' lo-' 10'

2.5 x 2.5 X 2.5 x 2.5 x

10-4 lop2 10" lo2

1.001 1.013 1.13 2.30

1.004 1.041 1.41 5.11

and G ( i s ) is the fluid shear rate. Typical values of shear rate in the ocean range from <10p7/s at midwater in deep oceans (McCave, 1984; Yamazaki and Osborn, 1988) to a maximum of up to 10is in estuaries (van Leussen, 1988). Inserting a value of D = 10-'cm2/s2 and values of G = 0.1 or 1.0is in eqs. 10 and 11 yields the Sherwood numbers for our four different algal sizes in Table 4. Only cells larger than about 100pm in diameter profit significantly in terms of nutrient uptake from turbulence. Lazier and Mann (1989) came to a similar conclusion along a somewhat different route. However, here again, the advantages of turbulence to large cells are only a partial and inefficient compensation for the disadvantages of being large in terms of nutrient uptake. This is true both in the surface-area-limited (i.e. high nutrient concentration) and the diffusion-limited cases (cf. eqs. 2 and 6, respectively).

F. Photosynthesis, Phytoplankton Cell Size and Turbulence While the foregoing Sections 1I.B-1I.E have dealt with nutrient uptake and cell size in the perspective of physical processes, we shall here consider the effect of turbulence on the light climate and the potential implications for phytoplankton cell size. Turbulent water motion influences the light environment experienced by suspended phytoplankters (i) by increasing the light intensity variability and (ii) by decreasing the average light intensity. The latter is an indirect effect due to the fact that turbulence is generally associated with a deep mixed layer. The photosynthetic rate depends on light intensity; i.e. below a certain threshold light intensity photosynthetic rate increases monotonically with light intensity to saturate at light intensities above the threshold intensity.

16

T. KIBRBOE

At very high light intensities the photosynthetic rate may become inhibited (photoinhibition). There is no a priori reason why larger cells should be better adapted to a fluctuating light climate than smaller cells; i.e. that they should have a larger range of light intensities at which the photosynthetic rate is maximum. However, there are large (apparently size-independent) differences between taxonomic groups (Richardson et a l . , 1983) and among the classes studied diatoms appear to be particularly well adapted to fluctuating light (Fig. 7). This is presumably a secondary adaptation to the characteristic turbulent environment of diatoms, and does not per se explain the dominance of diatoms in such habitats. Harvesting and utilization of light for photosynthesis by phytoplankters can be characterized by three parameters: viz. the quantum (or photon) efficiency of photosynthesis ( 4 ; mol Cimol photon); the absorption cross-section area (e.g. expressed relative to chlorophyll a; Achl;m2img chl;,, which can be considered an efficiency of light harvesting; and the relative concentration of chlorophyll a (Chl : C; mg chl,/mg C> (Geider et al., 1986):

where P J I ) is the carbon-specific gross photosynthetic rate ( i s ) , Z is a dimensionless constant equal to the atomic weight of carbon (0.012 mg Ci p m o l C ) and I is the photon flux density (light intensity) (pmol photonim’ls). Geider et at. (1986) reviewed the available laboratory reports on diatoms for these parameters and found that both the quantum efficiency and the relative concentration of chlorophyll appear to be independent of cell size per se. This is not to say that these parameters are constant; both can show (secondary) adaptation to the prevailing light climate. Thus, Chl : C ratios generally increase in light-limited cells (Fig. 8). Harris et al. (1983) found that the apparent quantum efficiency in phytoplankton field populations tended to increase with cell size (Fig. 9), presumably as a secondary adaptation to the deeper mixed layer and lower average light intensity in habitats dominated by large cells. In contrast the absorption cross-section in itself depends on cell size. This is because self-shading effects increase with increasing cell size. Geider et al. (1986), following Morel and Bricaud (1981), established the theoretical quantitative relationship between absorption cross-section and cell size (which we shall not repeat here), and verified this relationship with empirical data compiled from the literature. Combining this relationship with eq. 12 they found that carbon-specific photosynthetic rates decrease with cell size, both in light-saturated and light-limited cells.

T U R B U L E N C E , P I ~ IOPLANKTON Y CELL SIZE A N D P E L A G I C FOOD WEBS

Specific growth rate, relative scale

17

.................. ............ .... ...... ......................... Chlorophyta

1.0 -

...... 0.5

.......... ....... ...

-

\

.......... ....... ...... ....... 0

0

50

Eacillariophyta

\

Dinophyceae

150

100

200 250 Photon flux density, prnol/rnz/sec

FIG.7. Specific growth rates (relative scale) as a function of light intensity in four major taxa of marine phytoplankton. Note that diatoms (Bacillariophyta) grow at maximum rate over a wide rangc o f light intcnsities. After Raven and Richardson (1986).

g Chl-a/g C

0.06

i **

t

0’

100

300 500 Photon flux density, prnol/rn2/sec

FIG.8. Chlorophyll t o carbon ratio in four species of diatoms grown in the laboratory under continuous light at 18-22°C. After Geider ef ul. (1986).

18

T. KIORBOE

Quantum efficiency O9

r

0.091

-

0.073

-

0.054-

0.036

-

0.018 "

0

50

140

260

400 560 750 Mean cell volume, pm3

FIG.9. Photosynthetic quantum efficiency versus cell size in phytoplankton field populations (Lake Ontario). After Harris et al. (1983).

Although there is much variation within taxonomic groups and although phytoplankters appear to have fairly large adaptive capabilities to prevailing light conditions (see Figs 7-9) the outcome of the above short analysis nevertheless suggests that small size is superior to large size in both turbulent and stagnant waters, also when harvesting and utilization of light is considered.

G. Predation and Phytoplankton Cell Size The foregoing considerations of nutrient uptake and light harvesting capabilities by phytoplankters in contrasting environments show that small cell size is always superior to large size, even though the competitive pressure for small size may be somewhat weakened (but not offset) in a turbulent environment. Thus, although physiological and physical considerations are consistent with the empirical observation that large phytoplankton cells first of all occur in turbulent waters they do not explain the dominance of large cells in such environments. We, therefore, have to seek alternative explanations for the mere existence of large cells in the oceans and for their dominance in new, turbulent habitats. The most appealing idea that has been forwarded is that large size may

TURBULENCE. PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

19

provide a refuge from predation (e.g. Munk and Riley, 1952; Geider et al., 1986; Kiorboe, 1991). The following considerations lie at the basis of this hypothesis. There appears to be an almost constant predator-to-prey size ratio in planktonic food chains (10: 1 to 100: 1; linear scale) (e.g. Sheldon et al., 1977; Fenchel, 1987; Berggreen el al., 1988). Therefore, small phytoplankters are eaten by small predators (e.g. unicellular ciliates and heterotrophic flagellates) while large phytoplankters are consumed by large predators (e.g. metazoan copepods, euphausiaceans). For large phytoplankton size to provide a refuge from predation the relative density of predators must either decrease with increasing cell size and/or generation times of the predators must increase more rapidly with size than generation times of their phytoplankton prey populations, yielding a more lagged numerical response. Both appear to be the case. Sheldon et al. (1972) originally suggested that the average size distribution of organisms in pelagic ecosystems is such that the biomass is approximately constant in equal, logarithmic size classes. More recently it has been shown that the average biomass distribution actually has a negative slope (c. -0.25; Platt, 1985), implying that the overall average predation pressure decreases with size. The body mass dependency of intrinsic (i.e. maximum) specific growth rates (and other vital rates) in heterotrophic organisms appears to be characterized by a weight exponent of about -0.25 to -0.35 (e.g. Fenchel, 1974; Banse, 1982a). As is evident from the above, maximum phytoplankton growth rates also appear to decline with size, although there is much less consensus as to the magnitude of the weight exponent. Although several authors have found weight exponents in phytoplankters similar to those of heterotrophic organisms (e.g. Banse, 1976; Malone, 1980; Schleisinger et al., 198l), Banse's (1982b) review of the literature revealed large size-independent differences in growth rates between taxonomic groups. In general, diatoms grow much faster than dinoflagellates and other taxonomic groups grow at intermediate rates. Within taxonomic groups Banse found size dependency to be relatively weak, with weight exponents between -0.11 and -0.17. Blasco et ul. (1982) likewise found that growth in diatoms scale with rnass-".l4 while the weak size-dependency of growth in microalgae has more recently been confirmed by Sommer (1989) and Nielsen and Sand-Jensen (1990). In unicellular organisms the generation time ( T ) is simply the inverse specific growth rate (ig), while in multicellular organisms

'

T 3 In (Wad/ Wiuv)gp, where ( W2,d/W,L,L,) is the weight ratio between reproductive adults and

20

T. KIORBOE

reproductive products. In copepods, for example, this weight ratio is approximately 100. Thus, if the exponent for the growth-mass relationship in phytoplankton is indeed higher than for heterotrophic organisms and because small phytoplankters are consumed by protozoans and large cells by metazoans, it implies that the generation time increases faster with size in zooplankton predators than in their phytoplankton prey populations. Therefore, the numerical response of predators to blooms of phytoplankton will be increasingly lagged with increasing phytoplankton cell size, and mesozooplankters appear generally to be unable to control population sizes of the net-phytoplankton. Not all phytoplankton predators in the pelagic food web obey the “rule” of constant predator-prey size ratio of c. 10: 1. One of the most interesting examples of this is provided by the heterotrophic dinoflagellates. This group has hitherto been somewhat overlooked (ignored by both phyto- and zooplanktologists), but recent studies show that they can be quite numerous and presumably quantitatively important in the plankton (Hansen, 1991; Bjomsen and Kuparinen, 1991). Heterotrophic dinoflagellates may feed upon phytoplankton that are of a similar size to themselves; i.e. the predator-prey size ratio is close to 1 : 1 (Hansen, 1991), and the larger forms thus occupy the same feeding niche as the copepodshesozooplankton. Because the biomass of heterotrophic dinoflagellates may at times approach the equivalence of 25-5096 of the biomass of the “true” phytoplankton or the total biomass of other zooplankton groups (e.g. Smetacek, 1981; Hansen, 1991) one would expect that this group has the potential of controlling the net-phytoplankton. In fact, B j ~ r n s e n and Kuparinen (1991) suggested that small heterotrophic dinoflagellates may control nanophytoplankton populations in the Southern Ocean. However, maximum growth rates of heterotrophic dinoflagellates appear to be almost an order of magnitude less than that of similar-sized protozoans (e.g. ciliates); maximum growth rates at 20°C of 20 pm ciliates and heterotrophic dinoflagellates are about 0.2 and 0.03/h, respectively (Hansen, 1991). Thus, maximum growth rates of heterotrophic dinoflagellates are similar to maximum growth rates of small, neritic copepods; e.g. 0.025/h at 20°C in the copepod Acartia tonsa (Miller et af., 1977). It is therefore questionable whether the large heterotrophic flagellates are in general significantly more efficient than the mesozooplankton in controlling populations of net-phytoplankton. The predation hypothesis can explain several of the observed features of phytoplankton distribution in the oceans: (1) Small phytoplankters are consumed by protozoans with generation times of the same order of magnitude, allowing for predator

TURBULENCE, PHYTOPLANKTON CELL SIZE A N D PELAGIC FOOD WEBS

21

control of pico- and nanophytoplankton populations; this explains the strikingly constant and relatively low concentration of pico- and nanophytoplankton populations in the oceans. The same feature of bacterial populations has likewise been explained by predator control from heterotrophic flagellates (Andersen and Fenchel, 1985; Fenchel, 1982). (2) It explains the mere existence of large phytoplankters in the oceans despite their relatively low growth rates and poor nutrient uptake and light harvesting capabilities; their risk of being eaten is simply much lower. (3) It explains why the concentration of net-phytoplankton is typically highly variable and why blooms often occur in “new”, turbulent habitats at oceanographic discontinuities in water column structure. Injections of new nutrients to the euphotic zone due to temporarily or locally elevated turbulence and vertical mixing (e.g. caused by winds, currents or tides) give rise to blooms of large-sized phytoplankters that continue their growth largely unutilized by metazoan grazers until the inorganic nutrients have been exhausted and the majority of cells settle out of the water column. The relatively low growth rate and nutrient uptake capability of large in comparison to small cells are here offset by a much lower risk of being eaten because of the considerable timelag in the numerical response of their (mesozooplankton) predators. The considerations in this section have demonstrated that small size in phytoplankters is always superior to large size in terms of nutrient uptake and light harvesting, although the competitive pressure for small size may be somewhat relaxed in new, nutrient-rich, turbulent environments. The mere existence of net-phytoplankton in the oceans as well as the very variable (in time and space) concentration of net-plankton and the more constant concentration of pico- and nanoplankton is best explained by predation. The dynamic interactions between net-phytoplankton and their predators (the mesozooplankton) make the occurrence of netphytoplankton very episodic; typically net-phytoplankton form dense blooms at oceanographic discontinuities in vertical water column structure where new, turbulent habitats are created, and are rare in regions or periods with a permanently stratified water column. In contrast, populations of pico- and nanoplankton (as well as bacteria) are to a large extent controlled by their protozooplankton grazers and thus rarely form blooms (there are exceptions, though; e.g. blooms of toxic flagellates; see Nielsen ef ul. (1990) for an example).

22

T. KIGRBOE

Ill. Implications of Phytoplankton Cell Size and Turbulence for the Fate of Pelagic Primary Production This section deals with the implications of the effects and the combined effects of turbulence and cell size for the fate of pelagic primary production. The foregoing section attempted to explain empirical observations by theoretical considerations. The opposite approach will be adopted here: i.e. to make predictions from theoretical considerations, and subsequently provide empirical evidence to support them. Photosynthetates produced by phytoplankters may be phagocytized or engulfed by zooplankters as intact cells, or they may be excreted from the cells and assimilated by heterotrophic organisms, first of all bacteria, or they may sediment out of the water column as more or less intact cells. Each of these three processes is discussed in a separate section below. A.

Grazing, Phytoplankton Cell Size and Turbulence 1. Grazing and cell size

It has already been noted above that there seems to be an almost constant predator-prey size ratio in pelagic food chains and, therefore, that small phytoplankters are grazed primarily by protozoans and larger cells primarily by mesozooplankters. We shall first consider here the implications for grazers for these two phytoplankton size categories separately and subsequently discuss the more direct effects of turbulence on grazing processes. The episodic nature of the occurrence of net-plankton implies a pulsed availability of food to the mesozooplankton grazers. Since the marine mesozooplankton is frequently - if not always - limited by food availability (Boyd, 1985; Checkley, 1985; Frost, 1985; Runge, 1985), we would expect an immediate functional response in their feeding rate to variation in food availability and, hence, a similar spatio-temporal variability in their growth rate. In contrast, the numerical response in mesozooplankton biomass to variation in food availability would lag, and the abundance or biomass of the mesozooplankton would show little or no relation to small- and mesoscale variability in the concentration of net-phytoplankton. Fig. 10 demonstrates these points by seasonal data FIG.10. Seasonal cycle of phytoplankton (>11 p n ) biomass as chlorophyli,, (a), surface temperature (b), egg production rates (c. d) in two species of copcpods (Acarria clausi and Ccniropages hurnarus) and copepod biomass (e) at a shallow (28 m ) station in southcrn Kattcgat. Denmark. Note that copepod productivity closely follows net-phytoplankton concentration while copepod biomass follows the temperature. From Kiclrboe and Nielsen (unpublished).

TURBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

a

Chlorophyll

"C

Eggs I ?Id C Acartia

.*

15

Eggs/ ? I d

d Centropages 50

40

mgdw/rn* 2500

f

2ooot 1 1500 1000

500

1

1

'

..

. ~

..

*

.. -...

e Copepod biomass

.*

23

24

T. KIQRBOE

from a coastal station in southern Kattegat, Denmark. There is an immediate functional response in mesozooplankton productivity (here demonstrated by variation in the rates of egg production in two species of copepods) to the distinct spring and autumn blooms of diatoms, and the late summer bloom of dinoflagellates. The productivity of the copepods appears to follow quite closely the concentration of large-sized phytoplankton (>11 pm, Fig. lOa), and there is very little productivity in periods between net-phytoplankton blooms. The biomass of the copepods, on the other hand, appears to vary more or less independently of food availability on the mesoscale (week-month), although on the seasonal scale both phytoplankton and copepods are more abundant during the summer half-year than during winter. Ki0rboe (1991) provided several more examples of how episodic or localized blooms of netphytoplankton, typically generated by increased vertical mixing and enhanced availability of nutrients in the euphotic zone, give rise to locally or temporarily elevated mesozooplankton productivity but not to any obvious numerical response in the biomass (see also Figs 21, 24, 25). Kiorboe (1991) further argued that the majority of the mesozooplankton in (at least) temperate seas is in fact associated with such spatio-temporal oceanographic “events” or discontinuities in vertical water column structure, and that very little production occurs in between. Since the concentration of small phytoplankton cells in the sea is generally much less variable than is the concentration of large cells, we would by the same type of reasoning expect the availability of food to protozoan grazers, like ciliates, to be much more constant in time and space. Consequently, the growth rate of the protozoans should vary less than the growth rate of the mesozooplankton. This point is illustrated in Fig. 11, which shows the seasonal variation in the growth rate of the heterotrophic ciliate Lohmaniella spiralis at the same station and sampling period as considered above. Even though the growth rate of the ciliates varies seasonally, it does not have the same episodic character as the copepod growth rate, and 96% of the variability in ciliate growth rate can be explained by variation in temperature. When corrected for temperature dependency, ciliate growth rate thus appears to be almost constant during the year (Fig. l l d ) . 2. Grazing and turbulence It has recently become evident that turbulence also has more direct implications for the quantity of cells being eaten by predators than those being mediated by cell size. It is intuitively evident that turbulence may increase the contaa rate between planktonic predators and their (phyto-

TURBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

I

rng/rn2 200

>

25

200

a Chlorophyll 150

. loot

....

o.o*[ per h 0.05

q4

r

0.041

d Growth rate corrected to "C

FIG.11. Seasonal cycle of phytoplankton concentration (a) and surface temperature (b) as well as actual growth rate (c) and temperature-corrected growth rate (d) in the c. 50 Km heterotrophic ciliate Lohmuniellu spirulis at a shallow station in southern Kattegat, Denmark (same locality as in Fig. 10). For temperature correction a Q,,, = 2.9 was applied. Note that the temperature-corrected growth rate is approximately constant year round. From Nielsen and Ki@rboe (unpublished).

26

T. KIORBOE

plankton) prey, and Rothschild and Osborn (1988) made the first serious attempt to quantify the effects. For a predator moving with velocity v the predator-prey contact rate, Z , is

Z

=

rrR2vN,

(13)

where R = the predator’s reactive distance and N the concentration of Prey. Gerritsen and Strickler (1977) realized that it is the relative velocities of both predator and prey that determine the predator-prey contact rate, and modified the above expression to take prey swimming velocity into account:

Z

=

+

rrR2N(u2 3v2)/3v

(for v > u ) ,

(14)

where u is the velocity of the prey. Note that for u = 0 eq. 14 simplifies to eq. 13. The contribution of Rothschild and Osborn (1988) was to modify further this relation by taking turbulence into account. The effect of turbulence is to increase the relative velocities of both predator and prey and thus to increase the predator-prey contact rate. If w is the turbulent root-mean-square velocity of two particles separated by a distance a , u in the above equation should be replaced by (u’ + w2)’lrand v by (v’ w2)’”. Thus,

+

Z

=

+

+

r R 2 N ( u 2 3v2 + 4w2)(v2 w2)-1/23-1

(for v > u).

(15)

Note that for w = 0, eq. 15 simplifies to eq. 14. For realistic intensities of turbulence, quantified by the turbulent dissipation rate, E , from which w can be readily calculated (see below), they found that turbulence may increase the predator-prey contact rate by 50% or more (see also Yamazaki et al., 1991). In accordance with intuition they showed that the effect is largest for slowly moving predators and prey organisms, and that it increases with the intensity of turbulence. Subsequent modelling exercises by MacKenzie and Leggett (1991) have shown that for fish larvae preying upon copepod nauplii turbulence may increase the predator-prey contact rate by a factor of up to lo! The theoretical - and somewhat provocative - paper of Rothschild and Osborn stimulated research into the effect of turbulence on predatorprey interactions in the plankton. Thus, several recent experimental studies have demonstrated that feeding, growth and egg production may indeed be significantly enhanced in herbivorous copepods by the effect of

TUKBULENCE,

PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

27

Gut contents, average no. prey/gut 5 -

4 -

3 -

2 -

........... 2 e - - -1

0

I

I

10

20

I

I

1

40 50 Food concentration, nauplii/l

30

FIG.12. Gut contents of &10d old cod (Gudus morhua) larvae as a function of food density (nauplii of Culanus finmarchicus) in a Norwegian fjord. Data are grouped according to the average wind speed (W,) during the 8 h prior to sampling. I: W, = 2.0mis; 2: W, = 3.7 mis; 3: W, = 6.0 mis. After Sundby and Fossum (1990) by permission of Oxford University Press.

turbulence (Alcaraz et al., 1989; Costello et al., 1990; Marrase et al., 1990; Saiz and Alcaraz, 1991). Field evidence of turbulence-mediated elevated contact and feeding rates has been provided by Sundby and Fossum (1990) who reanalysed field data on gut contents in cod larvae by applying the model of Rothschild and Osborn. They found that the functional relationship between gut contents and food (copepod nauplii) concentration depended on the wind intensity and, hence, the intensity of the wind generated turbulence (Fig. 12), and that the quantitative dependency was consistent with the prediction of the Rothschild and Osborn model. A similar example may be provided by the observation of Kiorboe et al. (1988a,b) that the egg production rate and, therefore, the feeding rate of the planktonic copepod Acartia tonsa increased significantly subsequent to a severe October storm (25-30mis) in the northwestern North Sea, even though the concentration of phytoplankton remained constant (Fig. 13). This observation is in qualitative accordance with the Rothschild and Osborn idea, but it also appears that the observed factor of 8.5 increase in the slope of the functional relationship between egg production and phytoplankton concentration, from 3.0 eggsifemaleid (mg

28

T. KIORBOE

a

am,

0

a

0.5

I

1 .o 1.5 Chlorophyll, rng/rn3

0

0.5

1 .o 1.5 Chlorophyll, mg/m3

FIG. 13. Egg production rate ( E ) in the copepod Acurtia tonsa in relation to the concentration of chlorophyll, (CHL) before (a) and immediately after (b) a severe October storm in the northwestern North Sea. The regressions are: (a) E = 1.5 3.0 CHL and (b) E = -9.6 + 25.6 CHL. From Ki0rboc ct al. (1988a).

+

chlorophyll/m') before the storm to 25.6 immediately after the storm (Fig. 13), is largely consistent with the quantitative predictions. For this approximate estimate we assume that the phytoplankton is dominated by 10-20 p m cells occurring in a concentration of 102/ml;these are typical values for the northwestern North Sea in October (Richardson et ul., 1986 and K. Richardson pers. comm.) and consistent with the measured concentration of chlorophyll, about 1 mg/m'. We further assume a predator velocity (v) of 0.05 cm/s and a reaction distance ( R ) of 0.05 cm (Jonsson and Tiselius, 1990; Tiselius and Jonsson, 1990); this yields a clearance or volume swept clear (= n-R2v in the present terminology) equal to 34 mlid, which is reasonable for A . tonsa grazing on 1 0 p m cells (Berggreen et al., 1988). For the situation before the storm, w (and turbulence) as well as prey velocity ( u ) are assumed to be zero, and the predator-prey contact rate can be calculated after eq. 13:

Z

= Tx

0.052 x 0.05 x lo2 cells/s

= 0.039

cellsis.

For the situation during the storm we need an estimate of w , the uncorrelated root-mean-square turbulent velocity of particles separated by a distance a. Here we follow Rothschild and Osborn (1988) who provide equations to calculate w from a and the turbulent dissipation rate, F (their eqs. 3 and 5). The separation distance can be estimated as

TUKBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

29

N - ” 3 ( N = 102 cells/ml; i.e. a = (102)”3= 0.2cm) and the turbulent dissipation rate can be estimated from the empirical relation to wind speed (W, m/s) of Oakey and Elliot (1982), E = (W/91)’ = (25/ 91)’ = 0.02 watt/m3. From Rothschild and Osborn we then get w = 0.24cm/s and the predator-prey contact rate, Z , is (eq. 15):

+

Z = n-X 0.0!i2 X 102[(3X 0.052) (4 x 0.242)] x (0.052+ 0.242)p”2x 3-’ = 0.25 cellsis The predicted increase in feeding rate by a factor 0.25l0.039 = 6.4 is, thus, not very different from the factor of 8.5 increase in egg production rate actually observed subsequent to the storm. There is, thus, accumulating theoretical, experimental and field evidence that turbulence does indeed enhance plankton contact and, hence, feeding rates substantially. This, in turn, calls for a re-evaluation of (the applicability of) previous laboratory experiments aimed at determining the functional response in plankton predator feeding rates to prey concentrations, because most laboratory experiments have been conducted under non-turbulent conditions.

B. Excretion of DOM, Phytoplankton Cell Size and Productivity of Pelagic Bacteria A certain fraction of the photosynthetates may be excreted or lost from the phytoplankton cells to the surrounding water in dissolved form. This dissolved organic matter (DOM) cannot, of course, sediment out of the water column or be utilized by heterotrophs by phagocytosis or engulfment. To be degraded by heterotrophs it has to be assimilated directly by cell surfaces. Due to the low concentration of (degradable) DOM in sea water, and because of the strong size dependency of diffusion-limited uptake of dissolved compounds (see Section II.C), small suspended bacteria are the only heterotrophs that can efficiently utilize this source of organic carbon in the water column (Fenchel, 1987). By analogy with the analyses in Section I1 of nutrient uptake in microalgal cells, motility, fluid motion, etc., does not materially alter this conclusion. Conversely, it has been suggested that the major source of DOM for planktonic bacteria is Phytoplankton exudates (e.g. Azam et al., 1983). While it is now generally accepted that dissolved organic compounds leak out of even healthy phytoplankters (e.g. Mague et al., 1980; Larsson and Hagstrom, 1982; Fogg, 1983; Lancelot and Billen, 1985) the magnitude of leakage is still being debated. Empirical estimates range up

30

T. KIBRBOE

to >70% of total photosynthetic carbon fixation (Fogg, 1983; Lancelot, 1983). However, there are major technical difficulties in determining exudation rates from phytoplankters, in particular artefacts from filtering samples and from the presence of small grazers during incubations, and more recent - and presumably more reliable - estimates lie in the lower range of those previously reported; mainly below 10% (Larsson and Hagstrom, 1982; Lancelot and Billen, 1985; Zlotnik and Dubinsky, 1989; Lignell, 1990) of total primary production. The reasons behind, and the mechanisms of, phytoplankton exudation are also currently being debated. It has, for example, been suggested that exudation represents an active release of surplus organic carbon when inorganic nutrients are limiting biomass formation (e.g. Fogg, 1983). However, Bjornsen (1988) argued that this would lead to an apparently paradoxical situation in which the nutrient-limited phytoplankters would stimulate the growth of bacteria that, in turn, would compete (efficiently) with the phytoplankters themselves for the limiting nutrients. B j ~ r n s e n (1988) offered an alternative explanation by suggesting that exudation is simply caused by passive diffusion of low molecular weight organic compounds across the permeable cell membrane. An extension of his analysis also, once again, emphasizes the significance of phytoplankton cell size: The permeability ( P , cmls) of the cell membrane is given by:

where J is the flux of substance (mol/cm2/s) and C (mol/cm3) is the concentration difference across the cell membrane (- internal concentration, since external concentration 0). Since the intracellular pool, S (mol), of a particular compound is

-

s = cv,

(17)

where V (cm3) is the cell volume, and the leakage rate E (molls) is

E = JA,

(18)

where A (cm') is the cell surface area, then the fractional leakage rate, EIS (Is), is given by EIS = P A W = ( 4 n - ? P ) / ( 4 d / 3 ) - ' = 3Plr

(19)

for a spherical cell with radius r . Thus, the fractional exudation rate is

TURBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

31

inversely proportional to cell radius; i.e. small cells lose a larger fraction of their stored dissolved compounds than large cells. This is illustrated in Table 5. For the usual four size classes of spherical phytoplankters fractional exudation rates have been calculated on the assumptions that (i) P = lO-'ccm/s and (ii) that only low molecular weight compounds, constituting about 10% of the cell carbon, can permeate the cell membrane (Bjcjrnsen, 1988). According to this analysis exudation is insignificant for cells >10 p m and substantial for smaller cells. Thus, DOM release and bacterial processing of photosynthetates is expected to be relatively more important in oligotrophic, stagnant waters characterized by nano- and pico-sized phytoplankton than in new, turbulent habitats dominated by net plankton. Experimentally determined phytoplankton exudation rates are normally expressed relative to total carbon fixation rates (primary production). I have, therefore, in Table 5 also calculated the leakage rate relative to production rate (= E / ( S g ) ) , where g is the maximum intrinsic phytoplankton growth rate. I used the empirical growth rate - size relations provided by Banse (1982b) for the two extremes, namely fast growing diatoms and slow growing dinoflagellates. Here again the size effects are evident. Moreover, the predicted exudation rates relative to production rates, up to 23% in the extreme case, but mainly
TABLE5. LEAKAGERATESOF DISSOLVED ORGANIC MATTER (LOWMOLECULAR WEIGHT, LMW, COMPOUNDS)FROM SPHERICALPHYTOPLANKTERS OF DIFFERENT SIZES CALCULATED FROM EQ. 19 WITH P = 10-9cm/s A N D LMW = 10% OF CELLCARBON Cell diameter (Pm)

EIS,ld

(')E/(gS)

(2)E/(gS)

1 10 100 1000

5.2 x lo-' 5.2 x 5.2 x 5.2 x 10-4

13 x 2.7 X 0.58 X 0.12 x

23 x 7.5 x 10-2 2.5 x 0.80 x lo-'

Exudation rates have been expressed as fractions of biomass (E/S,/d) or carbon fixation rates ( E / ( g S ) , dimensionless). Phytoplankton growth rates have been calculated from the growth-cell size relations of Banse (1982b): (1): logg = 0.58-0.11 IogV (diatoms) or ( 2 ) : logg = 0.30 - 0.17 log V (dinoflagellates), where V is the cell volume in g m 3 and g is the instantaneous growth rate (id).

32

T. K1Q)RBOE

normally resource-limited, then bacterial production and growth rate are expected to vary in direct proportion to phytoplankton exudation rate. Several authors have related bacterial production in the sea to simultaneously measured phytoplankton biomasses and production rates (e.g. Fuhrman et al., 1980; Cole er a l . , 1988). Such correlations appear to depend on the spatio-temporal scale of observations (e.g. McManus and Peterson, 1988); i.e. they are fair on large scales but vanish on smaller scales (e.g. Ducklow, 1984). According to the above considerations and from eq. 18 it is evident, however, that phytoplankton exudation rate is proportional to phytoplankton surface area rather than to biomass or production (on larger scales these measures are, of course, intercorrelated). Thus, we would expect bacterial growth rate to be proportional to phytoplankton surface area, or bacterial generation time, T , to be inversely related to surface area ( T cc l/total phytoplankton surface area). Fig. 14 provides field data that fulfil this prediction. Even though the predicted release rates of exudates from phytoplankton (Table 5 ) are largely consistent with those more recently measured experimentally, both experimentally and theoretically determined rates (in the order of 10% of primary production, or less) often appear insufficient to support the estimated carbon requirements of pelagic bacteria. Measurements based on uptake rates of radio-labelled substances and other techniques have shown that the carbon demand of pelagic bacteria is often up to 20-50% of autotrophic carbon fixation or may even approach or exceed measured rates of primary production (Azam and Fuhrman, 1984; Hagstrom, 1984; Lancelot and Billen, 1985; Malone et al., 1986; Ducklow and Peele, 1987). If we accept that these rates and demands are not artefacts, significant alternative sources of DOM other than phytoplankton exudates must exist. Early experiments and observations, mostly on isolated animals, demonstrated a significant loss of DOM in association with meso- and macrozooplankton feeding on phytoplankton (Johannes and Webb, 1965; Corner and Newell, 1967; Webb and Johannes, 1967; Jawed, 1969; Szyper et al., 1976; Copping and Lorenzen, 1980), both by excretion and by leakage of DOM from faecal pellets (e.g. Lampert, 1978) and by ‘‘superfluous” or “sloppy feeding” (e.g. Marshall and Orr, 1962; Cushing and Vucetic, 1963; Conover, 1966); i.e. spillage of dissolved compounds from break-up of phytoplankton cells during feeding. Even though these early observations, as well as more recent ones (Fuhrman, 1987; Roy et al., 1989) together suggest that 10-30% of ingested phytoplankton carbon is typically lost as D O C during feeding, they have been largely ignored in the literature, presumably because, based on weight-specific vital rates, meso- and macrozooplankton are not likely to contribute significantly to

T U R B U L E N C E , PHYTOPLANKTON CELL SIZE A N D PELAGIC FOOD WEBS

33

Bacterial generation time, d 11 10 -

9 -

8 7 -

6 5 -

4 3 -

. .

2 1 -

01 0

I

500

I

1

I

1000 1500 2000 Particle surface area, 1 O3 prn2/ml

FIG. 13. The generation time ( T , d) of pelagic bacteria as a function of the surface area (SA, rnmz/ml) of suspended particles (2.5 - 60pm). The data are from the Skagerrak between Denmark and Norway in May. The regression is: T = llOISA~'lX'.After K i ~ r b o e et al. (1990a).

carbon flow. However, observations in mesocosm experiments (Eppley et al., 1981; Roman et al., 1988) and in the field (Williams and Poulet, 1986; Poulet et al., 1991) as well as the theoretical considerations of Jumars et al. (1989) have recently given a renaissance to the idea that zooplanktonmediated DOM production may be a significant source of dissolved organics for pelagic bacteria. Jumars et al. (1989) argued that optimal digestion theory (Penry and Jumars, 1986, 1987) predicts that in periods of plenty, zooplankters would decrease their assimilation efficiency to optimize the nutritional gain from ingested food and faecal pellets would, therefore, be rich in (dissolved) organics that, in turn. would leak rapidly out into the surrounding water. Their analysis is not restricted to the meso- and macrozooplankton but includes the micro- and na. x iz e d grazers. More direct evidence that meso- and macrozooplankton grazing activity may provide dissolved organic substrates for bacteria stem from mesocosm experiments; both Eppley et al. (1981) and Roman et al. (1988) observed that bacterial production and growth rates were substantially enhanced in the presence of zooplankton >183 pm and copepods (Acartia tonsa), respectively. Poulet er af. (1991) studied the vertical distribution

34

T. KIORBOE

of copepods and dissolved free amino acids (DFAA) at several locations in the Celtic Sea, the Irish Sea and in the Ushant front region off Brittany (France). They generally found a good correlation between copepod abundance and DFAA concentration when copepod abundance exceeded C . 1500 individuals/m3 (see also Fig. 15), while DFAA was negatively related to the concentration of chlorophyll a. This strongly suggests that the occurrence of DFAA is partly due to the activity of the copepods rather than to phytoplankton exudation. When differentiating between different groups of amino acids they further found that both those types that are representative of intracellular compounds of phytoplankton cells (Asp, Glu and Gln) and those that are the result of hydrolysis of proteins (Om) were related to the abundance of copepods, but that the former group showed stronger relation to copepod abundance than the latter. This is consistent with Lampert’s (1978) observation in Duphniu, that “sloppy feeding” appears more significant than excretion and leakage from faecal pellets for (meso- and macro-) zooplankton-mediated DOM production. Thus, both direct exudation of DOM from the phytoplankton as well as incomplete ingestion and digestion in zooplankton may provide organic solute substrates for pelagic bacteria. The latter source may be the most

Depth,

No. of copepods/rn3 0 1000 2000 3000

No.of copepods/rn3 0 1000 2000 3000

No. of Calanus/rn3 0 10 20 30 40

50

I

YK Copepods

0

2000

4000

0

1000

2000

500

600

700

800

Concentration of DFAA, nM/l FIG. 15. Vertical distribution of dissolved free amino acids (DFAA) and copepods of Calmus finrnarchicus at three stations in the Celtic Sea. Data compiled from Poulet rl a / . (1991) by permission o f Springer-Verlag, Heidelberg.

TUKBULENCE, PHYTOPLANKTON CELL SIZE A N D PELAGIC FOOD WEBS

35

important one, or at least more important than assumed during the last decade. We shall here finally discuss under what circumstances DOM “production” is expected to be most significant. It has already been noted above that phytoplankton exudation, expressed as a fraction of carbon fixation rate, is supposed to be most important when small cells characterize the phytoplankton; i.e. in stratified water columns. Jumars et al. (1989) suggested that zooplankton-mediated DOM flux would be most important during bloom conditions, because digestion and absorption is least efficient when food availability is high. However, because of the general temporal mismatch between net plankton blooms and peak abundance of meso- and macrozooplankton, and because microzooplankton is generally unable to feed upon net phytoplankton, this is not necessarily the case. I would rather suggest that exudation from zooplankton would be most important (relative to primary production rate) when a significant fraction of the primary production is in fact grazed by the zooplankton, and the nutrient cycle is relatively closed. This is generally most likely to be the case under non-bloom conditions, i.e. in stable stratified waters. Thus, by both of the considered processes we would expect highest fluxes of dissolved carbon to bacteria, and highest relative importance of microbial processes in non-turbulent , stratified, oligotrophic waters. Whatever the source of DOM there is ample field evidence from various environments that DOM flux and bacterial production indeed constitute a much larger fraction of the primary production in oligotrophic stratified waters than in eutrophic mixed waters (e.g. Hanson et al., 1986; Andersen, 1988; Fuhrman et al., 1989; Nielsen and Richardson, 1989; Kiorboe et al., 1990a; see also Kuparinen and Kuosa, this volume). This is also consistent with the general observation that bacterial production represents a decreasing fraction of the primary production with increasing primary production, with bacterial production in aquatic environments being approximately proportional to primary production raised to the power of 0.8 (Cole et al., 1988).

C. Sedimentation: Turbulence, Cell Size and the Formation of Phytoplankton Aggregates Phytoplankton cells that are not eaten or otherwise degraded by heterotrophic organisms in the water column will eventually sediment to the sea floor. In open oceans this is of course the only significant input of organic material to the sea bed, but even in neritic waters sedimenting phytoplankton - or material derived from phytoplankton, e.g. zooplank-

36

‘r. K I ~ R B O E

ton faecal pellets - is of major significance to benthic heterotrophic activity. Only in very shallow waters may benthic primary production and terrigeneous material exceed input from the water column. It is outside the scope of this paper to discuss the fate of the organic matter reaching the bottom, but we shall here consider the processes controlling this loss of organic material from the water column, in particular the significance of phytoplankton cell size and turbulence. We have already noted above (Section 1I.A) that large cells sink faster than small cells, and that the sinking rate is approximately proportional to the cell diameter squared (Stokes’ law; eq. 1). It is therefore somewhat trivial here to conclude that a greater fraction of the primary production that is due to large cells than of that due to small cells may sediment out of the water column. This also follows from the fact that blooms of net-phytoplankton are, among other things, due to the relatively low grazing pressure exerted by their metazoan grazers. Thus, sedimentation of phytoplankton to the bottom is generally most important in episodic, turbulent environments characterized by large cells, e.g. diatoms. The well described and classical example of this situation is the mass sedimentation of phytoplankton during and immediately subsequent to the spring diatom bloom in temperate, coastal seas. As much as 75% of the primary production during such blooms has been reported to reach the sea floor (e.g. Smetacek, 1980a; Forsskihl et al., 1982; Peinert et al., 1982; Meyer-Red, 1983; Conover and Mayzaud, 1984; Skjoldal and Wassmann, 1986; Laws et al., 1988). Even though such observations of sedimentation of spring blooms thus fit the above expectations, closer examination of such events has revealed significant quantitative deviations from predictions: sedimentation rates realized in the ocean are often very much higher than calculated from Stokes’ law (eq. 1) or measured on individual cells in the laboratory. For example, the common coastal diatom Skeletonema costarum often dominates spring blooms in temperate waters. Settling velocities according to Stokes’ law of these c. 5 pm diameter cells range between 0.06 and 0.13 m/d, depending on whether one assumes a constant differential density of 0.0Sg/cm3 or a size-dependent cell density (see Table 1). Settling velocities measured in the laboratory are similar (0.07 m/d, Riebesell, 1989) or somewhat higher (up to 1.35m/d, Smayda and Boleyn, 1966); the higher value may be caused by the fact that S. costatum is chainforming and possesses spines. However, sinking velocities measured in the ocean or in mesocosm experiments in this species may be very much higher; e.g. >4 mid (Riebesell, 1989) and 30-50 m/d (Bodungen et af., 1981). To account for such high settling velocities Smetacek (1984, 1985) suggested that the diatoms in the ocean may

TURBULENCE, PHYTOPLANKTON CELL SIZE A N D PELAGIC FOOD WEBS

37

aggregate into large, rapidly sinking flocs. Such phytoplankton aggregates have indeed subsequently been quite commonly observed in the ocean (e.g. Lampitt, 1985; Bodungen et al., 1986; Alldredge and Gotschalk, 1988; Riebesell, 1991b). Phytoplankton aggregate formation is now suspected to be the most important mechanism of vertical transport of phytoplankton in the ocean (review by Alldredge and Silver, 1988). More recently Jackson (1990) applied coagulation theory to suggest one potential mechanism for the formation of phytoplankton aggregates in the sea, a mechanism that - once again - emphasizes the significance of turbulence and cell size. The idea is that fluid shear, whether laminar or turbulent, brings about collisions between suspended phytoplankton cells, and that (a certain fraction of) the cells will stick together upon collision and, thus, gradually build up larger aggregates with enhanced settling velocities. This situation is equivalent to the mechanism of formation of “sedimenting” raindrops from suspended water particles in a turbulent cloud (Saffman and Turner, 1956). Other mechanisms may lead to particle collisions, viz. cell motility, Brownian motion and differential settling velocity. However, in the ocean Brownian motion is considered insignificant for particles larger than 1p m (McCave, 1984), and the two other mechanisms can - at least initially - be ignored if we consider the simple situation of a monospecific bloom of diatoms. According to classical coagulation theory the collision rate between suspended and, as usual, spherical cells in this scenario is given by:

where C1 is the concentration of individual cells, G is the (turbulent or laminar) shear rate and r the cell radius as usual. Not all cells stick together upon collision, but the probability of adhesion upon collision can be quantified by the stickiness coefficient, a. The initial coagulation rate, quantified as the rate by which single cells disappear from suspension and become incorporated into dimers, is then

This equation describes properly only the initial process, where collisions between individual cells dominate entirely. As the process proceeds collisions between dimers, trimers, etc., and all possible combinations, as well as collision caused by different settling velocities of differently sized aggregates, have to be taken into account, and the complete quantitative description consists of an infinite number of coupled differential equations of the same principal (albeit slightly more complicated) form as the

38

T. KIQRBOE

above one (see Jackson (1990) for an account). The interesting aspect in the present context is the dependency of aggregate formation on the intensity of turbulence (G) and on the size of the phytoplankton cells; the rate of aggregate formation depends on the cell radius cubed! Thus, in a given environment large cells aggregate much more rapidly than small cells, provided they are equally sticky and occur at similar concentrations. This is consistent with field observations that phytoplankton aggregates tend to be composed of large or chainforming diatoms rather than of small cells (Alldredge and Gotschalk, 1988; Alldredge and Silver, 1988; Riebesell, 1991b). Enhancement of settling rate by aggregate formation is therefore much more important for large than for small cells. The above considerations and those of Jackson (1990) assume that phytoplankters are indeed sticky. Kiqjrboe et al. (1990b) simplified the total set of equations by considering only the initial process and showed that an approximate description of the initial coagulation process could be given by: In C = -(a7.82+C/.rr)t

+ C,,",

(22)

where C is the total concentration of particles (single cells+ dimers trimers, etc.), Cl," is the initial concentration of single cells and the volume fraction of suspended cells. According to coagulation theory, therefore, particle concentration should initially decrease exponentially over time and average particle (aggregate) volume increase exponentially (since 4 is constant) if a suspension of sticky cells is subject to turbulence. In simple laboratory experiments Kicarboe et al. (1990b) showed that this was indeed the case (Fig. 16). Thus, the phytoplankton cells (Phaeodactyfum tricornutum) studied are significantly sticky, and aggregate formation can be properly described by coagulation theory. Since they were able to quantify the fluid shear in the experiments (the only unknown in eq. 22) they could provide quantitative estimates of phytoplankton stickiness. For several species of diatoms, including Skeletonema costatum, they found stickiness coefficients of up to 0.15; i.e. up to 15% of the collisions between cells result in adhesion. Jackson (1990) combined coagulation theory and simple algal growth kinetics by adding a growth term (g = specific growth rate) to the above eq. 21:

+

+

dClldt = gC1- 20.8a? GC:, and showed that coagulation may eventually put an upper limit to the magnitude of algal blooms, when growth (first term on right hand side of

TURBULENCE. PHYTOPLANKTON C E L L SIZE A N D PELAGIC FOOD WEBS

5X105-

'd

500

-

39

b

FIG. 16. Results of a typical laboratory experiment demonstrating formation of phytoplankton aggregates by coagulation. In a suspension of the diatom Phaeoducfylurn fricornutum (volume concentration = 33 ppm) subject to a fluid shear rate o f SOis the concentration of particles declines exponentially over time (a) and the average particle volume increases exponentially over time (h) as predicted by coagulation theory. From Kiarboe et al. (199Ob) by permission of Springcr-Verlag. Heidelberg.

eq. 23) and coagulation (second term) balance. At this critical concentration (Ccr) dC,/dt = 0, hence:

C,, = 0.048g(aG)-' F3.

(24)

Jackson showed this to be a two-state system in which coagulation is relatively unimportant at cell concentrations
40

T. KIORBOE

TABLE 6. C K I T I C A L PI IYTOPLANKTON C O N C E N T K A T I O N (ccr, CEl,l.S/ml) A N D BIOMASS (B,,, /Lg C A R H O N h ) F O K C O A G U L A T I O N CONTKOI. OF POPUL-ATION SIZE, CAl.CLJI.ATED F O R V A R I O U S CELI. SIZES A N D FLUID S H E A R RATES S h e a r r a t e (is)

0.1

1.0

10.0

Cell d i a m e t e r

(Pm) 1

10 100 1000

Ccr 1.2 X 5.6 x 2.6 x 1.2 X

Bcr

Ccr

Bcr

c,,

6.9 X 10' 1.2 X 10' 6.9 X lo3\ 1.2 X lo5 3.2 x lo4 3.6 x 10' 3.2 x ln'l 5.6 x 10' 1.5 x 10' 12.6 x ior-i.S-
B,, lo7 6.9 X lo2 lo3 3.2 x 10' 10') 1.5 x 102 lop37.2 X 10'

C,, = 0.48g(aC)-'r',(Y = 0.15 and g (specific growth rate, id) computed from the growth rate-size relation provided for diatoms by Banse (1982b): logg = 0.58 - 0.11 log V . where V is cell volume ( p m ' ) . Cell carbon calculated assuming 0.11 X pg Ciym' (Strathmann, 1967). Limitation of phytoplankton population sizes is potentially important only when C,, < 2 X 10'pg Cil, i.e. only for combinations of cell sizes and fluid shear rates to the right of the dotted line in the table.

several of the above theoretical considerations (Fig. 17). The concentration of phytoplankton aggregates peaked at the height of the bloom (Fig. 17a). Moreover, aggregate concentration was low and independent of phytoplankton abundance below a threshold concentration, but increased rapidly above this threshold (Fig. 17b), thus illustrating the two-state system predicted by Jackson. It is very likely that turbulence, flocculation and subsequent sedimentation, rather than predation or nutrient concentration, were the main factors responsible for the termination of the spring bloom. In summary, then, sedimentation is most likely to account for an increasing fraction of the primary production with increasing cell size and, thus, with increasing phytoplankton biomass, both because sinking rates increase with the cell size squared, and because larger cells aggregate more rapidly into sinking flocs than small cells. Because large cells dominate in turbulent environments this trend may be partly counteracted by resuspension of cells or flocs. Yet, sedimentation appears to represent an increasing fraction of the phytoplankton production with increasing primary production, with sedimentation being approximately proportional to the primary production raised to the power of 1.4 according to empirical estimates (Betzer et al., 1984; Wassmann, 1990). Sedimentation is, therefore, relatively more important in new, turbulent environments, and less so in stagnant waters.

TURBULENCE, PHYTOPLANKTON

Total aggregate volume, mm3/1

Total aggregate volume, mm3/1 L

1000- a

41

CELL SIZE AND PELAGIC FOOD WEBS

Chl. conc., mg/m3

t

1000- b

0 .

I

800 .

Phytoplankton cell number Chlorophyll-a

an ,

15 20 25 30 5 10 15 20 25 30 April I May

0 " " ' I 0 2 4 6 8 1 0 1 2 Chlorophyll concentration, mg/mi3 I

300

600 900 1200 1500 1800 Phytoplankton cell no., cells/ml

FIG.17. Observations on concentrations of phytoplankton and aggregates during a spring diatom bloom in the southern North Sea. (a) Temporal variation in chlorophyll concentration and total aggregate volume. (b) Relation between aggregate volume and phytoplankton concentration. Modified after Riebesell (1991a).

This section has documented that the fate of pelagic primary production is to a very large extent governed by phytoplankton cell size and turbulence. Loss of dissolved organics from phytoplankters depends directly on cell size and is significant only for very small cells. Potential grazers of phytoplankton cells are closely related to cell size, although there are noticeable exceptions to the 1: 10 predator-prey size ratio "rule" in plankton food webs. Rates of sedimentation and grazing may both be speeded up by the effects of turbulence due to enhanced plankton contact rates. The effect of turbulence is not linear with cell size, however, and is relatively most important for large phytoplankton cells. From the considerations in this section we would expect, therefore, that primary production due to large cells in turbulent environments will either sediment out of the photic zone or be eaten by mesozooplankton, while primary production due to small cells in stagnant waters will be processed by microorganisms, whether phagocytized directly by protozoans or the dissolved exudates assimilated by bacteria.

IV. Vertical Mixing and the Structure of Pelagic Food Webs In the two preceding sections a number of individual processes and components of pelagic food webs have been dealt with separately. The

42

T. K I a R B O E

theoretical considerations and empirical evidence presented so far have been largely in support of the emerging concept of pelagic food web structure and hydrodynamics as outlined in the introduction. In this section some case studies will be reviewed that - to a greater or lesser extent - consider all (important) components and processes simultaneously, and where pelagic community structure has been compared between stratified and (partially) mixed water columns or across spatio-temporal discontinuities in water column structure. Such studies - interdisciplinary by nature - are not common in the literature and we will, therefore, have to draw upon fragmented information as well. The following are organized as a series of cases, where distinct hydrodynamical events are treated.

A. Seasonal Events Temperate waters undergo a dramatic seasonality in plankton productivity. It has already been noted that the spring diatom bloom, initiated by the initial stabilization of the water column in deep waters, or by the increasing light intensity in shallow (mixed) waters, is associated with a distinct peak in productivity of the mesozooplankton (see Fig. 10) and in sedimentation of phytoplankton to the sea floor. It has also been noted for the Kattegat system that the productivity (growth rate) of one component of the microbial loop, namely the planktonic ciliates, is strikingly independent of the temporal variation in phytoplankton concentration across the temporal discontinuity to which the spring bloom is associated (Fig. 11). As a consequence of this it appears that microbial processes are relatively more important in the stratified, oligotrophic summer period, and export of photosynthetates via mesozooplankton and the “classical” food chain to higher trophic levels (e.g. fish), or by sedimentation to the sea floor, is more important during the spring bloom. The seasonal development of the plankton community in temperate waters is, thus, from a net-plankton community characterized by export production, with initial stratification to a nano- and picoplankton community characterized by regenerated production and more closed nutrient cycles in the subsequent strongly stratified period. This generally accepted temporal change in the relative importance of the “classical” and the microbial food webs associated with the seasonal change in vertical water column structure is also illustrated by the microbial biomass data from the North Sea compiled in Fig. 18 and Table 7 (data from Nielsen and Richardson, 1989 and Hay et al., 1991). The southern part of the North Sea is relatively shallow, t30-50 m, while the

TURBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

43

FIG. 18. (a) Depth contours in the North Sea and localization of the sampling regions considered in Table 7. The depth contours are: (....) 20rn; (------) 40m; (:.-.-.-.-) 100 m; (--) 200 m. Regions I and I1 were sampled in FebruarylMarch and region 111 in MayiJune. (b) Distribution of surface chlorophyll (rng/m3) in the North Sea during FebruaryiMarch 1988. Modified from Nielsen and Richardson (1989).

TABLE7. AVERAGEBIOMASSES (mg Urn2) OF PHYTOPLANKTON, PELAGic BACTERIA, HETEROTROPHIC NANOFLAGELLATES AND COPEPODSI N THE UPPER REGIONS/PERIODS IN THE NORTHSEA REPRESENTATiVE MIXEDLAYERIN THREE OF PRE-BLOOM (I), BLOOM(11) AND POST-BLOOM (111) COND~TIONS Region/ type

I Pre-bloom deep, mixed

I1 Bloom shallow, mixed

Phytoplankton Bacteria Flagellates Copepods

1580 200 10 260

8760 100 8 260

Bacteria/phytoplankton Flagellates/bacteria Copepodslphytoplankton

0.13 0.05 0.16

0.01 0.08 0.03 ~

111

Post-bloom deep, stratified 760 740 140 -

0.97 0.19 ~~~~~~~~~~~~~~

I: Deep (c. 100 m) mixed stations in FebruaryiMarch; 11: shallow (<30 rn) mixed stations in FebruaryiMarch; 111: Deep stratified stations in MayiJune (see also Fig. 18). Data from Nielsen and Richardson (1989).

44

T. KIBRBOE

northern parts are deeper, > l o o m . In the winter and early spring the entire North Sea is vertically mixed. By February the light intensity is sufficient to generate a modest diatom spring bloom (dominated by Rhizoselenia spp.) in the shallow southern parts (Fig. 18), while the spring bloom in the deep northern regions does not occur until the onset of weak temperature stratification in late April (e.g. Krause and Thrams, 1983). The three situations compared in Table 7, viz. northern and southern North Sea in February/March and the northern North Sea in May/June, thus represent pre-bloom (I), bloom (11) and post-bloom (111) conditions, respectively. The most striking pattern is the very insignificant contribution of microorganisms in the bloom situation, as judged from the biomasses of bacteria and heterotrophic nanoflagellates relative to phytoplankton biomass, in comparison to the post-bloom situation. The ratio of bacterial to phytoplankton biomass increases by a factor of c. 100 in the stratified post-bloom situation, where the biomasses of bacteria and phytoplankton are similar. This approaches the situation in the open stratified, oligotrophic oceans like the Sargasso Sea, where bacteria are supposed to be responsible for the majority of heterotrophic carbon processing in the plankton (Fuhrman et al., 1989). Thus, the microbial food web appears to be entirely dominating in the post-bloom situation, where the water column is vertically stratified, and insignificant during the spring bloom, where the water column is mixed. It is less easy to deduce anything about the relative significance of mesozooplankton from the information in Table 7, because zooplankton data are missing for the post-bloom situation, and because mesozooplankton production is generally unrelated to biomass (Kicjrboe, 1991). However, consistent with the observations in the Kattegat, there is no numerical response in copepod biomass to the spring bloom, and historical data show copepod biomass in the shallow parts of the North Sea to peak several months after the spring bloom (e.g. Roff et al., 1988). Copepod production measurements conducted in regions I and IT show the production to be three times higher in the bloom situation (Hay et al., 1991). Although mesozooplankton production thus appears to peak during the bloom, budgetary considerations suggest that in the shallow parts of the North Sea zooplankton grazing accounts for only a few per cent of the spring phytoplankton production and that the majority of the production sediments to the sea floor (Nielsen and Richardson, 1989). In the two examples considered above, mesozooplankton grazing only accounted for about 10% (Kattegat) or less than a few per cent (southern, shallow parts of the North Sea) of the spring bloom primary production. This is consistent with observations in other temperate shallow regions (e.g. Deason, 1980; Joiris et al., 1982; Nicolajsen et al.,

TURBULENCE, PIIYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

45

1983; Baars and Fransz, 1984; Roff et a f . , 1988; Tiselius, 1988) where the majority of the spring bloom sediments to the sea floor (Smetacek, 1980a; Forsskhhl et al., 1982; Peinert et al., 1982; Christensen and Kanneworff, 1986; Nicolaisen and Christensen, 1986). In these regions water temperatures are low (near 0°C) at the onset of the vernal bloom, the majority of the neritic copepods overwinter as resting eggs in the sediment (Uye, 1985; Lindley, 1986, 1990; Lindley and Hunt, 1989; Nass, 1991) and there is a considerable timelag in the numerical response of the mesozooplankton to the spring bloom (Kicirboe, 1991; see also Fig. 10). In more offshore, deeper regions, where a substantial copepod population (mainly Calanus spp.) overwinters at depth (Krause and Thrams, 1983; Colebrook, 1991), or in southern, warmer waters, where there is less lag in the numerical response (e.g. Sournia et al., 1987; Colebrook, 1991), the copepods may consume a substantial fraction of the primary production with zooplankton grazing accounting for up to 40-100% of the spring phytoplankton production (e.g. Gamble, 1978; Williams and Lindley, 1980; Joiris et al., 1982; Tiselius, 1988). There are, thus, considerable regional differences in the degree of temporal coupling between the spring bloom and the mesozooplankton. However, irrespective of which fraction of the spring primary production is consumed by the mesozooplankton, the mesozooplankton production associated with the spring bloom appears to be the most significant mesozooplankton production event on the seasonal scale in temperate waters (Kiorboe, 1991; see also Section 1II.A). The spring bloom in temperate waters typically exhausts all available inorganic nutrients in the euphotic zone and generally appears to be the single most significant event in which new nutrients are incorporated into particulate matter. In the stratified period subsequent to the spring bloom there is typically little net accumulation of particulate organic matter (Fig. 19) and diffusion of new nutrients across the pycnocline is, thus, balanced by sedimentary losses. Consequently, the amount of nutrients retained in the euphotic zone as particulate matter during the spring bloom is of importance also to the subsequently developing plankton community, which is based mainly on regenerated production. Copepods in particular, but also other mesozooplankters and perhaps large heterotrophic dinoflagellates, are the only important grazers of the spring diatom bloom. Their phytoplankton consumption during this period has, therefore, implications for the pelagic productivity beyond the immediate accumulation of zooplankton biomass. Smetacek and Pollehne (1986) suggested that because uneaten spring diatoms will eventually sediment out of the water column, grazing by these groups on the spring bloom will tend to retain particulate organic matter and essential elements in the

46

T. KIBRBOE

mg C i m 2

l6

r

12 10 8 6 4

2

0 Month Fit;. 19. Seasonal variation in phytoplankton carbon and total particulate carbon (POC) at a shallow station (28m) in southern Kattegat, Denmark. Data from Olesen and Lundsgaard (unpublished).

euphotic zone either as zooplankton biomass, excretion products or faecal pellets and thus enhance recycling of pelagic material and inhibit sedimentary losses. Packaging of phytoplankton into zooplankton faecal pellets has often been considered a process that will enhance vertical flux of particulate organic matter (e.g. Lorenzen and Welschmeyer, 1983). However, there is increasing evidence that faecal pellet residence times in the water column are considerably larger than anticipated from sinking rates (Smetacek, 1980b; Noji et al., 1991), that pellets are to a large extent consumed by copropagous mesozooplankton (Paffenhofer and Knowles, 1979) or fragmented by coprohexy (Lampitt et al., 1990; Noji et at., 1991) and that essential elements leak rapidly out of the pellets (Jumars et al., 1989). These processes will all tend to retain material in the water column (see also review by Noji (1991)) and provide a substrate for the subsequently developing regenerative plankton community. Thus. the mesozooplankton are actively conditioning the community that arises after passing of the bloom. The observations of Colebrook (1985) and Roff et af. (1988) that the size of the copepod summer populations in the North Atlantic and the North Sea is related to the size of the overwintering stock, rather than to the immediate food environment, was taken as evidence by Smetacek and Pollehne (1986). Along similar lines of reasoning, Colebrook (1991) recently suggested

TURBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC F O O D WEBS

47

-

FIG. 20. Decadal change in zooplankton and phytoplankton abundance in the North Sea, 1948-1987 After Colebrook (1991) by permiwon of Pergamon Press

that the similarity in the long-term variation in zoo- and phytoplankton standing stocks in the North Atlantic (Fig. 20) has its origin in the zooplankton (rather than vice versa) and is caused by recycling of nutrients from the zooplankton. The implications of this idea are that the larger the amount of phytoplankton consumed by the zooplankton during the spring bloom the larger the biomass of the pelagic community in the subsequent oligotrophic, stratified period. More generally, new nutrients that are transported into the euphotic zone by localized or temporary vertical mixing and that give rise to net-phytoplankton blooms, may be retained here by copepod grazing. These considerations place the copepods in a key position for structuring seasonal pelagic ecosystems and make their importance much larger than anticipated from their contribution to overall community biomass or metabolism (see also Roman et at., 1988).

B.

Wind Events

Wind-mixing events occurring in an otherwise stratified water column may temporarily enhance the vertical flux of nutrients across the Pycnocline to the surface layer (Klein and Coste, 1984; Eppley and Renger, 1988). They may also give rise to shortlived net-plankton blooms (e.g. Hitchcock et af., 1987) and a temporary shift in pelagic food web structure (Nielsen and Kicdrboe, 1991) with increased mesozooplankton production (Mullin et al., 1985; Cowles et al., 1987; Kiorboe et ai.,

48

T. K1Q)RBOE

Wind Depth, m

\

Wind

\

Arbitrary ilnits 30 r

2ol b Fluorescence > 1 I pm

10

:

Eggsl91d

Acartia

4 r

1

Eggs19 I d

24 October

30

1 5 November

10

FIG.21. The effects of a wind-mixing event on processcs in the plankton at a shallow (22 m) station in southern Kattegat. Denmark. Strong winds on 26 and 31 October eroded the pycnocline (a). This was followed by a moderate bloom of net phytoplankton (b) and subsequently by enhanced productivity (fecundity) of the copepods Acarria clausi (c) and Temora longicornis (d). After Kimboe and Nielsen (1990).

'TURBULENCE, PHYTOPLANKTON CELL SlZE AND PELAGIC FOOD WEBS

49

Wind velocity, mls

Mixed-layer depth, m

80

Particle flux, mg/m2/d 300

r

?. I . I.

. I

-

Shallow

Month

FIG.22. Sedimentation rate of particulate matter at two depths (shallow: 780 m; deep: 2900m) in the central Arabian Sea in relation to mixed layer depth and wind velocity. Strong monsoonal winds give rise to a deepening of the mixed layer, increased availability of new nutrients in the euphotic zone and enhanced primary productivity and sedimentation. After Nair et d.(1989).

1988a,b; Kiorboe and Nielsen, 1990; see also Figs 13 and 21) and sedimentation of particulate matter to the sea floor (Nair et al., 1989; Nielsen and Kiorboe, 1991; see also Fig. 22). Such a temporal discontinuity in water column structure may, thus, give rise to increased relative importance of the classical food web and, presumably, lower relative significance of microbial processes. While the response of the classical food chain to wind-induced changes in vertical water column structure must now be considered well documented (see review by Kiorboe, 1991) even at higher trophic levels (e.g. enhanced survival of planktivorous fish larvae, Thresher et al., 1989; Wroblewski et al., 1989) the response of microbial processes to such events appear poorly investigated. Nielsen and Kiorboe (1991) studied the response in abundance and growth of ciliates to a wind-mixing event

50

T. KIORBOF.

in the Kattegat that caused a factor of >3 increase in phytoplankton abundance. Although the surface concentration of heterotrophic ciliates decreased during the storm and subsequently increased to pre-storm levels within a few days, this was ascribed to advection and/or dilution due to vertical mixing because there was no response in ciliate growth rate. Even if “noise” due to advection/dilution is corrected for, the relative significance of microbial processes, as judged from ciliate productivity, diminished significantly during the wind-induced net-phytoplankton bloom. Only the autotrophic ciliate Mesodinium rubrum responded significantly and positively to the wind-mixing event, and “behaved” almost like the net-phytoplankton. Hanson et af. (1986) monitored the production of planktonic bacteria at a locality on the Spanish west coast during the decline of a shortlived wind-induced (upwelling) diatom bloom into the subsequent stratified period; bacterial production was almost constant and only weakly related to phytoplankton biomass, while the ratio of bacterial to phytoplankton production increased by a factor of c. 5 within 3-4 days. Both of the cited examples thus suggest that microbial processes respond only weakly to windinduced vertical mixing, and that their relative importance is therefore less during wind-generated net-phytoplankton blooms.

C. Fronts Fronts can generally be defined as regions of above average horizontal gradients in properties of the water (Le Fevre, 1986); here we shall in particular consider fronts that are characterized by horizontal discontinuities in vertical water column structure, such as tidal fronts, river plume fronts and salinity fronts. Fronts have long been regarded as regions of elevated biological activity although this is not necessarily true of all types of fronts (Le Fevre, 1986). There are several mixing mechanisms, operating on different time scales, that may enrich the photic zone of frontal regions with inorganic nutrients (Loder and Platt, 1985; Le Fkvre and Frontier, 1988) and, thus, give rise to net phytoplankton blooms. Some frontal types, in particular river plume fronts, are characterized by strong surface

FIG.23. The SkagerrakiKattegat salinity front. Distribution of salinity (a), fluorescence (b), primary production (c), assimilation index (d) and concentration of copepod eggs and nauplii ( e ) across the front in April. Both phytoplankton biomass and activity as well as abundance of copepod offspring peak in the transitional zone between mixed and stratified water. Modified from Richardson (1985).

TURBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

Depth, m 0 10 -

20

-

30 -

34

/

a Salinity, %.

40 50 -

60

-

d Assimilation index

60 50

I r-

~

45 nautical miles

I

51

52

T. KIBRBOE

convergence, which may lead to accumulation of buoyant material and positive phototropic animals in the frontal region, thus further enhancing the local concentration of particulate organic material and the production of heterotropic organisms. At other fronts surface convection can be ruled out (as at the tidal front illustrated in Fig. 24, which is mainly a bottom phenomenon) and phytoplankton blooms occurring here must be caused solely by locally elevated primary production based on enhanced availability of new nutrients. For a further discussion of fronts as “accumulation” versus “high production” biotopes see Le Fevre (1986). Figs 23-26 give several examples of how various components of pelagic food webs vary across horizontal transitions (fronts) in water column structure. Richardson (1985) studied the variation in phytoplankton biomass and production and abundance of copepod eggs and nauplii across the salinity front generated by outflowing brackish water from the Baltic and Kattegat into the Skagerrak in April (Fig. 23). At this time of the year the Skagerrak is still not temperature-stratified and the surface outflow of brackish water generates a distinct discontinuity in water column structure. Phytoplankton concentration and primary production both showed pronounced peaks at the immediate stratified side of the front. The coinciding peak in assimilation index (i.e. mg of carbon fixedlmg chlorophyll a/h) suggests that the elevated concentration of chlorophyll at the front is, at least partly, due to locally enhanced growth of the phytoplankton. The concentration of copepod eggs and nauplii, indicative of recent copepod secondary production, also peaked at the immediate stratified side of the front. Similar types of data from a tidal front in October in the northwestern North Sea off the Scottish east coast are presented in Fig. 24 (Kicbrboe el a f . , 1988b). Here again phytoplankton biomass peaks in the transitional region between mixed and stratified water and this pattern closely resembles the horizontal variation in egg production in the copepod Acartia tunsa; egg production is very low in both mixed and strongly stratified water and peaks at the front. Total copepod production shows a similar spatial pattern across the front (Kiorboe and Johansen, 1986). AS in the example above and consistent with the spatial variation in copepod productivity, the concentration of copepod eggs and nauplii is significantly elevated in the frontal region (Kiorboe and Johansen, 1986). In spite of the pronounced pattern in copepod productivity the distribution of total

24. Distribution of copepod productivity and copepod biomass across a tidal front in the northwestern North Sea in October. Distribution of temperature (a), chlorophyll, (b), egg production rate in the copepod Acartia tonsa (c) and biomass of copepods (d) across the front. Modified from Kiorboe et a / . (1988b). FIG.

TUKBULENCE, PHYTOPLANKTON CELL SIZE A N D PELAGIC FOOD WEBS

Depth, rn

0 Temperature,%

20

Depth, m

0

2

40 60

0

2

I

Chlorophyll, pgil

Eggs/? i d 16

-

c

12 -

-

84 -

-

0-

rng C i m 3

n

Copepod biomas

4

56"45'N 01'50'W

90 nautical miles

56"08'N OO"4O'W

53

54

T. KI0RBOE

copepod biomass is largely independent of water column structure at the scale considered. However, in this region mesozooplankton biomass peak further offshore in the stratified region (Kiorboe and Johansen, 1986). With a few exceptions (e.g. Floodgate et al., 1981; Le Fkvre and Frontier, 1988) researchers have generally been unable to relate distributions of copepods to fronts. This lack of distributional coincidence between mesozooplankton and fronts at many frontal types led Le Fkvre and Frontier (1988) to suggest that the structure of the pelagic food web at fronts is determined mainly by the time scale of the physical processes that enrich the photic zone with inorganic nutrients. They considered two examples, a tidal front, where the fertilizing mixing process occurs in a 14 d cycle (neap-spring tide), and a shelf-break front, where the fertilization process is of high frequency (12 h periodicity). Based on distributions of zooplankton biomasses they concluded that in the latter case enhanced productivity was in the form of a classical herbivore food chain, while in the former case primary production was consumed by microorganisms, because herbivorous copepods cannot adapt to short-lived, fortnightly phytoplankton blooms. However, even though advection may prevent accumulation of mesozooplankton biomass with long generation times at dynamic fronts, both copepod growth and production may well be elevated, and the only signal left behind is higher than average abundances of eggs and nauplii that do not contribute significantly to overall biomass. As in the example above it seems to be typical that copepod biomass peaks in stratified water well away from the discontinuity (e.g. Holligan et al., 1984; Kahru et al., 1984; Moal et al., 1985). This situation thus resembles the seasonal cycle in temperate waters, where zooplankton productivity is highest concurrently with the net-phytoplankton bloom occurring at the stratification-destratification interface, while the biomass lags behind and peaks well into the subsequent stratified period. The Skagerrak between Norway and Denmark is characterized by a dome shaped pycnocline (Pingree et al., 1982; see also Fig. 25); thus, vertical water column structure varies horizontally, with mixing regions occurring along the periphery of the area, distinguished from a strongly stratified central region. This makes this region ideal for investigating relations between water column structure and pelagic processes. Fig. 25 presents results from a transect study in the Skagerrak (Kiorboe et d., FIG.25. Horizontal variation in vertical water column structure across the Skagerrak and associated variation in properties of the pelagic community in May. (a) Water density as sigma-t units; (b) volume ratio of large (>Xpm) to small ( t 8 p m ) phytoplankters; ( c ) chlorophyll as fluorescence; (d) bacteria generation time; and (e) fecundities of the copepods Acurtitr cluusi and Ternoru longicornis. Modified from Kiorboe et a / . (1990a).

TURBULENCE. PIIYTOPLANKTON CELL SIZE A N D PELAGIC FOOD WEBS

Depth, 0 m 10

a

DENMARK 1 2 3 4 5

NORWAY 6

7

8

9

12 Station No. - 11

10 L

24 24 25 26

20

Sigma-t

30

27

40 0 10

b

20

30

40 0 05

10

0.5 0.3

20

01

C

30 40 0 10 20

d

2 3

30

5

generation time, d

40

Eggs/Q/d

50 Fecundity P Temora

40

e

0 Acartfa

30 20 10

0 1 2

3 4 5

55

6

7

8

9

10

11

12

Station No

56

T. KIORBOE

b

DOM

1

1 Sediment 1

a

DOM L

1 4

1

FIG.26. Comparison of primary production and pelagic food web structure in (a) rich frontal and (b) oligotrophic, stratified regions of the BeringChukchi Sea. In (a) the phytoplankton is entirely dominated by diatoms and in (b) by pico- and nanophytoplankton. Figures represent carbon flow rates as percentage of primary production rate. Modified from Andersen (1988).

TUIZBCJLENCE. PLiYTOPLANKTON CELL SIZE A N D PELAGIC FOOD WEBS

57

1990a) (see Fig. 4 for a map of the area and position of transect line). As expected, net-plankton blooms occur in the frontal regions at both ends of the transect, and copepod productivity peaks concurrently whereas bacterial growth rate is maximum in the central, stratified region characterized by nano- and pico-sized phytoplankton. Although the spatial pattern in pelagic processes is quite variable in this hydrographically complex region, basic patterns have been found again on subsequent sampling occasions (see Peterson ef al., 1991; Kahru and Leeben, 1991). Thus, it appears that a microbial type of food web characterizes the central, stratified part of the area and a more classical type of herbivorous food chain dominates in the frontal/mixing regions along the periphery. Pelagic food web structure in the Skagerrak thus depends on vertical water column structure in a predictable manner. Yet another example of pelagic food webs at fronts vs. stratified regions from the Bering Sea, perfectly consistent with the above, is illustrated in Fig. 26. The enhancement of net-phytoplankton and mesozooplankton producDepth, m

Larvae/m2

500

r

400 300 200 100

0

FK;. 27. Distribution of herring larvae across a tidal front in the northwestern North Sea in October (after Kiorboe er al.. 19XXb).

58

T. Kl0RBOE

FIG.28. Distribution o f sprat larval growth rate across a coastal front in the southeastern North Sea in August. Values of surface to bottom temperature diffcrenccs (delta t, "C) are contoured by full lines in (a) and larval growth rates (mmid) as inferred from otolith microstructures in (b). Note that growth rates are highest in mixed and frontal waters and decline in strongly stratified rcgions. Unpublished data of P. Munk.

tion at some types of fronts, as illustrated in the several examples above, may have implications at higher trophic levels (e.g. fish) as well. Larger scale fronts, such as shelf break fronts. are frequently associated with fisheries (e.g. Fournier, 1978). For smaller scaled fronts, such as river plume and tidal fronts, locally enhanced feeding and growth conditions at higher trophic levels are most easy to document for plankton predators such as fish larvae. Thus, several studies have demonstrated that the distribution of fish larvae is often related to such fronts (e.g. Townsend et al., 1986; Heath and MacLachlan, 1987; Kiorboe et ul., 1988b; Govoni et al., 1989; see also Fig. 27) and that feeding and growth of fish larvae are indeed enhanced here (Govoni et al., 1985; see also Fig. 28). Such observations are also consistent with the expectation of a classical grazing food chain at oceanographic discontinuities.

TURBULENCE, PIIYTOPLANKTON CELL SIZE A N D PELAGIC F O O D WEBS

Mixed (turbulent)

-

59

Stratified (stagnant)

Nutrient concentration

Phytoplankton biomass

Primary production

New/total production (f-ratio)

% netplankton

Mesozooplankton biomass

Mesozooplankton production and sedimentation

Microbial production

Time or space

-

29. Simple conceptual model of variation in pcAagic processes along spatio-temporal ting-stratification gradients. 'IG.

60

T. KIGRBOE

V. Summary and Conclusions This contribution has focused on the significance of hydrodynamic processes on various scales to patterns in pelagic food web structure. The basic relations between water column characteristics and pelagic food web structure, as schematically summarized in Fig. 29, appear to recur on a wide variety of spatio-temporal scales; from oceanwide and seasonal scales to the temporal scales characteristic of episodic wind-mixing events (days) or the horizontal scales of oceanic fronts (km), for example. It has here been suggested that the causalities behind the relation between water column characteristics and pelagic food web structure are mediated primarily by the effects of turbulence and phytoplankton cell size. The occurrence of significant concentrations of net-phytoplankton is restricted to turbulent, episodic environments occurring at spatio-tempora1 discontinuities in vertical water column structure. This is partly because turbulence both generally increases the availability of new, inorganic nutrients in the photic zone and enhances nutrient uptake in large cells, thus relaxing the competitive pressure for small size in turbulent environments. It is also partly due to the considerable timelag in the numerical response of the mesozooplankton predators of the netphytoplankton. Blooms of net-phytoplankton give rise to short, grazerbased herbivorous food chains and/or to elevated sedimentation of organic material to the sea-floor. Both grazing and sedimentation may be significantly enhanced by the effects of turbulence due to increased plankton contact rates. Nano- and pico-sized phytoplankton occur ubiquitously in the oceans while microbial processes appear less influenced by macro- and mesoscale physical processes and are more controlled by predator-prey interactions; microbial production is thus less variable in time and space. The relative contribution of microorganisms to the overall mineralization of the primary production is consequently most significant in oligotrophic, stagnant waters, where net-plankton is scarce. Because microbial food webs are typically long and primarily based upon regenerated phytoplankton production microbial production contributes insignificantly to fish production in the oceans. In contrast, classical herbivorous food chains are short and based on new nutrients and, thus, give rise to net accumulation of catchable biomass, also at higher trophic levels. Cushing (1989) and Legendre (1990) both emphasized the significance of net-phytoplankton blooms to the fisheries production in the ocean, having in mind first of all blooms associated with larger-scale physical processes, such as the major upwelling regions and the vernal temperature stratification in temperate waters. From the several examples

TUKBULENCE, PHYTOPLANKTON CELL SIZE AND PELAGIC FOOD WEBS

61

in Section IV it can be seen that t h e same processes recur on much smaller spatio-temporal scales. It thus becomes the spatio-temporal “frequency” of (large- or small-scaled) oceanographic discontinuities or mixing events, rather than total primary production, that eventually determines the magnitude of fish production in a particular region.

VI. Acknowledgements Thanks are d u e t o colleagues for permission t o utilize unpublished material a n d t o Professor J. H. S. Blaxter for inviting me t o write this paper.

VII. References Alcaraz, M., Estrade, M. and Marrase, C. (1989). Interaction between turbulence and zooplankton in laboratory mesocosms. Proceedings of the 21st E M B S , Gdansk 14-19 September 1986, Polish Academy of Sciences - Institute of Oceanology, pp. 191-204. Alldredge, A. L. and Gotschalk, C. (1988). In situ settling behaviour of marine snow. Limnology and Oceanography, 33,339-351. Alldredge, A. L. and Silver, M. V. (1988). Characteristics, dynamics and significance of marine snow. Progress in Oceanography, 20, 41-82. Andersen, P. (1988). The quantitative importance of the “microbial loop” in the marine pelagic: A case study from the North BerindChukchi seas. Archiv fiir Hydrobiologie Beiheft, 31,24S-251. Andersen, P. and Fenchel, T. (1985). Bacterivory by microheterotrophic flagellates in seawater samples. Lirnnology and Oceanography, 30, 198-202. Azam, F. and Fuhrman, J. (1984). Measurements of bacterioplankton growth in the sea and its regulation by environmental conditions. I n “Heterotrophic Activity in the Sea” (J. E. Hobbie and P. J. B. Le Williams, eds), pp. 179-196. Plenum Press, New York. Azam, F., Fenchel, T., Field, J. G., Gray, J. S., Meyer-Reil, L. A. and Thingstad, F. (1983). The ecological role of water column microbes in the sea. Marine Ecology Progress Series, 10,257-263. Baars, M. A . and Fransz, H. G. (1984). Grazing pressure of copepods on the phytoplankton stock of the central North Sea. Netherlands Journal of Sea Research, 18, 120-142. Banse, K. (1976). Rates of growth, respiration and photosynthesis of unicellular algae as related to cell size - A review. Journal of Phycology, 12, 135-140. Banse, K. (1982a). Mass-scaled rates of respiration and intrinsic growth in very small invertebrates. Marine EcoIogy Progress Series, 9, 281-297. Banse, K. (1982b). Cell volumes, maximal growth rate of unicellular algae and ciliates, the role of ciliates in the marine pelagial. Limnology and Oceanograp h y , 27, 1059-1079. Berg, H. C. and Purcell, E. M. (1977). Physics of chemoreception. Biophysical Journal, 20, 193-219.

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variability and coupling between primary and secondary production off Chile. Progress in Oceanography, 20, M 0 . Peterson, W. T., Tiselius, P. and K i ~ r b o e T. , (1991). Copepod egg production, moulting and growth rates, and secondary production, in the Skagerrak in August 1988. Journal of Plankton Research, 13, 131-154. Pingree, R. D., Holligan, P. M., Mardell, ti. T. and Harris, R. P. (1982). Vertical distribution of plankton in the Skagerrak in relation to doming of the seasonal thermocline. Continental Shelf Research, 1, 209-219. Platt, H. (1985). Structure of the marine ecosystem: Its allometric basis. Canadian Bulletin of Fisheries and Aquatic Sciences, 213, 5 5 4 4 . Poulet, S. A . , Williams, R., Conway, D. V. P. and Videan, C. (1991). Co-occurrence of copepods and dissolved free amino acids in shelf sea waters. Marine Biology, 108, 373-385. Raven, J . A. and Richardson, K. (1984). Dinophyte flagella: A cost-benefit analysis. New Phytologist, 98, 259-276. Raven, J . A. and Richardson, K. (1986). Marine Environments. In “Photosynthesis in contrasting environments” (N. R. Baker and S. P. Long, eds), pp. 337-398. Elsevier, Amsterdam. Richardson, K. (1985). Plankton distribution and activity in the North Sea/ Skagerrak-Kattegat frontal area in April 1984. Marine Ecology Progress Series, 26, 233-244. Richardson, K., Beardall, J. and Raven, J. A. (1983). Adaptation of unicellular algae to irradiance: an analysis of strategies. New Phytologist, 93, 157-191. Richardson, K . , Heath, M. and Pedersen, S. M. (1986). Studies of a larval herring (Clupea harengus L.) patch in the Buchan area. 111. Phytoplankton distribution and primary production in relation to hydrographic features. Dana, 6, 25-36. Riebesell, V. (1989). Comparison of sinking and sedimentation rate measurements in a diatom wintedspring bloom. Marine Ecology Progress Series, 54, 109-119. Riebesell, V. (1991a). Particle aggregation during a diatom bloom. 1. Physical aspects. Marine Ecology Progress Series, 69, 273-280. Riebesell, V. (1991b). Particle aggregation during a diatom bloom. 11. Biological aspects. Marine Ecology Progress Series, 69, 281-291. Roff, J . C., Middlebrook, K. and Evans, K. (1988). Long-term variability in North Sea zooplankton off the Northumberland coast: productivity of small copepods and analysis of trophic interactions. Journal of the Marine Biological Association of the United Kingdom, 68, 143-164. Roman, M. R., Ducklow, H. W., Fuhrman, J. A., Garside, C., Gilbert, P. M., Malone, T. C. and McManus, G. D. (1988). Production, consumption and nutrient cycling in a laboratory mesocosm. Marine Ecology Progress Series, 42, 39-52. Rothschild, B. J. and Osborn, T. R. (1988). Small-scale turbulence and plankton contract rates. Journal of Plankton Research, 10, 465474. Roy, S . , Harris, R. P. and Poulet, S. A . (1989). Inefficient feeding by Calanus helgolandicus and Temora longicornis on Coscinodiscus wailesii: quantitative estimation using chlorophyll-type pigments and dissolved amino acids. Marine Ecology Progress Series, 52, 145-153. Runge, J. A . (1985). Egg production rates of Cafanusfinmarchicus in the sea off Nova Scotia. Archiv f u r Hydrobiofogie, Supplement 21, 3340.

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Saffman. F. G. and Turner, J. S. (1956). On the collision of drops in turbulent clouds. Journal of Fluid Mechanics, 1, 16-30. Saiz, E. and Alcaraz, M. (1991). Effects of small-scale turbulence on development time and growth of Acartia grawi (Copepoda: Calanoida). Journal of Plankton Research, 13, 873-883. Schleisinger, D . A . , Molot, L. A. and Shuter, B. J. (1981). Specific growth rates of freshwater algae in relation to cell size and light intensity. Canadian Journal of Fisheries and Aquatic Sciences, 28. 1052-1058. Sheldon, R . W., Prakash. A. and Sutcliffe. W. H., Jr (1972). The size distribution of particles in the ocean. Limnology and Oceanogruphy, 17, 327-340. Sheldon, R. W., Sutcliffe. W. H. Jr, and Paranjafe, M. A . (1977). Structure of pelagic food chain and relation between plankton and fish production. Journal of the Canudian Fisheries Research Board. 34, 2344-2352. Skjoldal, H. R. and Wassmann, P. (1986). Sedimentation of particulate organic matter and silicium during spring and summer in Lindispallene, western Norway. Marine Ecology Progress Series, 30, 49-63. Smayda, T. J. (1970). The suspension and sinking of phytoplankton in the sea. Oceanography and Marine Biology Annual Review, 8, 353414. Smayda, T. J. and Boleyn, B. J . (1966). Experimental observations on rhe flotation of marine diatoms. 11. Skeletonerna costatum and Rhizosolenia setigera. Limnology and Oceanography, 11, 18-34. Smetacek, V. (1980a). Annual cycle of sedimentation in relation to plankton ecology in western Kiel Bight. Ophelia, Supplement I , 65-76. Smetacek, V. (1980b). Zooplankton standing stock, copepod fecal pellets and particulate detritus in Kiel Bight. Estuarine and Coastal Marine Science. 2. 477490. Smetacek, V. (1981). The annual cycle of protozooplankton in the Kiel Bight. Marine Biology, 63, 1-11. Smetacek, V. (1984). The supply of food to the benthos. In “Flows of Energy and Materials in Marine Ecosystems: Theory and Practice” (M. J. R . Fasham. ed.), pp. 517-548. Plenum Press. Smetacek, V. (1985). Role of sinking in diatom life-history cycles: ecological, evolutionary and geological significance. Marine Biology. 84. 239-25 1. Smetacek, V. and Pollehne, F. (1986). Nutrine cycling in pelagic systems: A reappraisal of the conceptual framework. Ophelia, 26, 401428. Sommer, U. (1988). Some size relationships in phytoflagellate motility. Hydrobiologia, 161, 125-131. Sommer, U. (1989). Maximal growth rates of Antarctic phytoplankton: only weak dependence of cell size. Limnology and Oceanography, 34, 1109-1 112. Sournia, A . (1982). Form and function i n marine phytoplankton. Biological Review. 57. 347-394. Sournia, A , , Birrien, J.-L., DouvillC. J.-L., Klein, B. and Viollier, M. (1987). A daily study of the diatom spring bloom at Roscoff (France) in 1985. I. The spring bloom within the annual cycle. Estuarine and Coastal Shelf Science. 25, 355-367, Steele, J. H. (1974). “The structure of marine ecosystems”, 129 pp. Harvard University Press, Cambridge, MA. Strathmann. R . R. (1967). Estimating the organic carbon content of phytoplankton from cell volume or plasma volume. Limnology and Oceanogruphy. 12, 41 1-418.

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Autotrophic and Heterotrophic Picoplankton in the Baltic Sea J. Kuparinen and H. Kuosa Finnish Institute of Marine Research, PO Box 33, SF-00931 Helsinki, Finland

Preface .. .. .. .. .. .. .. .. I. Introduction ., .. .. .. .. .. .. A. The Baltic Sea .. .. .. .. .. .. B. Picoplanktonic algae .. .. .. .. .. 11. Methods .. .. .. .. .. .. .. A. Autotrophic picoplankton . . .. .. .. .. B. Bacterioplankton .. .. .. .. .. .. 111. Phytoplankton Succession in the Baltic Sea .. .. .. IV. Autotrophic Picoplankton in the Baltic Sea . . .. .. A . Areal and vertical distribution .. .. .. .. B. Seasonal variation . . .. .. .. .. .. Bacterioplankton in the Baltic Sea .. .. .. .. A. Annual and seasonal variation of bacterioplankton production B. Distribution of bacterioplankton .. .. .. .. V l Factors Controlling Autotrophic Picoplankton .. .. A. Nutrients and temperature . . , . .. .. .. B. Grazing .. .. .. .. .. .. .. VII. Factors Controlling Bacterioplankton . . . . .. .. .. A. Nutrient- and carbon-limited bacterioplankton growth B. Predation control of bacterioplankton .. .. .. V11I. Bacteria in the Pelagic Food Web .. .. .. .. IX. Acknowledgements .. .. .. .. .. .. X. References .. .. .. .. .. .. ..

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Preface The introduction of the concept of a size-structured plankton food web (Williams, 1981; Azam et al., 1983) greatly stimulated studies of aquatic Copyrighr 01993 Acudemic Press Limrted All rightc of reproduction in any form reserved

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microbial ecology in the 1980s, and there was an outburst of publications on the oceans, brackish waters and lakes. In addition, the developing techniques of epifluorescence microscopy and the use of radioactive tracers have provided many new data. This review summarizes results obtained from various locations in the Baltic Sea, which has been described as one of the most intensively

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FIG. 1 . Map of the Baltic Sea. Study sites from which the majority of the data presented in this paper originate are marked on the map: Station 1 = 63"31'N, 19"48'E; 2 = 63"19'N, ZO"17'E; 3 = 59".50'N, 23"lO'E; 4 = S9"3S'N, 23"18'E; 5 = .59"26'N, 21"30'E; 6 = 59"02'N, 21"OS'E; 7 = S8"4S'N, 17"3S'E; 8 = S7"19'N, 20W2'E; 9 = SYlS'N, lS"S9'E; 10 = 55"00'N, IJOOS'E; I I = 54"36'N, lO"27'E.

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studied aquatic environments (Jansson, 1980). While Baltic Sea hydrography and plankton in general are well known, the category of “most intensively studied” does not yet apply to Baltic Sea picoplankton, on which few publications are yet available. To make good this deficiency we include in this review a substantial amount of new data.

1. Introduction A.

The Baltic Sea

The Baltic Sea is a large brackish water basin with limited connection to the North Sea from the southwestern end (Fig. 1). It comprises several more or less distinct basins or subareas (Fig. 1) with pronounced density stratification prevailing throughout the year (Kullenberg, 1981; Malkki and Tamsalu, 1985). The differences in density between the surface and more saline deep waters restrict exchange between the two layers. The salinity of the surface water decreases from more than 2 0 % ~in the opening to the North Sea to below 1%0in the extreme ends of the Bothnian Bay and the Gulf of Finland. In the Baltic Proper, surface water salinities are between 6 and 8700. The primary halocline is at a depth of 60 to 7 0 m in the Baltic Proper

Flc;. 2. Typical distributions of temperature (T), salinity (S) and density (D) in the Bothnian Sea (a), Gotland Deep (b) and the southern Baltic Proper (c).

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and 40 to 50 m in the Bornhom Basin (Fig. 2) (Kullenberg, 1981; Malkki and Tamsalu, 1985), below which salinities between 10 and 13%0are common. This layer receives new water irregularly from inflows through the Danish sounds (Grasshoff and Voipio, 1981). A weak secondary halocline, which separates the frequently anoxic bottom water from the overlying layers, can be detected at a depth of c . 110 and 150m. The extent of this area with insufficient oxygen for macofauna has fluctuated, but is approximately 70,000 km2, mainly in the deep parts of the central Baltic Sea (Andersin and Sandler, 1988). The bottom waters of the Baltic Sea are renewed only after exceptionally strong inflows of North Sea water from the Kattegat. Such inflows occurred in 1913, 1921, 1951 and 1976. Due to the lack of major inflows during the past 14 years, the salinity and density of the deep water have decreased continuously. In most parts of the Baltic Sea a thermocline develops at depths between 15 and 2 0 m in summer (Fig. 2). The layer of cold water from the previous winter can thus be found between the thermocline and the halocline. These two water masses of about the same salinity mix during the autumn turnover. The western Baltic Sea differs from most of the Baltic Sea in its stratification; due to the water exchange from the North Sea, it is salinity rather than temperature dependent (Fig. 2), and this has implications for the picoplankton in the area (Jochem, 1989). Another key factor influencing the Baltic Sea picoplankton is the fact that the Baltic is a northern sea, with Arctic characteristics, especially in its northern parts. The winter conditions emphasize differences between the subareas and their biology. The mean number of ice days varies from 190 in the northern end of the Bothnian Bay (Lepparanta et al., 1988) and more than 140 in the easternmost part of the Gulf of Finland to less than 10 days in the central Baltic Proper and in the Kattegat. The mean maximum annual ice thickness varies from more than 70 cm in the northern Bothnian Bay to less than 10cm in the southern Baltic Proper (Climatological Ice Atlas, 1982). These winter conditions contribute to the large seasonal temperature differences, from -0.3 to about 20°C. The numerous large rivers that bring fresh water and inorganic and organic compounds into the Gulf of Bothnia and to the Gulf of Finland impart special features to the biota of these areas. In particular the Bothnian Sea receives large quantities of allochthonous organics via the rivers (Fonselius, 1986). Due to the terrestrial origin, allochthonous material is highly refractory. Levels from 3.0 to 4.7g/m3 of dissolved organic carbon (DOC) have been reported from the Baltic Proper (Ehrhardt, 1969). Only a small fraction of this pool is liable for bacterial utilization (Bolter, 1981).

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Picoplanktonic Algae

1. Definition Pic0 is an epithet applied to pelagic organisms with a size less than 2 p m (Sieburth et al., 1978). The lower limit of pico-sized organisms, either bacteria, algae or protozoa, is 0.2pm. According to the thorough discussion by Raven (1986), the non-scalable properties of algae constrain their theoretical minimum size to just above 0.2 pm. It seems that in the pelagic environment only viruses and a small fraction of bacteria appear in the femtoplanktonic (0.02 to 0.2 p m ) size fraction. Thus according to the scheme of Sieburth et al. (1978) picoplanktonic organisms are those with cell size under 2 p m . However, this scheme is not totally straightforward when we consider algae. If algae were more or less spherical and if the cells of one species consistently showed very limited variability in size there would be few problems. However, as we know that the form of algae varies considerably, and that the size range of a given species is usually large, the precise definition of picoplanktonic algae in natural phytoplankton communities is difficult. The distinction of picoplanktonic algae as a separate group has clear ecological grounds. One of the most powerful is that it corresponds to a size fraction of pelagic organisms which is probably not effectively grazed by metazooplankton (rotifers, cladocerans and copepods) and, correspondingly, is effectively grazed by protozooplankton (see Section VI). The fraction of organic material produced by picoplankton is thus possibly an indication of the structure of the carbon transfer from primary producers to the higher predators (microbial loop vs. grazing food chain) as discussed by Azam et al. (1983); Ducklow et a f . (1986) and Sherr and Sherr (1988). If we confine ourselves to this ecologically based interest in picoplanktonic algae, the actual upper cell size becomes more a matter of choice than a strict definition. Eventually, it may be possible to choose the upper size limit according to the grazing structure in a given water body, and according to our knowledge of the particle capture ability of zooplankton species. Small algal cells also have other characteristics in common, such as Slow sedimentation rate and high nutrient uptake capacity. These characteristics are probably not as strictly correlated with cell size as grazing, but their existence further validates the separation of picoplanktonic algae as a single group. One reason for defining the fraction of the phytoplankton community to be examined in this review of the Baltic Sea is purely practical. Almost

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all the material from the Baltic Sea used for size-fractionated chlorophyll or production measurements has been gathered using 3 p m polycarbonate filters. The reason for this is not really important in the present context it may be the availability of filters or pure coincidence. At the moment we lack a definitive knowledge of the grazing properties of Baltic Sea zooplankton. Because of the nature of the existing material, and the fact that all size-limits in a phytoplankton community are only loosely defined due to the variable shapes of algal species, rather variable material is included in this review. 2. Organisms (a) Eukaryotic nlgae A number of eukaryotic algae belonging to variable algal classes are of picoplanktonic size or very near its upper end. As discussed above, there is no reason to regard 2 p m diameter as a strict limit when discussing picoplanktonic algae. Thus according to Thomsen’s (1986) excellent overview the scope of this introduction is also somewhat wider than would be required by pure picoplankton. The actual upper size limit for the species depicted in the following discussion is about 5 p m (“ultraplankton”). Because of their very small size many of these algae have certainly been overlooked in routine work (see Section 11). Their positive identification is possible only by electron microscopy and the routinely used Utermohl method gives few possibilities to count these very small cells in plankton samples. From t h e variety of algal classes and genera surveyed by Thomsen (1986). some with relevance to the Baltic Sea can be depicted. Very small Cryptophyceae have been found in the Baltic Sea. Thomsen (1986) presented a photograph of a very small Hemiselmis sp. (aff. anomala) from the Gulf of Bothnia. Hemiselmis virescens Droop is a very small cryptophycean algae (cell size 4 - 6 p m long and about 3 p m wide), which has been recorded in the Western Baltic Sea (Hill, 1992). Of the large class Chrysophyceae, Pedinella tricostata Rouchijajnen (4-6 pm) has been identified from Baltic Sea material (Edler et al., 1984). It is certain that many other small chrysophytes are also present in the Baltic Sea. Similarly, a number of very small solitary flagellated species of Chlorophyceae are probably to be found in the low salinity waters of coastal areas. Some very small centric diatoms (Bacillariophyceae) are found in Baltic Sea samples. The taxonomical work, using electron microscopy, has still to be done. From the genus Thalassiosira at least one very small species, T. pseudonunu. is present in the algal flora of the Baltic Sea (Edler et al.,

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1984). A very small, solitary Chaetoceros species was also found in many samples, but was probably overlooked like many other small diatoms (Kuosa, unpublished). However, even small Chaetoceros cells are a borderline case in picoiultraplankton. Although the cell size of Chaetoceros may be within the picoplanktonic size range, the seta will enlarge the effective size of the cells in grazing and in fractionation procedures. The algal class Eustigmatophyceae shows specific ultrastructure and pigment composition. An algal culture maintained at the Tvarminne Zoological Station has been assigned to the genus Nannochloropsis. This small species (2-3pm) may be common in the waters of the northern Baltic Proper, but as is the case in all other small species. we have very little knowledge of its areal distribution and abundance. Micromotzas pusilla (Loxophyceae) is a species within the same size range as Nunnochloropsis sp., but although it is flagellated, it is impossible to differentiate from Ncnnochloropsis sp. in normally preserved phytoplankton samples. Micromonas pusilla has been identified by electron microscopy in samples taken near Tvarminne (Thomsen, 1979). Micrornonas pusilla (1-3 x 1 p m ) has a wide distribution in the oceans (Throndsen, 1976), and it may commonly exceed cell numbers of 10h/l (Thomsen, 1986). Another very small species (1.5-2.5 pm) of the class Loxophyceae reported from the Baltic Sea is Pedinomonas micron (Thomsen, 1986). Of the related class Prasinophyceae some small species have appeared in samples studied by electron microscopy (Hallfors and Niemi, 1986). These are: Mantoniella squamata (3-4 pm), Nephroselmis minuta (<4 pm), Pseudoscourfieldiu marina (<4 pm) and Pyramimotzas virginica (<4 pm). According to Hallfors and Niemi (1986) about 25 species of the genus Chrysochromulina (Prymnesiophyceae) have been found from the northern Baltic Proper. The smallest of them belong to the ultraplankton, as does also Prymnesium parvum (Prymnesiophyceae), which is a member of the Baltic Sea phytoplankton (Hallfors and Niemi, 1986). In the Baltic Sea, as in many other marine areas. there is an almost total lack of integrated data on taxonomical composition and abundance of ultraplankton. The size of an alga is not the only factor determining its role in the ecosystem. The smallest size-class of phytoplankton may contain algae with very different morphological characteristics (flagellated cells, coccoid cells, diatoms with and without seta and cells with scales) and physiology (photosynthetic pigments and phagotrophy). One specific PhYsiological feature is the production of toxins. In 1988, the toxic bloom In the North Sea coastal waters of Sweden and Norway consisted of a small flagellate, Chrysochromulina polylepis (Rosenberg et al.. 1988), and PrYmnesium parvum is also known to be potentially toxic. Thus in order

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to fully understand the role of the smallest algae in the pelagic ecosystem we need cooperation between algal taxonomists, physiologists and ecologists.

(b) Picoplanktonic cyanobacteria Picoplanktonic cyanobacteria in the Baltic Sea mainly belong to two separate types: an ellipsoid or roundish type and a rod-shaped type. The small, almost round or ellipsoid coccoid cells have been recorded from the coastal and open waters of the whole Baltic Sea (Schmaljohann, 1984; Jochem, 1988; Kuosa, 1988a; Anderson et al., in prep.). The cell size of this type varies from very small (<1 pm) to relatively large (2-3 pm). However, the bulk of single coccoid cyanobacteria in the Baltic Sea belong to the true picoplankton. The cell division appears to be Synechococcus-type and these cells correspond to the existing group of oceanic coccoid cyanobacteria addressed to the genus (e.g. Waterbury et al., 1986). It is not known whether several taxa of small coccoid cyanobacteria exist within this group, or whether the coccoid cells with apparently different photosynthetic pigments belong to another taxon. Both Schmaljohann (1984) and Kuosa (1988a) observed the tendency of small coccoid cyanobacteria to form small cell groups. These colonylike assemblages contain up to ten cells in irregular order, many times several cells in a cell row. Their formation seems to be most pronounced during summer. These formations may be a consequence of rapid cell division and the subsequent loose attachment of daughter cells, and not true colony formation as in the cyanobacterial genera Merismopedia and Microcystis, which are also present in the area. However, if these cell groups are not easily broken by physical disturbance, they may have consequences for the ecological characteristics of the algae. First, these cell groups will be available to larger grazers. Secondly, in fractionation studies, cell groups may be retained by filters with large pore-size or they may be more or less broken by the filtration. We d o not yet know how these cell groups behave, and consequently it is unclear whether our results are biased to some extent. Other forms of picoplanktonic cyanobacteria reported from the Baltic Sea are rarer. Rod-shaped cyanobacteria have been reported from the Gulf of Bothnia (Anderson et al., in prep.) and the Gulf of Finland (Kuosa, 1988a). In the latter publication an unidentified species with long, twisted cells was also introduced. The systematic position of both taxa is still unclear, although the rod-shaped small cells may well be single cells of a very thin trichomatous cyanobacteria (Pseudanabaena sp.). Some specific characteristics (tendency to form short cell chains and sometimes tapering cell ends) point to this possibility.

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II. Methods A . Autotrophic Picoplankton 1. Counting The most widely used method of counting phytoplankton samples is inverted microscopy preceded by sedimentation in Utermohl chambers. Cell counting is usually performed with a 40x objective. This method has many drawbacks when it comes to counting pico- or ultraplankton. Firstly, there seems to be a clear tendency to obtain higher abundances if a small volume is sedimented (Huttunen and Kuparinen, 1986). This may be explained by coverage by other particles if larger volumes are sedimented or simply by the human factor of observing small objects more easily among fewer particles (Huttunen and Kuparinen, 1986). However, the sedimentation of small particles may also be incomplete (Kuosa, 1988b). Secondly, there are seldom possibilities to distinguish between very small colourless and chloroplast-containing cells. Thirdly, the smallest cells, picoplanktonic cyanobacteria, cannot be counted by inverted microscopy using a 40x objective. Epifluorescence microscopy has been used in counting picoplanktonic algae. Preservation of samples has normally been performed either with unbuffered glutaraldehyde or formaldehyde. However, glutaraldehyde was found to be superior in long-term preservation (Kuuppo-Leinikki and Kuosa, 1989). Picoplanktonic cyanobacteria can be counted from unpreserved samples due to their ability to withstand filtration without cell rupture. However, most eukaryotic cells will burst if filtered without aldehyde fixation. Black membrane filters (e.g. Nuclepore) with a pore size of 0.2 p m are used in epifluorescence counting. Picoplanktonic cells are usually counted with a 1 0 0 ~oil immersion objective. Epifluorescence counting is based on the autofluorescence of photosynthetic pigments of picoplanktonic algae. Different excitation wavelengths may be chosen to help the separation of picoalgal groups (Becker, 1985). Under blue excitation eukaryotic algae will fluoresce conspicuously. The cells with a high concentration of phycoerythrin (cyanobacteria) will have orange fluorescence under, for example, a Leitz filter set 12 (Fig. 3 ) . This can be used as a marker of small cyanobacteria. However, with blue excitation light the intensity of fluorescence of picoplanktonic cyanobacteria is relatively weak (Craig, 1987). Furthermore, cyanobacterial cells without phycoerythrin will not be observed under blue excitation. Thus blue excitation is most suitable for counting eukaryotic picoplankton only.

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R

FIG.3 . A: Chlorophyll a (Chl a), c-phycocyanin (PC), and c-phycoerythrin (PE) absorbance spectra in vivo. B: Transmission spectra of exciting wavelengths produced by Leitz G, I? and M2 filter blocks (Becker, 1985).

Green excitation (e.g. Leitz filter set M2) may be used in counting picoplanktonic cyanobacteria (Becker, 1985; Craig, 1987). Cyanobacteria containing phycoerythrin will be seen yellow-red and those containing phycocyanin will be red. Chlorophyll a fluorescence is also visible (red), although weak, but cyanobacteria containing phycocyanin are easily separated from eukaryotic chloroplasts by their disappearance under blue excitation. Cryptophycean algae also fluoresce under green excitation due to the phycobilins they contain, but they are usually not erroneously counted as cyanobacteria due to the size and form of the chloroplast. Cells and flagella of algae may be made visible by using different fluorochromes (e.g. Haas, 1982; Caron, 1983; Sherr and Sherr, 1983). The separation of autotrophic and heterotrophic cells is then also possible. Picoplanktonic cyanobacteria may be counted from the fluorochrome-stained filters using green excitation provided that the fluorochrome used does not mask the autofluorescence. For example, one useful fluorochrome, proflavine, which does not form significant background fluorescence in the northern Baltic Proper samples, somewhat masks chlorophyll autofluorescence (Kuosa, 1988b). A separate count for picoplanktonic eukaryotic algae from an unstained filter is therefore recommended. Although it is advisable to count the samples as soon as possible, it is

BALTIC SEA PICOPLANKTON

83

natural that in some cases they have to be kept for some time. Baltic Sea samples of picoplanktonic cyanobacteria may be kept for long periods at -20°C as a water sample without any preservative (Kuuppo-Leinikki and Kuosa, 1989). Other samples can be kept as prepared filters at -20°C (Booth, 1987; Kuuppo-Leinikki and Kuosa, 1989). Glutaraldehyde should then be used as preservative (Kuuppo-Leinikki and Kuosa, 1989).

2. Biomass, chlorophyll and production estimates Picoplanktonic biomass may be estimated from epifluorescence counts. However, in this case the general problem of estimating biovolumes must be faced. Estimation of the biovolumes of picoplanktonic cells may be carried out using an ocular grid, for example. It is advisable to use several standard mean biovolumes and count size-classes of cyanobacteria and eukaryotic algae, as their cell volume will vary considerably (e.g. Jochem, 1988). There is, of course, a contradiction between the number of size-classes and counting efficiency, which must be resolved in each study. It is not known how different preservatives will affect the biovolume estimates of picoplanktonic algae. If carbon estimates are standardized, conversion factors may be used. In this study, Jochem (1988) assumed a cell density of 1.1g/ml, dry weight 30% of wet weight and carbon content 50% of the dry weight. The percentage of dry weight from wet weight is probably the main source of error in the calculation. However, this calculation, giving a slightly higher carbon content than Edler (1979), but a considerably lower content than Strathmann (1967) and Eppley et al. (1970), is probably a reasonable estimate of the carbon content. We have no direct measurements of the carbon content of Baltic Sea picoplanktonic algae. In any case, a more critical step in estimating carbon biomass is volume calculation. Most of the problems in measuring picoplanktonic biomass, chlorophyll and production are met in the fractionation procedure. Both 2 and 3 pm filters have been used for separating the picoplanktonic fraction from the rest of the phytoplankton community (Table 7 ) . The choice of the filter is, as discussed earlier, at the moment rather subjective. Furthermore, relatively large cells will pass both 2 and 3 p m filters (Huttunen and Kuparinen, 1986), and some true picoplanktonic cells will probably be retained by these filters (Li, 1986). Li (1986) presented a good review of the effect of vacuum on the recovery of picoplankton in filtration, and his recommendation was to use a slight vacuum instead of gravity filtration if the intention is to get the bulk of picoplanktonic algae in the filtrate. Both Waterbury et al. (1986) and Kuosa (1990a) found that 1 pm fractionation is useful in separating picoplanktonic cyanobacteria from the rest of the

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J. KUPARINEN A N D H . KUOSA

TABLE1. AN EXAMPLE OF THE METHODSLJSED I N FRACTIONATED PRODUCTION MEASUREMENTS

Reference PreiPost

(1)

(2) (3) (4) (5)

Post Post Post Pre Post

Filter

Part/Total

Vacuum

Stopping Bacterial uptake

2 pm 3pm 3pm 3pm 3pm

Part (0.6 p m ) Part (GF/F) Part (0.2 p m ) Total Part (0.2 p m )

Not given <25 kPa Gravity Gravity Not given

No DCMU Formalin Formalin No

No No No -

Yes

“Pre/Post” indicates pre- or post-incubation fractionation, ”Filter” the filter type used in separating picoplankton, “PartiTotal” the type of production estimate (particulate production o r total production), “Vacuum” the amount of vacuum used in fractionation, “Stopping” how production measurements were stopped and “Bacterial uptake” if bacterial uptake of exudates was taken into account in the estimates of picoplanktonic primary production. (1) A n d e r s o n er al. (in prep.); (2) Jochem (1989); (3) Huttunen and Kuparinen (1986); (4) Kuosa (1990~);(5) Larsson and Hagstrom (1982).

algal community. A t the same time, unfractionated samples may be counted in order to estimate the amount of picoplanktonic cyanobacteria retained by the 1 p m filter and to correct the <1 p m measurements to total cyanobacterial community. Filtration as such is probably not very harmful to picoplanktonic cyanobacteria (Waterbury et al., 1986). However, filtration will evidently be harmful to eukaryotic cells (Goldman and Dennett, 1985; Kuosa, 1988b). The problem is present in all fractionated measurements, but is most pronounced in production estimates. According to Waterbury et al. (1986) cell fragments of large algal cells are caught by the filters with very small pore-size (in this case 0.2 p m Nuclepore polycarbonate filters), and they falsely increase the estimates of particulate production of the <1 pm fraction. This will be a problem as yet unsolved also in fractionated chlorophyll measurements. T h e choice between pre- and post-incubation fractionation in primary production measurements will affect the results considerably. In postincubation fractionation, cell fragments will possibly cause an overestimation of picoplanktonic production values. Pre-incubation fractionation will similarly cause fracture, but instead of a false size distribution the production of size fractions will be reduced, provided that the cell fragments are not photosynthetically active. One problem in postincubation fractionation is the uptake of algal exudates by bacteria, which is eventually included in picoplanktonic production. This characteristic was used by Larsson and Hagstrom (1982) when they estimated bacterial

BALTIC SEA PICOPLANKTON

85

uptake of algal exudates by the difference between post- and preincubation fractionated samples. Another problem present at least in the northern Baltic Proper is the difficulty in measuring particulate production. According to Lignell and Kuosa (1988), filtration with polycarbonate or membrane filters cause! considerable leakage of dissolved organic compounds from algae. Thi! leads to a highly unrealistic fraction of “net algal exudation”. Thu: post-incubation fractionation is of little use in production measurement: as it will give gross underestimates of those fractions susceptible to cel breakage in filtration. A pre-incubation fractionation procedure wil probably give somewhat more realistic (under)estimates of the productior of small size fractions. It will also give the opportunity to measure tota’ net production (particulate dissolved bacterial uptake of exudates: from the fractionated samples (Niemi e? al., 1983). Another positive characteristic of the pre-incubation fractionation procedure is the exclu. sion of the bulk of predators from the picoplanktonic size fraction. Both normal dark incubations and DCMU-treated incubations have been used as blanks in picoplanktonic production measurements. LI (1986) discussed the merits of both. More studies in the Baltic Sea should be carried out before any definite conclusions can be reached. However. Waterbury et al. (1986) used normal dark incubations in their studies.

+

B.

+

Bucterioplankton

Bacterioplankton cell count, biomass and production estimates were based on the following methodology, if not specified otherwise in the text. This applies to all studies quoted as “this study”, “recalculated from” and “unpublished cruise reports”. In the studies in which values for thymidine incorporation were given in “mole x unit volume x time”. recalculation was performed according to the conversion factor: presented here.

Bucterioplankton cell counts. Bacterioplankton cells were counted under an epifluorescence microscope (Hobbie et al., 1977). Water samples of 20ml were preserved with 1.01111 of 0.2pm filtered neutral formalin (39%). Subsamples of 1-2ml were mixed with 4 m l of 0 . 2 p m tiltered demineralized water and filtered onto black 0.2 pm Nuclepore filters (25 mm diameter). The filters were covered with particle-free acridine orange solution (1 gA). After 5 min of staining, the filters were sucked dry, air dried and stored in the dark. Bacteria were counted under blue light (filter set 12/3) with a Leitz Laborlux D epifluorescence microscope.

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J . K U P A R l N E N A N D H . KUOSA

[-'HI-thymidine incorporation into cold TCA precipitate (TTI). Duplicate or triplicate water samples of 10-20ml were incubated with about 10 nmol/l of methyl-['HI-thymidine (Amersham, 1.5-2.2 TBqimmol, 4060 Ciimmol) for 3&120 min. Incubation with 10 nmol/l of thymidine is likely to be sufficient to saturate uptake in the open sea area of the Baltic Sea (Heinanen, 1992b, submitted) except in the northern Bothnian Sea, where >20 nmolil concentrations are needed (Anderson et al., in prep). Incubations were terminated by adding 100 p l 39% formalin. A prekilled sample was used as blank. The samples were filtered onto 0.22 pm cellulose acetate or cellulose nitrate filters, rinsed 5-10 times with 1 ml 5% ice-cold trichloroacetic acid (TCA) and radioassayed in an LKB-Wallac 1209 RackBeta liquid scintillation counter, using PCS (Amersham) as scintillator. Bacferioplankton production. Data on ['HI-thymidine incorporation into macromolecular material was converted into carbon production using the following factors: (1) from incorporation into cell production by an empirical factor of 1.1 x 10'' cellsimol (Riemann et al., 1987); (2) to biovolume production by mean cell volumes of 0.052 pm3 in samples from the Gulf of Bothnia (Heinanen, 1992a), 0.059 pm' from the Baltic Proper during the spring period and 0.032 pm3 during the summer period Heinanen and Kuparinen, 1991); and (3) to carbon net production by assuming a cell carbon content of 0.35pg C/pm3 (Bjcirnsen, 1986). The value of B j ~ r n s e n (1986) was chosen because it has been used in monitoring studies in the Baltic Sea and is close to the mean value measured in temperate and arctic seas. (Bratbak, 1985; Nagata, 1986; Kuparinen, 1988; Lee and Fuhrman, 1987; Bjornsen and Kuparinen, 1991). Conversions (2) and ( 3 ) were also applied to calculations of bacterial biomass values. Batch culture experiments. Bacterioplankton batch cultures were prepared for the conversion factor and for the enrichment experiments by diluting natural bacterial assemblages in filtrates (800 ml; 0.8 p m Nuclepore filter) of sea water with filtered (7200ml; 0.2p.m Nuclepore filter) sea water from the same site. T h e 0.8pm filtration removes the majority of predators. Small flagellates may, however, penetrate through the filter and therefore when bacteria were observed microscopically, flagellate appearance was also recorded to see whether flagellate growth occurred in the batches. The seawater cultures were divided into four 2.5-litre glass or polycarbonate bottles in aliquots of 2 litres. One bottle was kept as a control and the remaining were enriched with nutrients (20pg/l of P as KH2P04 and 80 of pg/l of N as NH,Cl) and/or carbon (200pgC/1 as

BALTIC SEA PICOPLANKTON

87

C12H2201 The nutrient and carbon enrichments represent levels that bacteria may experience during bloom periods and/or upwelling. The batch cultures were incubated in the dark at in situ temperature (f2"C).

111. Phytoplankton Succession in the Baltic Sea The Baltic Sea shows remarkable variability in both seasonal and areal physical characteristics of the environment (Section I). This is also reflected in the phytoplankton succession, which varies greatly between sub areas. Fig. 4 shows the general annual cycles of phytoplankton development in four parts of the Baltic Sea. In spring, phytoplankton development starts after the incoming solar radiation increases to a level at which it can sustain algal growth in deep mixing water. Thus the onset of the vernal bloom tends to be earlier towards the southern Baltic Sea. The vernal phytoplankton community consists mainly of cold-water diatoms and dinoflagellates. After thermal stratification develops, and surface water is physically cut off from the deeper nutrient-rich water layer, the spring bloom declines as a result of the removal of inorganic nutrients from above the thermocline. The summer stage is characterized by low algal biomasses, moderate productivity and the dominance of small algae. In late summer, a dense cyanobacterial bloom of large trichomatous species may develop, but its intensity and areal distribution varies greatly from year to year. Autumnal cyanobacterial blooms do not occur in the Bothnian Bay due to the high inorganic N : P ratio (Niemi, 1979), as these species possess heterocytes to fix molecular nitrogen. Phytoplankton development in the Bothnian Bay is also generally limited by the low availability of phosphorus together with the short average growth season. In the southern and western Baltic Sea autumnal diatom maxima are regular. Dinoflagellates also appear in the area which is heavily influenced by oceanic water.

IV. Autotrophic Picoplankton in the Baltic Sea A. Areal and Vertical Distribution Although not well documented, there are some indications of an omnipresence of picoplanktonic algae in the Baltic Sea. The first microscopical observations of very small algal cells were from the early 1980s (Larsson and Hagstrom, 1982; Schmaljohann, 1984; Huttunen and

88

J . KUPARINEN A N D H . K U O S A I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

BB

J

I

I

I

I

I

I

I

I

F M A M J J A S O N D

FIG.4. Generalized annual cycles of phytoplankton development in different parts of the Baltic Sea. Vertical scale arbitrary. BB = Bothnian Bay. NB = northern Baltic Proper, the Gulf of Finland and the Bothnian Sea, SB = southern Baltic Sea, WB = western Baltic Sea (redrawn from Hallfors and Niemi, 1986).

89

BALTIC SEA PICOPLANKTON

Kuparinen, 1986). Picoplanktonic cyanobacteria were evidently not included in the data of Larsson and Hagstrom (1982) and Huttunen and Kuparinen (1986), as these were obtained by the Utermohl technique. Thus Schmaljohann (1984), who used electron microscopy, was the first to document picoplanktonic cyanobacteria from the Baltic Sea. Recent studies have used epifluorescence microscopy for counting both eukaryotic and prokaryotic algae. Most of the studies on picoplanktonic algae in the Baltic Sea have been made at coastal stations (Table 2). The data from the Bothnian Sea indicates almost total dominance of cyanobacteria in the picoplanktonic size-class (Anderson et al., in prep.). Eukaryotic picoplankton is abundantly present ( 103-104 cells/ml) in other coastal stations of the Baltic Sea (Tvarminne, Ask0 and Kiel). In a north-south transect through the whole Baltic Sea, eukaryotic picoplankton was present in abundance at all stations in early summer (Tanskanen et al., in preparation). Picoplanktonic coccoid cyanobacteria have been reported from all stations studied along the Baltic Sea coast, from the Gulf of Bothnia to the western Baltic Sea (Jochem, 1988; Kuosa, 1988a, 1991; An d e r so n et

TABLE 2. GENERAL ESTIMATES OF THE NUMBEROF PICOPLANKTONIC CYANOBACTERIA ( i d ) AND THEIR GENERATION TIMES(id) AT THREECOASTAL STATIONS OF THE BALTICSEA AND AT ONE TRANSECT IN THE OPENBALTIC

PROPER Area

Season

Abundance

Growth

Umea (1989, 1990)

Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Summer Autumn

104 1o4-1oS 1 4 x lo5 105-1 o4 104 104-105 104-106 5 x 105-104 n.d. 104 104-2 x 105 105-104 10"

148 28 5 4 ? (very long) zero-growth-5 days 2 n.d. n.d. n.d. n.d. n.d. n.d.

Tvarminne (1986, 1988) Kiel (1986) Baltic Proper (June 1987)

Data from A n d e r s o n ef al. (in prep.), Kuosa (1991), Jochem 1988) and Tanskanen et al. (in Prep.). n.d. = not determined.

90

J . KUPARINEN A N D H. K U O S A

al., in prep.). Similarly, two as yet unpublished cruise studies revealed high numbers of picoplanktonic cyanobacteria in the open sea area of the Baltic Proper (Tanskanen et al., in preparation) and the Gulf of Finland (Kuosa, unpublished). The transect study of the low-salinity waters of an inner archipelago bay, Pojo-bay, also revealed the existence of small cyanobacteria (Kuosa, 1988a). However, single cruise reports provide little information o n the role of picoplankton in the Baltic Sea. The occurrence of cyanobacteria with different fluorescence characteristics (yellow-red/red under green excitation) has not yet been reported. However, according to some data sets from the Gulf of Finland (Kuosa, unpublished), it seems that cyanobacteria containing phycocyanin (red under green excitation) are common only in very diluted low-salinity waters of the inner archipelago and river outlets. Jochem (1988) and Kuosa (1988a, 1990b) have studied the vertical occurrence of picoplanktonic algae. The absolute maximum was found at the surface in both studies. This is not surprising as in the Baltic Sea the euphotic layer is usually relatively shallow (10 to 20 m), and no real deep maximum can be expected under normal circumstances. However, we still lack systematic studies of the thermocline and pycnocline regions of the Baltic Sea, which may both possess deep chlorophyll maxima apparently due to algal growth at these depths (Jochem, 1989; Kuosa, 1990b). Although the maximum abundance of both eukaryotic and prokaryotic picoplankton is in surface water, they are both also found in relatively high numbers (compared with larger algae) at greater depths (e.g. Kuosa, 1988a). It is still unknown whether this deep population is active, and whether it may function as a seed population after upwelling events, which are common in some areas of the Baltic Sea. Meromictic lakes, which were earlier connected with the sea, but which are now isolated by rising land level, are specific ecosystems along the coasts of the Baltic Sea. They still possess a deep saltwater layer, which prevents normal circulation in the lake. Craig (1987) found picoplanktonic cyanobacteria in abundance in one of those lakes in Aland. They occurred as a thin layer just under the halocline.

B . Seasonal Variation 1. Abundance There is limited seasonal variation of eukaryotic picoplankton. These plankton appear to be present in low numbers (10'/ml) during the winter (Kuosa, 1991), and to increase in abundance during early spring. Both

BALTIC SEA PICOPLANKTON

91

Jochem (1988) and Kuosa (1991) recorded the dominance of eukaryotic picoplankton in cold water. Kahru et al. (1991) reported the growth of “unidentified small-sized ( 1 4 p m ) phytoplankton” in early spring. This description fits well with the eukaryotic picoplankton fraction which was studied by both Jochem (1988) and Kuosa (1991). Huttunen and Kuparinen (1986) also reported high abundance of “Nannochloropszs sp.” during spring. Another season of high relative eukaryotic abundance of the picoplanktonic fraction is autumn, at least at the Tvarminne station (Kuosa, 1991). The abundance of eukaryotic picoplankton during the growth season is about 1-3 x 10‘lml at Tvarminne (Kuosa, 1991). This compares well with the values from oceans, which range up to 3 4 X 104/ml (Joint, 1986). Picoplanktonic cyanobacteria show wider seasonal variation. All existing material indicates a clear positive correlation between cyanobacterial number and water temperature (Jochem, 1988; Kuosa, 1991; Andersson et al., in prep.). Temperature has been proposed as a controlling factor of cyanobacterial growth in all these studies. This also fits the general picture arising from the existing literature (Krempin and Sullivan, 1981; El-Hag and Fogg, 1986; Joint, 1986; Waterbury et al., 1986). There is, however, one reference (Shapiro and Haugen, 1988) to a cold-water race of Synechococcus. Strangely, it seems that the Baltic Sea cyanobacteria do not incorporate this characteristic, which would be expected due to the long annual cold season. The number of picoplanktonic cyanobacteria (Table 2) has been found to be high at all those stations from which we have proper data sets (Jochem, 1988; Kuosa, 1988a, 1991; Andersson et al., in prep.). Generally, Baltic Sea values are of the same order of magnitude as oceanic coastal abundances (Joint, 1986). There seems to be a maximum at the Tvarminne station, in which summer abundances are nearly an order of magnitude higher than at two other stations. Even worldwide data of the cyanobacterial numbers show the northern Baltic Proper station at Tvarminne to be at the higher end of the range (Joint, 1986). The highest values at Tvarminne can only be matched by the values from the tropical Pacific (Li er al., 1983). 2. Biomass and production Almost all values from spring indicate low contribution of picoplankton to the total algal biomass and production (Jochem, 1989; Kuosa, 1990a; Andersson er al., in prep.). However, the fraction of picoplanktonic biomass and production has been more intensively measured during summer. The available data indicate the high contribution of picoplank-

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J . KUPARINEN A N D E l . KUOSA

TABLE3. ESTIMATES OF T H E S H A R E OF PICOPLANKTONIC ALGAE T O T H E TOTAI. BIOMASSES AND PRODUCTION I N SUMMER, WHICHIS THE ONLYSEASON WITH A NUMBEROF MEASUREMENTS.DUE TO SEVERALMETHODOLOGICAL DIFFERENCES THE VALUES A R E Nor FULLYCOMPARABLE

Area

Biomass

Production

Umei Tvarminne Ask0

25% (bm) 2(1-50% (chl) 10-25% (bm) 2-20% (chl)

3&70% 2&50% 20 % 7-38%

Kiel

Data from Anderson et al. (in prep.), Kuosa (1990a). Larsson and Hagstrom (1982) and Jochem (1989). bm = the share estimated from phytoplankton biomasses and chl = the share estimated from chlorophyll values.

ton in either biomass (or chlorophyll) values or production. Thus, although the methodology varies, it may be concluded that picoplanktonic algae are of considerable importance in the Baltic Sea during the stratified low production period in summer. In one study (Kuosa, 1990a), the fraction of picoplanktonic cyanobacteria of the total algal biomass and production was studied (Table 3). In those samplings in summer from which the estimation could be made, the fraction was 2 5 4 0 % of the chlorophyll and 1.5-50% of the production. The fraction was consistently well over SO% of the total chlorophyll and production in the < 3 p m fraction, and in one sampling it even exceeded the <3 Fm production due to the abundant cyanobacterial cell groups in the sample. Generally, picoplanktonic cyanobacteria thrive in low-nutrient environments in the Baltic Sea. They are a typical component of the regenerated production period of stratified warm water. There are clear indications of the decreasing importance of the picoplanktonic fraction toward the eutropic end of the oligotrophic gradient. Larsson and Hagstrom (1982) estimated picoplanktonic biomass to account for up to 10% at a eutrophicated station and 2.5% at the control area in Himmerfjarden, and Jochem (1988) found that the relative fraction of picoplankton decreased in the more eutrophic waters near Kiel.

V. Bacterioplankton in the Baltic Sea A. Annual and Seasonal Variation of Bucterioplunkton Production Seasonal succession of bacterioplankton production (mg Clm'lday) on the coast of the Gulf of Finland during different years is presented in Fig. 5.

BALTIC SEA PICOPLANKTON

93

a 1

..._.,,......................

-.,.,.!.?

b

C . . ..: :., . .. ......... ... ... .;;.. .. .. . . .. ...

-.,, ,

;

l,jlj

JFMAMJ JASOND FIG.5 . Bacterioplankton production (mgC/m7/d) on the coast of the Gulf of Finland during 1985(a), 1986(b) and 1988(c). Figure a is redrawn from Kivi et al. (submitted), b from Kuosa and Kivi (1989) and c from Lignell et al. (submitted) and Autio (1992). Temperature IS given as the dotted lines.

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J . KUPARINEN A N D H . KUOSA

Lines were not drawn between the data points (Fig. 6) because only in the study of Kuosa and Kivi (1989) (Fig. 5 ) was the sampling frequency high enough in relation to the time-scales of bacterial growth (doublings in days), and to physical forcing such as water mass movements (in days), for the data actually to describe seasonal features at the study site. These data from the Gulf of Finland (Fig. 5 ) show some general features of bacterioplankton seasonality. As a response to phytoplankton spring bloom, bacterioplankton cells (Vaatanen, 1976; Virtanen, 1985; Kuparinen, 1988), cell volumes and biomass (Kuparinen et al., 1984; Virtanen, 1985; Kuparinen, 1988; Lahdes et al., 1988) and production (Kuparinen, 1988; Kuosa and Kivi, 1989; Lignell, 1990a) start to increase, reaching their maximum values about 2 weeks after the phytoplankton peak (Kuparinen ef al., 1984; Kuosa and Kivi, 1989; Lignell et al., submitted). This response of bacterioplankton to the spring bloom has been recorded throughout the Gulf of Finland (Heinanen and Kuparinen, 1991), in a cruise study in which all stations showed elevated production values along the Gulf (Fig. 6) during phytoplankton spring bloom development (Leppanen et al., 1991). Moreover, two of the quasisynoptic transects through the central line of the Gulf of Finland showed low values in the northern Baltic Proper and elevated values at the entrance to, and in the eastern parts of the Gulf of Finland (Fig. 6), parallel to the trends found in phytoplankton production (Lassig et al., 1978; Leppanen et a[., 1991). The spring bacterioplankton peak is followed by a decline in early summer. In late summer, with increasing temperature and with the development of cyanobacteria, another peak in bacterioplankton variables is usually observed (Kuparinen et al., 1984; Virtanen, 1985; Kuosa and Kivi, 1989; Kivi et al., 1992). During years with favourable hydrographic and illumination conditions, a phytoplankton autumn peak may develop, which gives rise to autumnal bacterioplankton production as seen in the data from Kuosa and Kivi (1989) (Fig. 5 ) . Some general features in the seasonal course of bacterioplankton can also be identified in the northern and in the southern Baltic Sea. In the Bothnian Sea, which is characterized by long winter seasons with ice coverage of about 5 months (December-April), bacterioplankton growth starts after the initiation of phytoplankton spring bloom (Fig. 7), followed by a decline and another rise with the increasing temperature in late summer (Hagstrom, unpublished data). Due to the climatic conditions, the late autumn phytoplankton peak does not occur in the northern Baltic. The southwestern Baltic Sea is most complex with regard to the features of seasonal bacterioplankton development due to physical

95

BALTIC SEA PICOPLANKTON

12.5 I

10 -

7.5 -

. U

-=;

0

cn

=L

5 -

2.5 -

0

20

I

I

I

I

I

1

21

22

23

24

25

26

27

Longitude FIG.6 . Bacterioplankton production (pg Cilld) during the spring period in a transect running from the western part of the northern Baltic Proper to the eastern part of the Gulf of Finland in 1990 (a map of the transects is presented in Heinanen and Kuparinen, 1991).

forcing (the inflow of North Sea water, numerous freshwater affluents, etc.) resulting in instability of stratification and its annual development. However, the response to phytoplankton spring development, and the high productivity period during the late summer months (Fig. 7) are similar to other areas in the Baltic Sea (Gocke et af., 1990). The longer summer period in the southern Baltic Sea, with moderately high light levels compared with northern areas, frequently allows an autumn phytoplankton bloom to develop, which in turn gives rise to autumn bacterioplankton development (Rheinheimer, 1981; Kirstein, 1991). Annual variation, measured with the same methodology and applying similar conversion factors, can be compared with the data of bacterioplankton productivity from the Gulf of Finland (Fig. 5 ) . Similar maximum values in spring were recorded each year, but the summer values differed considerably in different years. In 1986, production remained at a low level throughout the summer compared with 1985 and 1988. In

96

J . KUPARINEN AND H . KUOSA

C 75

-

50-

FIG. 7. Bacterioplankton production (mgC/m3/d) in the Bothnian Bay (a, b) (from different years) and in the Kiel Bight (c) during the late 1980s. Figures a and b are redrawn from Hagstrom (unpublished data) and c from Gocke ef a / . (1990).

BALTIC SEA PICOPLANKTON

97

1988, high summer values appear to follow temperature changes during the year, but the low values in 1986 cannot be explained by low temperatures (Fig. 5 ) . Primary production was on an average level in 1986 (Kuosa and Kivi, 1989) compared to long-term series (Gronlund and Leppanen, 1990) and thus cannot alone explain the low bacterial production. However, the spring phytoplankton development, in terms of exudate production into the photic zone and the degree of sedimentation from the photic zone, may contribute to the difference in bacterioplankton production between years. The mobilization of particulate and dissolved material from below the thermocline during the weakly stratified early summer period (Laakkonen et al., 1981), may also markedly contribute to this difference in bacterioplankton production between years.

B.

Distribution of Bacterioplankton 1. Vertical distribution

Vertical distribution of bacterioplankton generally follows the distribution of food or energy sources available in the water column, with maximum values found in the photic zone and in the transition zones such as the pycnocline, the halocline and the chemocline. Bacterioplankton cells, heterotrophic activity and bacterioplankton production have been shown to follow the vertical distribution of phytoplankton production and biomass (chlorophyll a), with peak values recorded in the upper photic layer above the thermocline (Virtanen, 1985; Rheinheimer et al., 1988; Autio, 1990; Gocke et al., 1990; Cruise report 1990, unpublished; Heinanen, 1991). In the eutrophication gradient near Ask0 (Fig. 1) Larsson and Hagstrom (1982) measured some 80% of the annual bacterial production in the 0-20 m trophic layer compared with the 2G35 m atrophic layer. In the more eutrophicated stations, in which the trophic layer was thinner than the atrophic layer, values of 6&70% of the annual production in the trophic layer were recorded. In regions affected by stratification forces other than temperature, high values of bacterioplankton variables may be obtained from below the photic layer. In the deep parts of the Baltic Sea, where anoxic waters are encountered, Gast and Gocke reported the existence of regions in which the total bacterial number as well as the bacterial activity show well-defined peaks around the oxiclanoxic interface, with significantly higher values than in the oxic water above or anoxic water below (Gast and Gocke, 1988; Gocke et al., 1990).

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J . KUPARINEN AND 14. KUOSA

2. Horizontal distribution As shown in Fig. 7, an order of magnitude difference prevails in the maximum values of surface water bacterioplankton production in the northern (Bothnian Sea) and southern (Kiel Bight) Baltic Sea. In the Gulf of Finland maximum values are higher by a factor of 2 than in the Bothnian Sea. When seasonality in the data is taken into account, differences of more than two orders of magnitude in the production rates (0.1-74mg C/m3/day) have been recorded in the Baltic Sea (Table 4). The highest variability has been recorded at the study sites near the Finnish (Tvarminne, Fig. 1) and Swedish (Asko, Fig. 1) coasts. The southern part of the Baltic Sea is the most productive area. This part of the Baltic has on average the highest surface water temperatures and also the highest phytoplankton productivity (Schulz et al., 1990). High values, up to 30.4mg C/m3/day, have also been recorded from the entrance to the Gulf of Finland. This locality is a biologically active site most probably because of quasi-stationary fronts appearing in the mouth of the Gulf. The formation of fronts is related to the bottom topography and circulation patterns of the Gulf (Kononen and Nommann, 1992). The lowest values in the Baltic Proper were measured in the northern and central Baltic Proper, which are least subject to nutrient and organic loading from land. The exceptionally high values recorded from the Baltic Proper have been related to dense cyanobacterial blooms (Gocke et al., 1990; Heinanen and Kuparinen, 1991). Total bacterioplankton cell counts vary by two orders of magnitude (0.1-10 x lo9 cellsil) in the surface waters of the Baltic Sea (Table 5). Most of the variation has been measured in the Gulf of Finland, where seasonal cycles have been adequately covered to detect short time fluctuations. In the southern Baltic Sea, the overall variation in total cell counts is low compared with other localities (Table 5 ) . The central Baltic Proper also exhibits little variation, except for the unusually high value of 10 x 10' cells/l associated with the heavy cyanobacterial bloom of 1984 (Gocke et al., 1990) and the low value measured by Autio (1990) in the early summer of 1987. Bacterioplankton cell volumes vary from 0.015 to 0.125 pm' in the surface waters of the Baltic Sea (Table 6 ) . In general the highest values have been recorded from spring samples throughout the Baltic Proper, except for the high values from the Bothnian Sea (0.114pm') and Kiel Bight (0.1OH. 124 pm3). In epifluorescence microscopy the phytoplankton spring development has a visible effect on bacterioplankton cells, which are well stained and give bright fluorescence (Virtanen, 1985; Kuparinen, 1988) compared with the small and faintly fluorescing cells

99

BALTIC SEA PICOPLANKTON

TABLE

4. REGIONAIDISTRIBUTION OF SURFACE WATER BACTERIOPLANKTON

PRODUCTION (mg C/m3/d) IN mg C/m3/day Area

THE

BALTICSI-.A

Season

Reference

Winter Summer Summer All

Heinanen, Heinanen, Heinanen, Hagstrom,

Spring Spring Spring Spring Summer Summer Summer Summer Autumn Autumn All All

Lignell, 1990b Kuparinen, 1988 Heinanen and Kuparinen, 1991 This study Lignell, 1990a This study Autio et al., 1988 Larsson and Hagstrom, 1982 Autio et al., 1988 This study Virtanen, 1985 Larsson and Hagstrom, 1982

0.0M.1 3.2-6.1 3.5-17.5 0.3-7.5

BS

1.4-8.4 0.9-23 3.M.2 0.2-1 3.8 5.0-14.9 0.2-12.1 6.5 10-15" 5.3 0.2-1.9 M.2" 0-24"

Tvarminne

2.9-9.3 6.8-10.1 30.4 4.0 13.1

GOF

Spring Summer Summer Summer Summer

This study Heinanen and Kuparinen, 1991 Gocke et al., 1990 Heinanen, 1991 Cruise report 1990 (unpublished)

2.6 2.M.6 3.9 2.8 6.2-12.4

Teili NBP

Spring Spring Early summer Summer Summer

Lahdes et ul., 1988 This study Autio, 1990 Heinanen, 1991 Heinanen and Kuparinen, 1991

BP

Early spring Early summer Summer Summer

Heinanen and Kuparinen, 1991 Autio, 1990 Gocke et al., 1990 Cruise report 1990 (unpublished)

SBP

Early summer Summer Summer Summer Summer

Autio, 1990 Gocke et al., 1990 Heinanen, 1991 Cruise report 1990 (unpublished) Heinanen and Kuparinen, 1991

Kiel Bight

Summer All

Gocke et a / . , 1990 Gocke et al., 1990

0.3-2.5 1.9 14.4 7.4 3.7 12.9- 19.0 2.5 10.0 6.2-1 2.7 13.9 6-74

BS-coast

Asko Tvarminne Ask0

1992a 1991 1992a unpublished data

BS = Bothnian Sea, GOF = Gulf of Finland, NBP = northern Baltic Proper, BP = Baltic Proper, SBP = southern Baltic Proper. For stations Tv&rminne,Ask0 and Teili see Fig. 1. "FDC technique.

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J . KUPARINEN A N D H . KUOSA

TABLE5. REGIONALDISTRIBUTION OF SURFACE WATER BACTERIOPLANKTON x 10’) IN THE BALTICSEA (CELLS/~ Area

Season

Reference

0.5-1.0 1.7-1.8 1.2-3.3 3.3 3.6 3.1-4.7 2.C3.6

BS

Winter Summer Summer Summer Summer Summer Summer

Heinanen, 1992a Wikner et al., 1986 Wikner et al., 1990 Gocke et al., 1990 Gocke and Rheinheimer, 1991 Heinanen, 1991 Heinanen, 1992a

0.8-3.4 1.C5.3 1.5-5.8 2.3-5.9 4.6-10 1.5-4.5 6.1 1.2-4.8 3.2 0.8-1.2 0.4-2.8

Tvarminne

Spring Spring Spring Spring Summer Summer Summer Summer Autumn Autumn All

Virtanen, 1985 Kuparinen, 1988 This study Heinanen and Kuparinen, 1991 This study Virtanen, 1985 Autio et al., 1988 Kuuppo-Leinikki and Kuosa, 1990 Autio et a/., 1988 Virtanen, 1985 Vaatanen, 1976

1.9-3.5 0.1-1.8 2.9-7.7 8.2 0.1-2.1 3.5 4.64.6

GOF

Spring Early summer Summer Summer Summer Summer Summer

This study Kiinnis and Saava, 1990 Gocke et a / . , 1990 Cruise report, 1990 (unpublished) Kiinnis and Saava, 1990 Heinanen, 1991 Heinanen and Kuparinen, 1991

0.1-1.1 1.0-2.8 2.2 2.8 3.7-5.4

Teili NBP

Spring Spring Early summer Summer Summer

Lahdes et al., 1988 This study Autio, 1990 Heinanen, 1991 Heinanen and Kuparinen, 1991

0.8 3.3-10 5.0 5.34.4 4.0 3.1-3.9

BP

Early summer Summer Summer Summer Summer Autumn

Autio, 1990 Gocke et al., 1990 Cruise report, 1990 (unpublished) Gast and Gocke, 1988 Gocke and Rheinheimer, 1991 Wikner et a / ., 1986

0.2-0.7 0.5 3.H.9 2.3 2.G5.1 3.6

SBP

Early spring Early spring Spring Early summer Summer Summer

Gocke and Hoppe. 1982a Gocke and Hoppe, 1982b Gocke el a[. , 1990 Autio, 1990 Gocke et al.. 1990 Gocke and Rheinheimer. 1991

cellsil x lo9

BALTIC SEA PICOPLANKTON

101

Table 5 - c o d .

celldl x 10’

Area

3.3 4.2 3.67.2 3.5-7.1

3.6-4 Kiel Bight 4.0 4.7“ 2.Ob

Season

Reference

Summer Summer Summer Autumn

Heinanen, 1991 Cruise report, 1990 (unpublished) Heinanen and Kuparinen, 1991 Gocke et al., 1990

Summer Summer Summer All

Gocke et al., 1990 Gocke and Rheinheimer, 1991 Galv&o, 1990 Galvao, 1990

“Maximum value. hMean of the period September 1987-May 1989.

during the summer period (Virtanen, 1985). This spring bloom effect of producing larger cells was also recorded in the scanning electron micrographs of samples from the northern Baltic Proper (Lahdes et al., 1988). The biomass estimates of the surface water bacterioplankton in the Baltic Sea show a variation of two orders of magnitude (Table 7). However, different study sites do not follow the same coherence in values as do the cell number and production data. The biomass determinations are based on cell counts, but involve cell volume measurements and carbon conversion factors (Table 7). The variation is partially reduced if carbon conversion factors are unified, but nevertheless it is higher than expected from cell number measurements. This fact indicates the difficulty of accurate cell volume measurement in epifluorescence microscopy. Even when similar procedures, e.g. standard ocular grids, are used in cell measurements, the difference between operators may be significant (unpublished comparisons). New methodology that is less sensitive to personal judgement should be developed in order to obtain more reliable estimates of bacterioplankton biomass.

VI. Factors Controlling Autotrophic Picoplankton A. Nutrients and Temperature Theoretically, picoplanktonic algae have the ability to take up nutrients effectively due to their small cell size. In the Baltic Sea, picoplanktonic algae are a principal component of the stratified summer period, which

102

J . KUPARINEN AND H . KUOSA

TABLE6. REGIONALDISTRIBUTION OF SURFACE WATER BACTERIOPLANK~ ON CELLVOLUMES( p d ) IN THE BALTICSEA ~

Pm

Area

Method and season

Reference

0.038-0.090 0.0460. 072 0.018-0.080 0.114

BS

EFM - winter EFM - summer EFM - summer EFM - summer

Heinanen, 1992a Heinanen, 1991 Heinanen, 1992a Gocke et al., 1990 Gocke and Rheinheimer, 1991

0.03Y-0.079 0.032-0.112 0.018-0.048 0.0 15-0.033

Tvarminne

EFM - spring EFM - spring EFM - summer EFM - autumn

Virtanen, 1985 Kuparinen, 1988 Virtanen, 1985 Virtanen, 1985

0.058-0.072 0.088 0.046 0.022-0.026

GOF

EFM - spring EFM - summer EFM - summer EFM - summer

This study Gocke ef al., 1990 Heinanen, 1991 Heinanen and Kuparinen, 1991

0,033-0.125 0.047-0.052 0.019 0.029-0.038

Teili NBP

SEM - spring EFM - spring EFM - summer EFM - summer

Lahdes et a / . , 1988 This study Heinanen, 1991 Heinanen and Kuparinen. 1991

0.0940.099 0.094

BP

EFM - spring EFM - spring

Gocke et al., 1990 Gocke and Rheinheimer, 1991

0.091-0.114

SBP

EFM - summer EFM - summer EFM - summer EFM - summer

Gocke et a / . , 1990 Gocke and Rheinheimer, 19Y1 Heinanen, 1991 Heinanen and Kuparinen, 1991

Kiel Bight

EFM - winter EFM - summer EFM - summer EFM - all

Galviio, 1990 Gocke et al., 1990 Gocke and Rheinheimer, 1991 Galviio, 1990

0.017 0.026-0.047 0.134" 0.1 06-0. 124 0.124 0.091'

EFM = epifluorescence microscopy, SEM = scanning electron microscopy. "Maximum value. "Mean of the period September 1987 to May 1989.

relies strongly on regenerated nutrients. Picoplanktonic algae do not appear to be limited by nutrient availability or, at least, they can cope with the nutrient limitation better than larger algae during summer. In the Baltic Sea, in which surface water temperatures vary greatly, cold water may be an important controller of picoplankton growth. It seems likely that water temperature controls the growth of picoplanktonic

BALTIC SEA PICOPLANKTON

103

TABLE 7. REGIONAL DISTRIBUTION OF SURFACEWA.IER B A C IERIOPLANKTON BIOMASS (mg C/m3) IN THE BALTIC S h A mg C/m3

131

Area

Carbon conversion pg C/pm3

Reference

BS

0.35 0.35

Gocke et a / . , 1990 Gocke and Rheinheimer, 1991 Heinanen. 1991

0.11 0.12 0.11

Virtanen, 1985 Kuparinen, 1988 Larsson and Hagstrom. 1982

77-86 1.6-19.3 4.2-51.7 (5-35

Tvarminne Ask0

49.8-79.7 88.0 55.0

GOF

0.35 0.35 0.35

This study Gocke et al., 1990 Heinanen, 1991

20. Ck59.2 21.0 3.7-5.7 1.7-19.1

NBP

0.35 0.35 0.10 0.35

This study Autio, 1990 Rheinheimer et al., 1989 Lahdes et al., 1988

0.35 0.35 0.35

Gocke et al., 1990 Autio, 1990 Gocke and Rheinheimer, 1991

Teili

114-132 4 132

BP

0.8-2.3 1.7 117-144

SBP

0.35

19 26 51-91 11-173 173 45.8" 102b

-c -c

Kiel Bight

Gocke Gocke Gocke Gocke

and Hoppe, 1982a and Hoppe, 1982b et al., 1990 and Rheinheimer, 1991

0.35 0.35 0.10

Heinanen, 1991 Autio, 1990 Rheinheimer et a/., 1989

0.35 0.35 0.35 0.35

Gocke et al.. 1990 Gocke and Rheinheimer, 1991 GalvBo, 1990 GalvCo, 1990

"Mean of the period September 1987 to May 1989. hMaximum value. 'Not known.

cyanobacteria. However, there is evidence for the growth of eukaryotic picoplankton in cold water (e.g. Kahru ef a t . , 1991). We do not have growth estimates of eukaryotic picoplankton in order to assess the actual growth rate of eukaryotes during the spring bloom. However, the increase of their abundance during the growth season is not comparable

104

J . KUPARINEN A N D H . KUOSA

with that of cyanobacteria, which may be a consequence of decreased growth rate or increased loss rate. The reason why eukaryotic picoplankton are not a dominant algal group during the spring bloom and later during the summer is not clear. Their growth, although starting at an early stage, may be slow at least in cold environments, or they may be grazed, or some other factor (growth-inhibiting substances from other algae?) may be responsible for their slow increase. However, we lack completely the requisite knowledge of the physiology of picoplanktonic algae in the Baltic Sea. The growth rate of picoplanktonic cyanobacteria is quite high in the Baltic Sea. The mean generation time is estimated to be 2 d at Tvarminne and 4 d at Umei. Growth rate may be controlled by the availability of regenerated nutrients, The number of picoplanktonic cyanobacteria is probably tightly grazer-controlled throughout the summer. Protozoan grazers are evidently actively grazing on cyanobacteria. In eutrophic waters, it may be that even at their maximum growth rate, picoplanktonic cyanobacteria consume only a small proportion of the available nutrients due to their low grazing-controlled biomass. This would explain the small relative fraction of picoplanktonic algae in eutrophic areas.

B.

Grazing

As discussed earlier, the contribution of picoplanktonic algae to the algal biomass and production is of considerable significance. Sedimentation has been estimated to account for about SO% of spring production in the Kiel Bight (Smetacek et a f . , 1984) and S&70% in Tvarminne (Kuparinen et al., 1984; Lignell el al., submitted). Thus the fraction of picoplanktonic production in the total primary production actually consumed at the surface water layer is of even greater importance. Grazing is in fact one of the decisive factors in the ecological significance of algal size fractions. Some very large algae (e.g. colonial and trichal cyanobacteria) are not grazed by zooplankton, which leads to intense algal blooms in the Baltic Sea. Similarly, the more intensive is the grazing control of algae and the more rapidly is primary production consumed, the less chance is there for an algal bloom. Carbon cycling through the microbial loop consists of many individual steps before the carbon enters the metazooplankton, and respiration losses are high. Consequently, the more carbon is cycled via the microbial loop the less energy is received by the higher predators. Thus there is great interest in the fraction of primary production incorporated via the microbial loop, either directly or indirectly via bacteria.

BALTIC SEA PICOPLANKTON

105

Picoplanktonic primary production may enter the microbial loop by protozoan grazing. Either protozoa may compete with metazooplankton for the prey or metazooplankton may be incapable of grazing on small algae. The latter explanation seems to fit picoplanktonic cyanobacteria, which are known to enter the guts of copepods, but which are not harmed by the passage through the gut (Johnson et al., 1982; Caron et al., 1985; Iturriaga and Mitchell, 1986). There are also indications of similar phenomena in the Baltic Sea (Kuosa, 1990~). Protozoan grazers are known to graze effectively on picoplanktonic algae (Landry et al., 1984; Iturriaga and Mitchell, 1986; Hagstrom et al., 1988; Nagata, 1988; Rassoulzadegan et a [ . , 1988). However, the only study from the Baltic Sea is that of Kuosa (1991) concerning fractionated incubations. In this study the grazing impact of nanoflagellates on picoplanktonic cyanobacteria was estimated. Nanoflagellates were found to be effective consumers of cyanobacteria, with 1 to 205 cells grazed/ nanoflagellateid. The number of grazed cells was a function of cyanobacterial abundance as flagellate clearance rates varied less (0.4 to 11.4 x 10-6mVflagellate). It was concluded that flagellates grazed on a large fraction of the primary production during summer and autumn (32 and 42%, respectively), but only a considerably smaller fraction during the winter and spring (6 and 396, respectively). Other grazing measurements with independent methods should be obtained before the validity of these figures can be confirmed. A qualitative study revealed great differences between the particle capture abilities of ciliate species (Kuosa, 1990~).The ability to graze on picoplanktonic cyanobacteria was not a simple function of the predator’s cell size. Some medium-sized ciliates apparently grazed heavily on small particles (e.g. Cothurnia maritima, Strombidium sp., Tintinnopsis lobiancoi and Vurticella sp., Fig. S), but the smallest common ciliate did not ingest cyanobacteria at all. In the same study some chloroplast-containing flagellates were found to graze on particles. However, quantitative estimates of the grazing of picoplanktonic algae either by ciliates or by chloroplast-containing flagellates are not presently available from the Baltic Sea.

VII. Factors Controlling Bacterioplankton A.

Nutrient- and Carbon-limited Bacterioplankton Growth

Batch experiments without the presence of predators were performed to study nutrient and carbon limitation of bacterioplankton growth during

106

J . KUPARINEN A N D H . KUOSA

A

B

D FIG.8. Examples of pelagic ciliates found to graze on picoplanktonic algae and cyanobacteria. A = Cothurnia maritirna (on Chaetoceros sp.), 35-45 p m ; B = Strombidiurn sp.. 3@40 p m ; c = Vorricella sp., 35-55 km; and D = Tintinnopsis lobiancoi, 50-70 p n .

different seasons. In low temperature water (
107

BALTIC SEA PICOPLANKTON

100 r

"0

1

2

3

4

5

6

7

Days FLG.9. Thymidine incorporation rate (pmol/l/h) in early spring and summer batch cultures. Cultures were enriched with 20 pgil of P as KH2PO4 and 80 of pg/l of N as NHdCI and 200pgil of C as Cj2H120,,.

carried out during the phytoplankton spring pre-bloom, experiments 2-5 during the post-bloom period, experiment 6 during late summer and experiments 7-10 during autumn, Experiment 3 (TV-89) was performed about 2 weeks after the phytoplankton chlorophyll-a maximum, when bacterioplankton growth was intensive as indicated by the high integrated value of the control sample in Fig. 10.

108

J . KUPARINEN A N D H . KUOSA

0 control EZ

nutrients

sucrose

nutrients+sucrose

500 400

2 300

1

%

8 200 100

0

6

3

4

E c

2 Mav

0

Nov. June

Oct.

GF-90 TV-87 N-89

Oct.

I TV-87 TV-88 GF-90 TV-87 K-87

K-87

Experiments FIG. 10. Effects of inorganic nutrient ( N + P) and sucrose-carbon (C) enrichment (96 of the control) on hacterioplankton thymidine incorporation integrated (5-6 samples in 12 h or 24 h interval) over the growth period (upper graph) and integrated thymidine incorporation of control samples in different experiments (lower graph). Enrichments were the same as in Fig. 9. The location and the year of the experiments is given below the bars, G F = Gulf of Finland, TV = Tvarminne and K = Kiel Bight.

BALTIC SEA PICOPLANKTON

109

In all experiments in which inorganic nutrients were added alone, a moderate increase in production was detected. The single carbon (sucrose) addition markedly stimulated bacterioplankton growth only in the pre-bloom experiment and in the autumn experiment which showed very low bacterioplankton productivity. In other experiments the single carbon addition had only a minor effect or no effect at all (Fig. 10). In all experiments the combined inorganic nutrient and sucrose manipulation had the greatest effect on bacterioplankton productivity, 450% in the pre-bloom and about 300% in autumn experiments (Fig. 10). These manipulation experiments reflect some general features of the Baltic Sea water and its nutritional status with respect to bacterial growth. During the pre-bloom period in spring, when inorganic nutrient concentrations are high, single inorganic nutrient manipulation has little effect on bacterial growth. In fact the slight stimulation of productivity in the pre-bloom experiment (Fig. 9) was more probably due to preference for the added ammonium nitrogen over the nitrate nitrogen which was present in the water column. Due to low phytoplankton biomass and growth, bacteria need carbon sources for their growth, in which case the single sucrose addition could provoke substantial growth. Similarly, during low phytoplankton activity in autumn, the combined inorganic nutrient and carbon manipulations could substantially stimulate bacterial growth. During the post-bloom period when the dissolved products from phytoplankton bloom development have accumulated in the water column, bacteria are least affected by the manipulations (experiments 2-5 in Fig. 10). As suggested by the substantial bacterial growth of the control batch in experiment 3 , and the minor effects of sucrose manipulations (Fig. lo), good substrate availability prevails during the post-bloom period. Some rough estimates of the amount of organic substrates produced by phytoplankton during the bloom periods have been presented (Larsson and Hagstrom, 1982; Kuparinen et al., 1984; Lignell, 1990a; Lignell et al., submitted), suggesting that bacterioplankton growth could be sustained for several days at cell densities of 1-2 x 109/1 and at growth rates of 0.254.50/d. During the late summer period, in experiment 6, sucrose addition had a negative effect on bacterioplankton growth (Fig. 9). However, when nutrients and sucrose were added together, a clear stimulation of growth was observed. This experiment may demonstrate the need for a correct C : N : P ratio in substrates required by bacteria for growth, as suggested by several authors (Goldman et al., 1987; Tezuka, 1990; Goldman and Dennett, 1991). Similar results were obtained in the post-bloom experiment (Fig. 11) in which the individual effects of nitrogen, phosphorus and

110

J . KUPARINEN A N D H . KUOSA

1.5

0.5

Man ipuI ations Fic,. 11. Effects of inorganic nutrient (N, P) and sucrose-carbon ( C ) enrichment on bacterioplankton thymidine incorporation integrated ( 5 samples in 12 h interval) over the growth period. Enrichments were the same as in Fig. 9.

sucrose-carbon were tested. The results from individual manipulations suggested that phosphorus was the limiting factor for bacterioplankton growth during this period. The combined sucrose-carbon and phosphorus resulted in a slightly lower growth than the combined phosphorus and nitrogen. However, when sucrose was supplied together with nitrogen and phosphorus, a substantial stimulation of growth was obtained. The results of these experiments suggest that during periods other than phytoplankton blooms, when exudates and products from sloppy feeding of micro- and mesozooplankton provide substrates for intensive growth even at low temperatures, Baltic Sea bacterioplankton is predominantly limited by inorganic nutrients in a proper C : N : P ratio. When the size of the labile DOC pool originating from the plankton community is small, bacteria must adapt to the use of the large refractory DOC pool (Ehrhardt. 1969), the utilization of which can be stimulated by inorganic nutrient addition. In the latter process, temperature plays an important role and may explain the large annual variation in the summertime productivity found at t h e entrance to the Gulf of Finland.

BALTIC SEA PICOPLANKTON

B.

111

Predation Control of Bacterioplankton

Since the introduction of the microbial loop concept by Azam et al. (1983), microbial ecologists have been concerned with the role of bacterioplankton as a food source for higher trophic levels. Heterotrophic nanoflagellates are the main organisms controlling bacterial assemblages by predation in temperate marine (e.g. Fenchel, 1982; Andersen and Fenchel, 1985; McManus and Fuhrman, 1988) and freshwater (Bloem et al., 1989; Sanders et al., 1989) habitats. The close interaction between flagellates and bacteria has been demonstrated by coupled oscillations of the two groups (Andersen and Fenchel, 1985; B j ~ r n s e net al., 1988). In the Baltic Sea, the coupled oscillation of bacteria and heterotrophic nanoflagellates has been demonstrated by Wikner and Hagstrom (1988) and Galvlo (1990). Coupled oscillation is also evident between bacterioplankton thymidine incorporation rate and flagellate numbers in 30 m3 plastic enclosures (Fig. 12). Only a few studies of heterotrophic flagellate predation on bacteria are hitherto available from the Baltic Sea (Table 8). In the western Baltic Sea the mean ingestion rates, 28-38 bacteria/flagellate/h, were similar to maximum values of the whole range of 2-37 bacteriaiflagellateih obtained from Tvarminne, in the Gulf of Finland (Kuuppo-Leinikki and Kuosa, 1990; Kuuppo-Leinikki, 1990). In the Bothnian Sea the values were somewhat higher (Wikner et al., 1990) than those found in the Tviirminne area, although when a similar methodology was applied to both locations a comparable range in the die1 predation rates was obtained (Wikner e f al., 1990). Areal comparison is difficult because different methods for measuring predation were applied in different localities except in the study of Wikner et al. (1990). In the Tvarminne study (Kuuppo-Leinikki, 1990), ingestion and clearance rates were low compared with values obtained using labelled bacteria as food sources for natural assemblages (Sherr e f al., 1987; Wikner et al., 1986, 1990). However, they were comparable with several other studies in which natural water samples were used (Wright and Coffin, 1984; Coffin and Sharp, 1987; Bjornsen et al., 1988), suggesting that the fractionation method gives lower values than the method in which labelled bacteria are used as food. The ingestion rates obtained from diffusion chamber experiments (Galvlo, 1990) were even higher than the rates obtained using fluorescently labelled bacteria and fluorescent particles (Sherr et al., 1987; McManus and Fuhrman, 1988). When a similar diffusion chamber technique was used, comparable ingestion rates were obtained (Landry et a[., 1984). From the above comparisons it is quite evident that several

112

J . K U P A R I N E N AND 11. K U O S A

MHNAN

12

n Q

4

I

Bag2

8

0

5

2 I

Bag 3

0

-12

T

1 0

7

25

Bag5

5

10 Days

15

20

FIG.12. Bacterioplankton production (cellsimlih x lo4) and number of heterotrophic flagellates (iml x 10’) in 30 mi plastic bags. Bags 1-4 were manipulated with nutrients (NH,-N and PO,-P) and small fish (stickleback fry) in a cross-over experiment. Bag 5 is the control unit. Kuuppo-Leinikki (unpublished).

113

BALTIC SEA PICOPLANKTON

TABLE 8. Rl-GlONAI DISTRIBUTIONC)C SURFACE WATER NANOFIAGL-I PREDATION O N BACTERIOPLANKTON I N THE BALTIC SEA Area

Ingestion rate bact.iflag.1h

Bothnian Sea: range" medianh Tvarminne: range" daily mean Tvarminne: range'' Kiel Bight: maximum" minimum' mead

3-22 2-37 6690 13-14 28-38

I ATt

Clearance rate Predation rate nllflag.1h bact.lml/h

Reference

&34

1-20 x 104 3.0 x lo4

(1) (1)

1-20 x lo4 8.0 x 104

(1)

1-6 1-5

1-7

X

lo4

(2) (3)

(4)

"Die1 study. "Median daily. 'Different calculation methods. "Diffusion chamber experiments in May 1988. 'Diffusion chamber experiments in July 1988. 'Diffusion chamber experiments 1987-1988, without the zero value in January 1958. (1) Wikner et ui., 1990. (2) Kuuppo-Leinikki and Kuosa, 1990. ( 3 ) Kuuppo-Leinikki. 1990. (4) GalvBo, 1990.

methodological calibrations and refinements are needed to obtain better geographical comparability of the ingestion and clearance rates of bacteria by heterotrophic flagellates. Die1 periodicity of bacterioplankton and flagellates has been analysed by Wikner et af. (1990). Bacterial mortality (Pace, 1988) other than predation has been neglected from the estimates since practically no data exist on mortality rates in the Baltic Sea. In comparison with predation vs. bacterial growth, heterotrophic nanoflagellates were able to remove more than 100% of the bacterial production at maximum ingestion rates (Wikner et al., 1990; KuuppoLeinikki, 1990; GalvHo, 1990) in all studies. Wikner et al. (1990) found predation to exceed production in several study sites by a mean factor of 2.6 (s.e. = 0.7). Since no net decrease in bacterial numbers was observed to explain the imbalance, they focused on methodological errors involved in the measurements. They concluded that bacterial production was most probably underestimated by the ?TI method due to the use of a

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conservative conversion factor of 1.7 X 10" cells produced per mole of thymidine incorporated. Conversion of tritiated thymidine incorporation into bacterial cell production is the first step in the TTI method. This conversion, unlike the conversion of biovolume production to carbon production, is well established and has been shown to lie near to the value of 1.1 x 10'' cells/mole as calculated by Riemann et al. (1987) in many marine environments. In freshwater environments and in eutrophic marine environments higher conversion factors have been reported (Smits and Riemann, 1988). The low salinity environment in the northern, coastal stations may give rise to the conversion factor value obtained in the Baltic Sea. With regard to the Baltic Sea bacterioplankton, the use of a single conversion factor obtained from marine environments may not be justified. Galvgo (1990) found greater disagreement between production and ingestion rates estimated by the TTI method when using a single empirical conversion factor than when using experimentally determined conversion factors in the Baltic Sea. Experimental conversion factors were lower in the unfiltered samples (mean 0.5 x 10" cells/mole) than in the filtered samples (mean 1.7 x 10" celldmole). Several conversion factor experiments have been made with Gulf of Finland waters in connection with the nutrient and carbon enrichment experiments. The overall mean of the factors was 1.8 ( n = 27) (Fig. 13), with no significant deviation between the values from manipulated and unmanipulated enclosures. The mean conversion factor is somewhat higher than the mean of 1.1x 10'' cells/mole reported by Riemann et al. (1987), but still within the range reported by several authors (e.g. Kirchman et al., 1982; Bell, 1990; Riemann and Bell, 1990; Bjornsen and Kuparinen, 1991). Conversion factors from 0.6 to 3.9 x lo1* cells/mole have been reported earlier from the same locality (Bell, 1986; Kuparinen, 1988). Autio (1992) obtained conversion factors well above 2 from the same locality at low temperatures. "Near natural" conversion factor values could be determined in 30 m3 experimental enclosures due to the coupled oscillation of heterotrophic nanoflagellates and bacteria (Fig. 12). During the latter part of the experiment cell numbers and thymidine incorporation rates increased for several days, most probably due to the low number of predators (Kuuppo-Leinikki, 1990). The conversion factor values calculated from two of the five experimental units were 1.6 x 10" cells/mole and 1.4 x 10" cells/mole. These values must be considered as conservative estimates since a minor predation impact was observed during the exponential growth period, decreasing the accumulation of cells in the

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0

6)

0

0 0 I

I

I

1 1.5 Specific growth rate, l/d

0.5

2

FIG. 13. Conversion factors of thymidine incorporation to cell production (10'' cellsimol) in different batch culture experiments. Conversion factors were plotted against specific growth rates. Samples were taken from the Gulf of Finland and from the Tvarminne sea area (see Fig. 1 for the location).

enclosures. These experiments suggest that a higher conversion factor value of, for example, close to 2 x 10" cellshole could be used for Baltic Sea samples.

VIII. Bacteria in the Pelagic Food Web In many aquatic ecosystems, bacteria have been considered to be the major decomposers of organic matter (Steele, 1974; Fenchel and Blackburn, 1979). Due to the introduction of new techniques to measure growth rates of bacteria (Hagstrom et al., 1979; Fuhrman and Azam, 1980; Moriarty, 1986), many investigators have suggested that production of particulate heterotrophic bacterial biomass provides an important link between dissolved organic matter, detritus and higher trophic levels (e.g. Pomeroy, 1974; Williams, 1981). The growth of bacterioplankton and the level of their standing stock are controlled by several abiotic and biotic factors such as concentrations of organic substrates and inorganic nutrients and predation. In open ocean ecosystems, where the bulk of organic carbon comes via phytoplankton production, close coupling between phytoplankton and bacterioplankton

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production can be expected (Cole et af., 1988). In a cross-system overview Cole et af. (1988) found that 57% of the variance in bacterial production was explained by primary production on a volumetric basis in the photic zone. When bacterioplankton cell numbers were added to the model, 73% of the variance in bacterioplankton production was explained. In the Baltic Sea, considerable loading from land-based organic and inorganic matter reaches the gulf areas, the Gulf of Bothnia, the Gulf of Finland, the Gulf of Riga and the western Baltic Sea. In these areas, as in estuarine systems (Findlay et af., 1991), bacterioplankton production may be based on carbon sources other than those arising from primary production. Bacterioplankton production values measured in the Baltic Sea are within the range of 0.4 to 153 mg C/m3/d reported by Cole et af. (1988). The majority of the Baltic Sea values fall below the average value of 26.4mgC/m3/d obtained by Cole et af. (1988), but are close to their median value of 11.5 mg C/m3/d (Table 4). The fragmentary primary production data from the Baltic Sea (Lassig ef af., 1978; Schulz et af., 1990) does not allow comparison with the regression line of Cole et af. (1988), but their general trend between bacterioplankton and phytoplankton production is evident in the Baltic Sea data (Fig. 5 , Table 4). The low values recorded in the northern and central Baltic Proper for bacterioplankton (Table 4) also correspond with the primary production of the area (Lassig et af., 1978; Schulz et af., 1990). This trend agrees with the data of Cole et af. (1988) suggesting that phytoplankton production is the primary basis of bacterioplankton growth in the open Baltic Sea. Cole et af. (1988) concluded that bacterial production averaged 20% (median = 16.5%) of primary production for the pelagic data on a volumetric basis. When they considered the entire water column on an areal basis, bacterial production was even more significant, averaging 30.6% (median = 27.1%) of primary production. In the Baltic Sea, Larsson and Hagstrom (1982) measured bacterial production with the FDC (frequency of the dividing cells) technique in the southern Stockholm archipelago (Asko, Fig. 1) as 24% of phytoplankton primary production. In a similar study site near the coast of the Gulf of Finland (Tvarminne, Fig. 1) Kuosa and Kivi (1989) measured annual bacterial production with the thymidine technique as 15% of net primary production. The batch experiments (Fig. 10) support the conclusions of Larsson and Hagstrom (1982) that phytoplankton exudation is the primary basis of bacterioplankton growth in spring. The sucrose additions had little effect on bacterioplankton growth after the peak of the spring bloom, whereas substantial stimulation by carbon sources was observed during the initial

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phase. In the northern Baltic Proper, where a substantial part of the phytoplankton spring bloom escapes from the photic layer by sedimentation (Kuparinen et al., 1984; Lignell et al., submitted), bacterioplankton plays an important role in transferring the energy lost by phytoplankton exudates and sloppy feeding to succeeding microbial communities. In many areas of the Baltic Sea, where the phytoplankton spring bloom ends with the removal of inorganic macronutrients from the stratified upper water column, the scarcity of labile carbon compounds begins to limit bacterioplankton growth. In the northern Baltic Proper this process occurs simultaneously with the development of heterotrophic nanoflagellate communities, making predation one of the main controllers of bacterioplankton standing stocks (Lignell et af., submitted). Bacterioplankton and heterotrophic nanoflagellate growth rates reported from different areas of the Baltic Sea (Wikner et af., 1990; Lahdes et af., 1988; Galvlo, 1990; Heinanen and Kuparinen, 1991) are quite similar, or even higher for heterotrophic nanoflagellates. This suggests the possibility of a rapid response from heterotrophic nanoflagellates to changes in bacterioplankton standing stock, as demonstrated by the coupled oscillations in experimental ecosystems (Fig. 13). Moreover, as demonstrated by, for example, Kuosa and Kivi (1989), flagellate carbon demand may exceed the supply from bacterioplankton, making heterotrophic nanoflagellates potentially reactive to increases in bacterioplankton standing stock. Very few data exist concerning heterotrophic flagellate communities in the open Baltic Sea. However, the results of a few experiments suggest that the control of bacterioplankton by heterotrophic nanoflagellates may explain the low variability found in the late summer stage in the standing stocks of bacteria in the whole Baltic Sea (Autio, 1990; Gocke et af., 1990; Gocke and Rheinheimer, 1991; Heinanen and Kuparinen, 1991). Low variability during summer has also been found in cell volume measurements in the Gulf of Bothnia (Fig. 14). This low variability in cell volume may be explained as selective predation by heterotrophic nanoflagellates (Anderson et al., 1986; Gonzales et af., 1990; KuuppoLeinikki, 1990; Monger and Landry, 1991). When the variability in cell volumes in spring, and the large cells found in winter when predation is negligible (Fig. 14), are taken into account, the explanation of predation control is even more attractive. However, the present data do not provide a straightforward answer to this speculation because other factors could explain the same observations of cell volume seasonality. The high surface-to-volume ratio of small cells may be an adaptation to low nutrient concentrations typical in the summer period (van Gemerden and Kuenen, 1984). Exceptionally high bacterioplankton counts of 8 to 10 X lo9 cells/l

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0.12 -

0.08

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found even in open areas of the Baltic Sea during cyanobacterial blooms in summer may have been a result of a simultaneous lack of predation control and high substrate availability. Ignoring these extreme cases, but assuming cell number of 5 x lo9 celldl as an average for pelagic waters in

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the Baltic Sea and using a conservative cell volume estimate of 0.032 (Table 6) and the carbon conversion of 0.35 pg CIpm3, bacterioplankton biomass would be 56 mg C/m3. This is within the range reported from the Baltic Sea (Table 7). In the carbon-limited summer situation indicated by the batch experiments (Fig. 9) and confirmed in many aquatic environments (Nagata, 1986; Kogure and Koike, 1987; Kirchman el al., 1990), the bacterial C : N ratio by atoms would be 4.5 : 1 (Goldman and Dennett, 1991). For an average bacterial biomass of 56 mg C h 3 , this would mean that 1 mmol or 14 mg of nitrogen/m3 is reserved in bacterioplankton biomass. During warm water periods in summer, bacterioplankton growth rates of 0.5 to lid are common (Galvfio, 1990; Kuparinen and Heinanen, 1992; Wikner et at., 1990). If predation equals (or even exceeds) bacterioplankton production, then more than half of the total nitrogen reserve in bacteria is continuously transferred to higher trophic levels for regeneration. The low ambient nitrogen concentrations found in the stratified upper water column in the Baltic Sea during summer periods (Nehring et uf., 1990) and the low C : N ratio (Goldman and Dennett, 1991) make bacterioplankton effective concentrators of inorganic nitrogen. For phytoplankton production, the close coupling between bacterioplankton and other heterotrophs is thus essential.

IX. Acknowledgements This study is a contribution to the research project PELAG under which most of the data have been obtained. The Finnish Academy, University of Helsinki, Maj and Tor Nessling Foundation, Walter and Andree de Nottbeck Foundation and the Finnish Institute of Marine Research have provided funding for this study. We are grateful to Tvarminne Zoological Station, to the University of Helsinki and to the Finnish Institute of Marine Research for providing facilities to carry out these studies. We also want to express our gratitude to our colleagues in the project PELAG and in the Finnish Institute of Marine Research for inspiration and feedbacks to continue our efforts in the field. The work of Mr Michael Bailey for checking the language of this article is greatly appreciated.

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Spermatophores and Sperm Transfer in Marine Crustaceans T. Subramoniam Department of Zoology, University of Madras, Guindy Campus, Madras 600 025, India

.. .. .. .. .. Introduction .. .. .. Spermatophore Morphology, Composition and Transfer .. .. .. .. .. .. .. .. A. Decdpoda . . .. .. .. .. .. B. Copepoda . . .. .. .. C. Euphausiids . . . . .. .. .. .. .. .. .. .. .. .. .. .. D. Stomatopoda .. E. Mysidacea and other spermatophore producing marine crustaceans 111. Spermatophore Hardening .. .. .. .. .. .. IV. Cryopreservation of Spermatophores . . .. .. .. .. V. Spermatophores and Artificial Insemination . . .. .. .. VI. Spermatophore Pathology .. .. .. .. .. .. VII. Comparison with Other Spermatophore-producing Marine Invertebrates .. .. .. .. .. A. Polychaeta .. .. .. .. .. .. B. Pogonophora .. .. .. C. Chaetognatha .. .. .. .. .. .. .. .. .. .. .. D. Mollusca .. .. .. .. .. .. .. .. .. VIII. Conclusion .. .. .. .. .. .. Ix. Acknowledgements .. .. .. .. .. .. .. .. X. References ., .. ..

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1. Introduction Reproductively unspecialized marine animals discharge their gametes into the medium, where fertilization occurs. In contrast, species living in fresh water or on land adopt other methods such as direct or indirect insemination into the female reproductive tract. Gametes released into Copyright 01993 Academic Press Limited All rights of reproduction in any form reserved.

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the sea survive for a long period of time, by virtue of being iso-osmotic with the ambient medium. In fresh water, however, the released gametes, especially the sperm, would quickly swell by water entry, unless suitably protected by mucoid envelopes. The land habitat is also hostile to unprotected sperm, which would rapidly desiccate if released into the outside. A common means that has evolved among invertebrate species for sperm transfer is the production of sperm packets called spermatophores. Spermatophore production is habitat-related. In the marine media they appear to have evolved to minimize the sperm loss in broadcast fertilization; in terrestrial forms such as land arthropods, spermatophores protect the delicate sperm cells from drying (Schaller, 1980). Spermatophores may have evolved prior to copulatory organs as a means to transfer semen. Crustacea, enjoying diversified distribution in various marine habitats, have developed many modes of sperm transfer. The majority, however, produce spermatophores which are either deposited externally onto the female body or inserted into the seminal receptacles by way of intromittent organs. The occurrence of spermatophores in different groups of aquatic Crustacea is listed in Table 1. Direct sperm transfer without spermatophore formation occurs in cirripedes (Barnes and Barnes, 1977), but the sperm cells or spermatophores of crustaceans are never discharged freely into the seawater medium. Although crustaceans are primarily aquatic, some exhibit insemination tendencies suited to terrestrial conditions, as exemplified by certain crabs and by wood-lice, which possess specialized gonopods for sperm/spermatophore transmission. The use of spermatophores as the main mode of sperm transfer by crustaceans is important in two respects. First, most crustacean spermatozoa, and all decapod spermatozoa, are aflagellate and non-motile (Felgenhauer and Abele, 1991) and hence need a vehicle for their transference to females in the absence of a copulatory organ. Secondly, crustaceans have employed spermatophores as an effective method for sperm transfer in aquatic media inasmuch as the cryptozoic arthropods have used them in land habitat. This review examines the morphological diversity of spermatophores and the mechanism of sperm release from spermatophores. Special attention is paid to the origin of spermatophoric components in the male reproductive tract, spermatophore hardening, biochemistry of the seminal plasma with reference to sperm survival, and maintenance during storage inside the female reproductive tract. This review deals first with the brachyuran crabs, whose breeding is best known, and then goes in the reverse of the usual taxonomic order to the lobsters and prawns and less studied orders of Crustacea.

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TABLE1. AQUATICC R UI ACEANS ~ I N WHICH SPERMAT OPHORES A R E R ~ P O R T E D . THE TAXONOMIC A R K A N G ~ M EISNTAKEN T FROM BOWMAN A N D ABELE (1982)

Higher taxa and family Class Remipedia, Yager Family Speleonectidae, Yager Class Maxilliopoda, Dahl Subclass Mystacocarida, Pennak and Zinn Subclass Copepoda, Milne-Edwards, Order Calanoidea. Sars

Order Harpacticoida, Sars Order Cyclopoida, Burmeister Class Malacostraca, Latreille Subclass Hoplocarida, Calman Order Stomatopoda, Latreille Subclass Eumalacostraca, Grobben Order Mysidacea, Boas Family Mysideae, Dana

Order Euphausiacea, Dana

Order Decapoda, Latreille Sub-order Dendrobranchiata Bate Family Penaeidae. Rafinesque

Species

Speleonectus lucayesis Yager 1981 S. benjamini Yager 1987 Derocheilocaris typicus Pennak and Zinn 1943

Acartia tonsa Dana 1849 Cnlanus finmarchicus Gunnerus 1770 Candncia armata Dana 1846 Centropages furcatus Dana 1849 C. typicus Kroyer 1849 Euchaeta norvegica Boeck 1872 Labidocera aestiva Wheeler 1900 L. jollae Esterly 1906 Pseudodiaptomus coronatus Williams 1906 Diuptomus (Cyclops) castor Jurine 820 Diarthrodes cystoecus Thompson 1882 Lepeophtheirus pectoralis Muller 1776 Tisbe holothuriae Humes 1957 Cyclops americanus Marsh 1892 Pachypygus gibber Thorell 1859 Squilla holochista Kemp 1911 Leptomysis lingvura Sars 1868 Mysis relicta Loven 1862 Neomysis integer Leach 1814 Praunuspexuosus Miiller 1776 Thysanopoda aequalis Hansen 1905 T. orientalis Hansen 1910 T . tricuspidata Milne-Edwards 1837 Stylocheiron longicorne Sars 1883 Penaeus uztecus Ives 1891 P. duodarum Burkenroad 1939 P. japonicus Bate 1888 P . indicus Milne-Edwards 1837

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TABLE1 - contd. Higher taxa and family

Species ~

Family Sicyoniidae, Ortmann Infraorder Caridia, Dana Family Palaemonidae Rafinesque

Superfamily Crangonoidea, Haworth Infraorder Astacidea, Latreille Family Nephropidae, Dana Infraorder Palinura, Latreille Family Polychelidae, Wood-Mason Family Palinuridae, Latreille

Family Scyllaridae, Latreille Infraorder Anomura, MilneEdwards Family Coenobitidae, Dana Family Diogenidae, Dana Family Paguridae, Latreille

~~

P. kerathurus Forskil 1775 P. monodon Fabricius 1798 P. orientalis Kishinouye 1900 P. pencillatus Alcock 1905 P. schmitti Burkenroad 1934 P. setiferus Linnaeus 1761 P. stylirostris Stimpson 1871 P. vannamei Boone 1931 Metapenaeus monoceros Fabricius 1798 Sicyonia ingentis Burkenroad 1938 Palaemonetes sp. Heller 1869 P. pugio Holthuis 1949 P. vulgaris Say 1818 Macrobrachium acanthurus Wiegmann 1836 M . asperulum Von Martens 1868 M . carcinus Linnaeus 1758 M . formosense Bate 1868 M. nipponsense de Haan 1849 M . rosenbergii de Man 1879 M . shokitai Fugino and Baba 1973 Crangon crangon Linnaeus (1758) Enoplometopus occidentalis Randall 1840 Homarus americanus Milne-Edwards 1837 H. gammaraus Linnaeus 1758 Jasus lalandii Milne-Edwards 1837 Palinurus gilchristi Stebbing 1898 Panulirus homarus Linnaeus 1758 P. interruptus Randall 1839 P. penicillatus Oliver 1791 P. angulatus Bate 1888 Potamobius Fabricius 1819 Linuparus trigonus Siebold 1824 Thenus orientalis Lund 1793 Parribacus antarcticus Lund 1793 Coenobita rugosa Milne-Edwards 1837 Clibanarius longitarsus de Haan 1849 Diogenes pugilator Roux 1828 Anapagitrus hyndmani Thompson 1843 Birgus latro Linnaeus 1767 Dardanus asper de Haan 1839

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TABLE1 - contd.

Higher taxa and family

Species

D. punctulatus Hilgendorf 1869 Eupagurus bernhardus Linnaeus 1758 Pagurus novae-zealandiae Dana 1852 P. prideauxi Leach 1865 P. bernhardus Linnaeus 1865 Pleuroncodes planipes Stimpson 1860 Albunea symnista Linnaeus 1758 Emerita asiatica Milne-Edwards 1837 Hippa pacifica Fabricius 1787

Family Galatheidae, Samouelle Family Hippidae, Latreille Infraorder, Brachyura, Latreille Family Dromiidae, de Haan Family Homolidae, de Haan Family Raninidae, de Haan Family Majidae, Samouelle

Family Portunidae, Rafinesque

Family Goneplacidae, MacLeay Family Xanthidae, MacLeay Family Ocypodidae, Rafinesque

Dromia personata Linnaeus 1756 Homola barbata Fabricius 1793 Ranina ranina Linnaeus 1758 Lyreidus tridentatus de Haan 1839 Chionoecetes opilio Fabricius 1788 Inachus phalangium Fabricius 1775 Libinia emarginata Leach 1815 Macrocoeloma trispinosum Rathbun 1900 Callinectes sapidus Rathbun 1896 Carcinus maenas Linnaeus 1758 Portunus pelagicus Linnaeus 1758 Ovalipes ocellatus Herbst 1799 P. sanguinolentus Herbst 1796 Scylla serrata Forski1 1775 Geryon fenneri Manning and Holthuis 1984 Paratelphusa hydrodromus Herbst 1794 Menippe mercenaria Say 1818 Rithropanopaeus harrisii Gould 1841 Ocypoda platytarsis Milne-Edwards 1852 Uca lactea de Haan 1835

II. Spermatophore Morphology, Composition and Transfer A. Decapoda 1. Brachyura Brachyurans, true crabs, produce the simplest type of spermatophore found among Crustacea. In general, the spermatophores of true crabs a r e ovoid, ellipsoidal or round and contain varying numbers of sperm. T h e

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spermatophores are carried in a fluid medium of granulated seminal plasma. Light microscopic studies on the morphology of the spermatophore reveal the presence of a single layer enveloping the sperm mass as reported in Carcinus maenas (Spalding, 1942), Callinectus supidus (Cronin, 1947), and Portunus sanquinolentus (Ryan, 1967). A distinctive spermatophore envelope layer may be lacking in some crabs, in which cases, sperm aggregates retain their integrity merely by differential viscosity of the surrounding seminal fluid. Examples are the Hawaiian crab, Ranina ranina (Ryan, 1984) and the ocypod crab, Ocypoda plutytarsus (Varadharajan, 1982). Such simplification of spermatophore structures is consistent with the statement of Spalding (1942), that brachyuran spermatophores are degenerate structures which have lost their original function of protection, serving merely to keep the sperm together during transmission to the female. The spermatophores of brachyurans are microscopic; for example in Scylla serrafa they measure 3 4 p m diameter with radiating arms of 2-5 p m . Such spermatophores can enclose varying numbers of spermatozoa agglutinated in a viscous seminal plasm. Hinsch (1973) reported that in the oxyrhynchan, Macrocoeloma trispinosum, a spermatophore contains only one sperm with its nucleated arms projecting through the spermatophore wall. In others, the arrangement of sperm within the spermatophore is irregular and does not follow any definite pattern. In Ovalipes ocellatus, the sperm are closely packed within the spermatophore leaving little fluid space between adjacent sperm, whereas in Libinia emarginata the sperm are less closely packed (Hinsch, 1986). In the mud crab Scylla serrata, the spermatozoa within the spermatophores are found to be embedded in a viscous fluid with their multistellate arms folded on the surface of the main body (Uma and Subramoniam, 1979). In S. serrata each spermatophore is enveloped by an outer thick and an inner thin layer, the latter being confluent with the inner sperm mass (Uma and Subramoniam, 1979). A two layered spermatophore has also been reported in the golden crab, Geryon fenneri (Hinsch, 1988). This crab is a non-swimming oceanic form and the spermatophore layer is significantly thicker. The size of the spermatophore varies considerably in an individual and hence the number of spermatozoa enclosed within them also differs. Recently, Jeyalectumie and Subramoniam (1991) reported an increase in the size of the spermatophores during the maturation of the male S. serrata. The size increase of the spermatophore is consistent with the increase in the organic components of t h e spermatophores. In some populations of this crab, a polymorphic condition of the spermatophore, ranging in shape from dumb-bell to boomerang, has been reported

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recently (Bhavanishankar and Subramoniam, 1992). While the vesiculate spermatophores bathed in the seminal plasma are characteristic of the advanced brachyurans, in two primitive groups, Gymnopleura and Dromiacea, only a single sperm mass surrounded by viscous seminal secretions is produced (Hartnoll, 1975). A distinctive spermatophore layer is lacking. (a) Origin of spermatophores In decapod Crustacea, the inner epithelial cells lining the vas deferens are highly glandular and secrete a variety of substances that make up the spermatophore proper. In the brachyuran crabs, the proximal vas deferens is secretory in nature, whereas the distal vas deferens is storage and ejaculatory in function (Spalding, 1942; Cronin, 1947; Ryan, 1967). In Scyffa serrata, the anterior portion of the proximal vas deferens secretes substance “A”, which agglutinates the sperm and forms the spermatophore layer (Uma and Subramoniam, 1984). The secretory nature changes in the distal part of the proximal vas deferens in that a granular substance “B” is released to constitute the medium for storing the completed spermatophore. The middle vas deferens is membranous and acts as the storage region for the seminal products. The distal vas deferens again becomes secretory with its cuboidal epithelial cells. Its fluid-like secretion “C” is not mixed with the rest of the semen stored in the mid vas deferens until the time of spermiation. In view of the mucoid nature of this secretion, it may possibly form the material for sperm plugs in the mated females, as reported in several other brachyuran crabs (Spalding, 1942; Ryan, 1967). Following the distal vas deferens is a short ejaculatory duct, filled with a secretion similar to that found in the distal vas deferens. In other crabs such as Carcinus maenas (Spalding, 1942) and Uca factenus (Uma, 1978) a distinct dilated portion in the form of a seminal vesicle is found in the distal vas deferens, serving to store the spermatophores. Electron microscopic studies on the vas deferens of the spider crab, Libinia emarginatu, reveal that the cytoplasm of the vas deferens contains vast arrays of rough endoplasmic reticulum and Golgi complexes with few mitochondria (Hinsch and Walker, 1974). In this crab, sperm enter the anterior vas deferens individually and become surrounded by secretion products. They are then compartmentalized into spermatophores of varying sizes. The middle vas deferens secretes the seminal fluid, surrounding the spermatophores. The posterior vas deferens functions Primarily as a storage centre for spermatophores until they are released at the time of ejaculation. The cells of this region are also secretory, contributing further to the seminal plasm.

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In Dromia personata, representing the primitive superfamily Dromioidea, the vas deferens is divisible into three zones, based on the structure of its wall and on the basis of the contents and secretions (Hartnoll, 1975). In the first zone, the sperm are aggregated into a white mass which stains blue in Mallory’s triple stain. This secretion also forms a layer around the central core of sperm. In the second zone, a central core of sperm is again surrounded by a thick layer of vacuolated blue-staining secretion. The remainder of the vas deferens, comprising the third zone, is filled with a translucent secretion, which is devoid of sperm. The ejaculatory duct also contains a secretion similar to that found in zone three. Similarly, in another gymnopleuran, Homola barbata, a single sperm mass is embedded in a blue-staining medium. This central core of sperm is surrounded by a firm red-staining rubbery layer which is again ensheathed by a thin layer of blue-staining secretion. Evidently, there appears to be no spermatophore wall enveloping the spermatophoral fluid. However, the secretion ensheathing the central sperm mass is so viscid that, even after the forcible removal of spermatophores from the vas deferens, the shape of the spermatophore is not altered. Evidently, the spermatophore and its formation within the vas deferens of these brachyurans are similar to those of macruran Astacidea where the spermatophore is a single mass of sperm surrounded by various secretions, lacking an enveloping layer (Hartnoll, 1975). (b) Chemical composition of sperrnatophores and seminal plasma Extensive histochemical studies made on the spermatophore of the mud crab Scylla serrata reveal the presence of a structural polysaccharide, chitin, in the outer wall. The chitinous nature of the spermatophore wall has also been indicated in another crab, Carcinus maenas (Spalding, 1942) and in the harpacticoid copepod, Tisbe holothuriae (PochonMasson and Gharagozlou-van-Ginneken, 1977). In addition to chitin, the outer layer of the S. serrata spermatophore is rich in sulphated acid mucopolysaccharide (sAMPS). In the inner layer, the AMPS, rich in carboxylated groups, predominate; chitin is absent in this layer. Protein components of the two layers appear to be rich in tryptophanyl reactive groups, but tyrosyl and other phenolic substances are lacking. Brachyuran crabs secrete copious quantities of fluid semen to carry the spermatophores. The heterogeneity of the seminal plasma was first recognized by Spalding (1942) who indicated by staining properties two secretion products in Carcinus maenas. From a positive reaction to alcian blue and bromophenol blue, Hinsch and Walker (1974) suggested that the seminal plasma of Libinia emarginata contained a mucoprotein. In S. serrata, the sperm-bearing substance, as well as the spermatophore layer,

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stain blue with Mallory's triple stain, whereas the granulated seminal plasma found in the proximal vas deferens stains red. The agranular secretion of distal vas deferens as well as the gelatinous substance secreted by the ejaculatory duct are eosinophilic (Uma and' Subramoniam, 1984). A recent study on the spermatophores and seminal plasm of the field crab, Paratelphusa hydrodrornus, has revealed that they are rich in protein, free carbohydrates and lipids (Jeyalectumie and Subramoniam, 1987). The occurrence of much carbohydrate in spermatophores corroborates earlier findings that sperm cells of many decapod crustaceans are endowed with glycogen stores (Pochon-Masson and Gharagozlou-van-Ginneken, 1977). The spermatophores also show high activity of the enzyme lactate dehydrogenase (LDH) (Jeyalectumie and Subramoniam, 1989). A recent biochemical study on the mud crab S. serrata provides insight into the type of sperm metabolism inside the spermatophores during their storage in the female reproductive tract pending ovulation and egg fertilization (Jeyalectumie and Subramoniam, 1991). The seminal plasma and the spermatophores are rich in protein, carbohydrate and lipids with their relative concentrations very high in spermatophores (Table 2). Surprisingly, succinate dehydrogenase activity is very low, whereas fumarate reductase and lactate dehydrogenase exhibit high activity. Electrophoretic analysis of the isoenzyme pattern of LDH reveals six fractions, the most conspicuous of them being a fraction homologous to the LDHx of mammalian spermatozoa. LDHx of the spermatophore is more related to M4 (LDH5) than to H4 (LDH,) (Clausen and Ovlisen, 1965) suggesting that the spermatozoa inside the spermatophore primarily undergo anaerobic metabolism utilizing glycogen-derived glycosyl units or free sugars. Evidently, the seminal plasma of S. serratu serves the nutritional requirement for the metabolism and storage of spermatozoa both within the male and female reproductive tracts. This is especially important for sperm storage in the spermatheca, as the interval between mating and the first ovarian maturation, and hence ovulation is rather long (Ezhilarasi and Subramoniam, 1982). The male reproductive tissues as well as their secretions (including the spermatophores) contain a smaller amount of organic components in the immature crabs; during maturation, there is not only an increase in concentration of organic substances but also an increase in size of spermatophores. These data indicate a biochemical maturation of spermatophores within the vas deferens during sexual maturation. Further, the inorganic ions, such as Na, K, Ca and Cu, also increase in the seminal substances as sexual maturation proceeds in the male S. serratu (Table 3). Mature decapod sperm are characterized by a lack of mitochondria or, when present in the mature cell, they are ill-defined and degenerated

138

T. S U B R A M O N I A M

TABLE2. BIOCHFMICAL COMPOSITION (mg/100 mg Tissue) OF SEMINAI SECRETIONS OF S C Y L L A SERRATA AT DIFFER~NT MATURITY STAGES

Tissue

Total

Total free Protein

protein sugars

Senzinal plasma I 1.012 f

I1 111

IV

0.290 3.205 k 0.586 5.397

* 0.449

5.792 f 0.780

0.075 f 0.0083 0.138 f 0.0094 0.185

+

-

0.0070 0.253 f 0.0257

Spermatophores I 2.011

I11

0.017 f 0.517 0.0039 9.039 0.078 + i 1.082 0.0088 10.052 0.105

IV

0.683 0.0085 10.243 0.150

f

I1

*

+

-

* *

1.163 0.0148

Glycogen

bound sugars

0.16

+

-

0.0024 0.139 f 0.0220 0.158 f 0.0133 0.166

* 0.0098 0.024

+

-

0.0036 0.554 f 0.0566 0.525 f 0.0489 0.495

+ -

0.0214

Total

Water

lipid

content

0.032

95.33 f 1.119 93.49

0.011 f 0.0002 0.0103 f 0.0007 0.0115

0.0069 0.074

0.0013 0.0109

*

0.117

0.0007

0.0165

0.0072 f 0.0009 0.0199 k 0.0030 0.0246

0.077 f 0.0051 0.153

89.15 f 2.372 86.32

t

i

+ -

*

0.0033 0.0280 f 0.0029

-t

0.0039 0.070 +

* 0.0059

*1.610

90.25 k 1.797 NA

k

0.0218 1.702 0.176 84.12 f f 0.0038 3.270 0.219 NA

+ -

0.0209

Each value is mean +s.e. NA indicates not analysed. From Jeyalectumic and Subramoniam, 1991.

(Pochon-Masson, 1983). In the crab, C. maenas, Pearson and Walker (1975) could not demonstrate the enzymes (e.g. cytochrome C oxidase) associated with oxidative phosphorylation in the lamellar complex, which according to Anderson and Ellis (1967) is a functional homologue of sperm mitochondria in decapods. Thus, both enzyme histochemistry and the biochemical studies discussed above, strongly favour the predominance of anaerobic respiration in decapod sperm. The only other group of crustaceans to receive attention on sperm metabolism is the cirripedes. However, the motile spermatozoa of the cirripedes utilize mainly endogenous substrates such as lipids (Barnes,

139

S P E R M A T O P H O R E S IN M A R I N E CRUSTACEANS

TABLE3. CONCENTRATION OF INORGANIC IONS (/.Lg/g) OF S. SERRATA SECRETIONS Seminal plasma

IN THE

SEMlNAI

Spermatophores

Metals

Na K Ca cu Mn Zn Mg

Stage I

Stage I1

Stage I11

Stage I

Stage I1

Stage I11

11.05 8.09 <1.0 <2.0 <1.0 <1.0 <0.10

9.85 2.74 t1.0 <2.0 <1.0 <1.0 <0.10

48.58 10.41 <1.0 15.01

6.97 5.13 <1.0 t2.0 <1.0 t1.0 <0.10

5.30 3.27 3.08 7.65 <1.0 <1.0 <0.10

7.24 7.45 9.77 7.05 <1.0 <1.0 <0.10

t1.0 (1.0 <0.10

From Jeyalectumie and Subramoniam, 1991.

1962b). The seminal plasma of Balanus balanus is highly proteinaceous and rich in amino acids, but meagre in lipids. Unlike mammals, cirripede semen is devoid of fructose, but is high in glycogen, glucose and a small quantity of lactic acid. The low oxygen uptake of B. balanus spermatozoa together with low mortality and ATP content indicate glycolysis under anaerobic conditions for energy production in sperm metabolism (Barnes, 1962a,b; Barnes and Blackstock, 1974). (c) Sperm transfer in Brachyura In the Brachyura, sperm transfer and storage mechanisms are related to the type of fertilization. In all the advanced brachyuran families, fertilization is internal whereas in the primitive families it is external. In both the types, however, there is true copulation, using a penis or accessory copulatory organs such as the modified pleopods. (d) Copulatory organs The brachyuran crabs invariably employ copulatory organs in the transference of spermatophores and semen. The right and left gonopods (modified 1 and I1 pairs of pleopods) and their respective penes form a complete insemination unit. In mature males, the vasa deferentia open on the coxae or sternum of the last thoracic segment (Drornia personata of Dromiidae- fifth segment - and in all advanced brachyurans - eighth segment) by short muscular penes (Hartnoll, 1975). In the majid crab, Chionoecetes opilio, the first gonopod consists of a basal protopodite and an elongated endopodite, in which the cuticle is infolded the entire length, forming a tube which functions as an ejaculatory canal (Beninger

140

T. SUBRAMONIAM

et al., 1991). The second pleopod fits into this cuticular fold, while the penis fits into a slit on the lateral side at the base of the endopodite. During coitus, seminal substances are ejected by muscular action through the penis into the ejaculatory canal of the first pleopod. The ejaculate is further pushed down the canal by the pumping action of the piston-like second pleopod. There is a general similarity in the functional morphology of the first and second gonopods among the advanced brachyurans (Spalding, 1942; Cronin, 1947; Ryan, 1967; Elner et al., 1985). Notwithstanding the similarity among the advanced brachyurans, the primitive forms show considerable variation in the insemination morphology. These variations include: (i) the size of the penis; (ii) length of the first and second gonopods; (iii) prominence of the appendix musculina and (iv) number of segments in the pleopod (see Hartnoll, 1975). Such differences are bound to reflect on insemination efficiency as seen from the duration of mating in different brachyuran forms. For example, in the primitive forms mating lasts for only a few minutes, whereas in advanced forms it may last for several hours. More importantly, there emerges an evolutionary trend in the higher brachyurans with a tendency towards tubulation of the endopodite of the first gonopore and the reduction and fusion of the appendix masculina with the endopodite of the second gonopod (Hartnoll, 1975). The setal types as well as arrangements in the apical region of the first gonopod are presumed to assist in stabilizing its insertion in the vagina during copulation in the snow crab, Chionecetes opilio (Beninger et al., 1991). A mechanoreceptory function, possibly in positioning of the first gonopod at the onset of copulation, has also been ascribed to these setae. Several studies have shown the occurrence of rosette-shaped glands inside the first gonopod of brachyuran crabs (Spalding, 1942; Ryan, 1967; Diesel, 1989). Their ultrastructure, as well as the nature of the secretions, have been investigated recently in the lobster pleopods (Johnson and Talbot, 1987). In the snow crab C. opilio, Beninger et al. (1991) also studied their distribution pattern within the first pleopod. Based on the finding that the complex duct network arising from these glands leads to pores in the ejaculatory canal and not to the general cuticular surface of the gonopod, the authors maintain that they produce a seminal fluid, a suggestion earlier supported by Spalding (1942). Several structural studies, however, indicate a similarity between the pleopodal rosette-glands and the tegumental glands of Crustacea (Johnson and Talbot, 1987; Diesel, 1989). It is well known that the enzyme phenol oxidase, released by the tegumental glands at ecdysis, brings about tanning and melanization of the new cuticle (Krishnan, 1957; Stevenson, 1961). In addition, pleopodal tegumental glands of Homarus arnericunus show cyclical activity with moulting (Talbot and Zao, 1991).

SPERMATOPHORES IN MARINE CKUSTACEANS

141

Hence, the secretory role, if any, of the rosette-glands in male reproduction can be resolved only by direct biochemical studies. (e) Sperm receipt and storage in Brachyura Just as the male gonopods show complexity, the sperm storage structures of Brachyura show elaboration. In general, the true crabs receive copious quantities of seminal fluid during mating, and store them in special seminal receptacles, viz. spermatheca, for a prolonged period. In the primitive brachyurans, Dromiacea and Gymnopleura, the spermathecae are invaginations of the sternal integument having no internal connection with the oviducts. In all other advanced brachyurans, spermathecae are dilations of the oviduct. Differences also exist in the location of the oviducts; in primitive brachyurans they open on the coxae of the third pereiopod, whereas in the advanced forms they open on the sternal plates of the sixth thoracic segment (Hartnoll, 1979). Since the sperm-storing spermatheca is separate from the oviducts, fertilization is external in the primitive brachyurans. In the Dromiacea, the sternal spermatheca are paired structures with their openings independent of each other (Hartnoll, 1975). In the Gymnopleura there is usually only a single spermathecal opening on the anterior portion of the seventh thoracic sternite (Gordon, 1963, 1966). However, in a raninid, Lyreidus tridentatus, Hartnoll (1979) found paired oval spermathecal openings. The cryptic spermathecal structure found in the primitive brachyurans imposes restrictions on the internal fertilization. The sperm-storing capacity within the cryptic spermatheca is increased by the nature of the sperm mass, which is a white mass consisting of sperm embedded in a viscid medium. In Dromia personata, the spermatheca consists of a chitin-lined, rigid canal which leads into a spermathecal chamber, lined by a flexible wall. After mating, the sperm mass substance at the entrance to the spermatheca hardens to form plaques (Hartnoll, 1975). In the primitive Brachyura, all the spermathecal content is shed at each moult with the cast cuticle and hence the female must mate again before ovulation. The mechanism of sperm evacuation during spawning to effect external fertilization is, however, not known for any primitive Brachyura. Equally unknown is the histology of their spermathecae. The spermatheca of the advanced brachyuran crabs has been described for many species (Cronin, 1947; Spalding, 1942; Ryan, 1967; Ezhilarasi and Subramoniam, 1982). The histological description of the spermatheca of brachyuran crabs shows a striking similarity among the species described so far. The spermatheca is a dilation of the proximal region of the oviduct connecting it to the ovary. The vagina leads from the base of the spermatheca and opens outside the ovipore. The spermatheca proper

142

T. SUBRAMONIAM

comprises the anterior glandular portion and the posterior chitinous region which is continuous with the vagina, lined again with a chitinous intima. In the unmated condition, the spermatheca looks like a very thin, white, translucent sac flattened in the longitudinal plane of the crab (Ezhilarasi and Subramoniam, 1982). The anterior glandular portion is made up of two layers; the outermost is very thin and membranous and is made up of nucleated epithelial cells, whereas the inner layer is very thick and is composed of two types of highly secretory cells. The lumen of the proximal glandular spermatheca is spherical whereas that of the distal portion is elongated and flattened and devoid of any secretory material. In the mated females, the spermatheca shows hyperactivity of the secretory cells and a consequent surge of the secretory products into the lumen. After this, the secretory cells lining the lumen are gradually abolished, whereas the inner cells undergo cellular stratification. Concurrently, the posterior chitinous spermatheca shows characteristic foldings of the inner chitinous layer giving rise to deep furrows. In Scylla serrata, t h e seminal fluid as well as the spermathecal secretions are found in the inner lumen of the swollen spermatheca. A few spermatophores have been found in the chitinous furrows in the posterior region of the spe rmatheca. The seminal secretions delivered during mating have been shown to undergo a kind of solidification inside the spermatheca, giving rise to a structure called “sperm plug”. In some species the sperm plug extends into the vagina and even protrudes from the vulva (Hartnoll, 1969). In Curcinus maenus, the sperm plug fits the spermatheca very closely so that its shape is roughly spherical. Two portions with differential staining can be discerned in the sperm plug, with the spermatozoa lying on the top of the plug as a thin layer. The sperm plug can be retained for varying periods of time. The role of a sperm plug is not certain, but it has been assumed that it helps to prevent the loss of sperm after copulation. Hartnoll (1969) theorizes that in the ancestors of Brachyura the spermatozoa were almost certainly not deposited in the oviducts, but were probably either glued to the sternum or, as in the primitive Brachyura, deposited in shallow integumentary spermathecae. This would involve the sperm being transported in a medium which would harden and retain them until fertilization, and the sperm plug is perhaps the remnant of this mcd i u m . The spermathecae of the higher brachyurans show glandular activity, which is closely correlated to the ovarian cycle (Anilkumar and Adiyodi, 1977). I n Puratelphusu hydrodromus, the spermathecal epithelium is composed of columnar cells. The epithelium is externally protected by a coat of muscle with spindle-shaped nuclei. Between the two layers is

SPERMATOPIIOKES IN M A R I N E CKUSTAC'EANS

143

found a connective tissue layer. In the mated females, the sperm seem to aggregate near the apical border of the epithelial cells. The spermathecal secretion, which is largely proteinaceous, with periodic acid Schiff (PAS) positive materials, could provide the proper fluid medium for prolonged sperm storage, in addition to the seminal plasma. In the mud crab, S . serratu, mating in the puberal female influences the secretory activity of the spermatheca, which in turn initiates vitellogenesis in the ovary (Ezhilarasi and Subramoniam, 1982). This may indicate a common endocrine mechanism, which is set off by the mating process, regulating spermathecal as well as ovarian activity, at least in the first ovarian cycle (Subramoniam, 1981). Although the lower part of the spermatheca and the oviduct is of integumental origin, the upper portion of the spermatheca is free from cuticular lining and hence sperm storage in this region is not affected by moulting. Cheung (1968) provided histological evidence for the transmoult retention of sperm in the spermatheca of the stone crab, Menippe mercenarza. Understandably, the free spermatozoa tend to aggregate towards the apical portion of the spermatheca which is devoid of chitinous lining. Cheung (1969) also demonstrated the presence of the old invaginating exoskeletal wall between the sperm and the new cuticle in M . mercenaria, even after the ecdysis. This condition is similar to the supernumerary moults without casting off the old cuticles in certain insects (Richards, 1951). The sperm viability during prolonged storage is an interesting question; but the seminal substances, together with spermathecal secretions, could provide nutrients for sperm maintenance. In a recent study on the biochemistry of seminal secretions of the crab S. serratu, Jeyalectumie and Subramoniam (1991) determined the activity ratios of both succinic dehydrogenase (SDH) and fumarase reductase (FR) enzymes together with substrate changes in the spermathecal content of the mated and unmated females. The spermathecal content of the unmated female crabs is low in organic constituents. After mating, their contents are enriched by organic substances derived from contributions of the seminal substances. As sperm storage in the spermatheca advances, there is subtle utilization of carbohydrates, with lipids and proteins remaining untouched. Furthermore, there is very low activity of SDH and a moderate level of lactate dehydrogenase (LDH) activity in the spermathecal content. However, the FR activity is exceedingly high, again suggesting the continued anaerobic metabolism of sperm by utilizing carbohydrates, during their storage in the female. FR catalyses the reduction of fumarate to succinate during the reversible TCA cycle. The reversible step in the TCA cycle is predominant among helminth parasites (Barrett, 1981).

144

T. SUBRAMONIAM

suggesting an interesting comparison between the anaerobic sperm metabolism in the crab and parasitic metabolism in the host. Further evidence for the utilization of carbohydrates for sperm metabolism is derived from the in vitro storage of spermatophores of S. serrata at very low temperatures such as -4°C (Jeyalectumie and Subramoniam, 1989). In the anecdysic brachyuran crabs, which cease to moult after sexual maturity, multiple spawning with single mating is common (Berry and Hartnoll, 1970). In such anecdysic crabs, mating in a hard shell condition becomes a necessity for effective fertilization of successive batches of eggs (Kundsen, 1964; Hinsch, 1968). Berry and Hartnoll (1970) have even reported mating in a stone crab carrying brooding eggs in the abdomen. Multiple ovipositions from a single mating have also been reported in other crabs such as Rhithropanopeus harrisii (Morgan et al., 1983). Hartnoll (1969) classified the brachyuran crabs into two groups based on whether the female mated in “soft” or “hard” shell condition. He found a relationship between this and “simple” and “concave” patterns of the vulvae. Hazlett (1975) has given a long list of crabs which are hard or soft shelled at the time of mating. In general, all the majid crabs cease moulting at maturity and enter the terminal anecdysis during which the female incubates successive batches of eggs. The female snow crab, Chionecetes opilio, cannot mate prior to their terminal moult to maturity, since, until then, the gonopore is inflexible and sealed (Adams and Paul, 1983). C. bairdi produces one clutch per year, and the embryos are incubated for up to a year. This crab either mates each year or makes use of sperm stored in spermathecae from previous year’s mating (Paul, 1984). However, laboratory observations on isolated females after a single mating reveal a reduced fecundity and higher percentage of non-viable clutches, indicating the necessity for repeated mating for reproductive success (Paul and Adams, 1984). Even in R. harrisii, which continues to moult after sexual maturity, Morgan et al. (1983) reported deterioration of stored sperm with time, and hence the number of the eggs produced and their hatching success decreased with each successive egg clutch fertilized with stored sperm. Brachyuran crabs, in general, are solitary crustaceans and hence mate-finding is difficult. Therefore, mating occurs soon after t h e puberal moult, when the ovary is not even mature. Evidently, the brachyuran crabs have evolved a highly efficient sperm storage mechanism in the spermatheca coupled with the copious quantity of energy-providing seminal plasma that is transmitted to the female during each mating. In insect species where multiple mating occurs, there is sperm competition for fertilization. Parker (1970) defined sperm competition as the competition between ejaculates of at least two males for fertilization

SPEKMATOPHOKES IN MARINE CRUSTACEANS

145

of a female’s eggs. Unfortunately, sperm competition studies in brachyuran crabs are meagre although they possess excellent sperm storage facilities to sustain viable sperm for extended periods in the spermathecae and mate with more than one male before eggs are fertilized. Recently, Diesel (1990, 1991) demonstrated the possible occurrence of sperm competition in the spider crab, Inuchus phalangium. In this crab, insemination and fertilization occur in the seminal receptacle, which is placed ventrally (ventral type). Male spider crabs displace rivals’ sperm dorsally and seal it with hardening seminal plasma, leading to last male sperm precedence. Further studies are, however, desirable to shed more light o n sperm competition in brachyuran crabs in general. (f) Spermatophore dehiscence The process of dehiscence in the spermatophore of brachyuran crabs has been studied in Scylla serruta (Uma and Subramoniam, 1979). The sperrnatophores tend to swell gradually in tap water and a cone-like process forms from the sperrnatophore layers. Eventually, the cone-like process gives way to the release of the spermatozoa in a stream of viscous fluid. Incubation studies using several vital dyes also indicate the permeable nature of the S. serrata spermatophore layers. Presumably, sperm release from spermatophores stored within the spermatheca may be brought about by the absorption of low molecular weight substances from the spermathecal fluids by acidic rnucopolysaccharide substances of the sperm mass. In S. serruta, spermatophores are intact inside the sperrnatheca until the time of ovulation; free sperm have been encountered only after the egg release suggesting that ovulation releases metabolites that cause the spermatophore to swell by osmotic pressure and finally release the spermatozoa (Ezhilarasi and Subramoniam, 1982). The mechanism, as well as the time of sperm release from the spermatophores stored within t h e spermatheca, have not been determined for other brachyuran crabs, but invariably the presence of free spermatozoa has been reported in the spermatheca of other crabs (Diesel, 1991). 2. Anomura Unlike the brachyuran spermatophores, which are simple vesiculate structures, the spermatophores of anomuran crabs are highly complex. They are generally pedunculate (Calman, 1909) and are structurally species-specific. The complexity of anomuran sperrnatophores is related to the type of fertilization. Invariably, the spermatophores are attached to the female body to effect epizoic fertilization. The spermatophores also have a variety of accessory mucoid structures which not only serve the

146

T. SUBRAMONIAM

function of attachment but also protect the inner sperm mass from environmental hazards during the prolonged storage in the female body. (a) Morphological diversity (i) Pugurids. The spermatophoric structure in anomurans can be broadly categorized into two major types: the pagurian (hermit crab) type and the mole crab (Emerita) type. In the pagurids, the spermatophore consists of three distinct parts viz. the sperm containing ampoule, peduncle or stalk and a glutinous pedestal to fix the spermatophore on the sternal region of the female. In mole crabs, the spermatophore is ribbon-like, bearing a resemblance to the macruran type of spermatophoric mass (Subramoniam, 1984). Furthermore, among the hermit crabs, there occurs a near species-specific morphological diversity pertaining to the number of spermatophores in the ribbon, shape of the ampoule and the length of the peduncle. Figs 1 to 8 illustrate some of the pagurian spermatophores. In two terrestrial hermit crabs, Coenobita rugosus and Birgus larro, Matthews (1956b) found a similarity in the spermatophore structure with that of the aquatic form Durdanus punctulutus. In a recent ultrastructural study, Tudge and Jamieson (1991) described the spermatophore of B. latro consisting of a laterally compressed heart-shaped ampulla, a stalk and a broad pedestal. In Dardanus sp. the spermatophores are arranged in a single file with a pear-shaped ampoule kept aloft by the long stalks whose other ends are attached to the pedestal. The whole file of spermatophore is ensheathed in a non-sticky veil (Matthews, 1953, 1956a). The similarity in the development of spermatophores in the terrestrial C. rugosus and B. latro and the truly aquatic D. punctulatus tends to suggest that copulation and the subsequent process of fertilization are also aquatic in these terrestrial species (Matthews, 1956b). Although reports of spermatophore transfer in the hermit crabs are scarce, both the marine form, Pagurus prideauxi (Mouchet, 1931), and the terrestrial species, C. rugosus (Matthews, 1956b), are found to have spermatophores deposited on the female body as well as on the internal wall of the gastropod shell. In these forms the elevation of the spermatophore ampoule enclosing the sperm mass on a stalk may be

FIGS1-8. Spermatophores of anomuran crabs. (Redrawn from various sources.) 1 and 2, Before and during sperm release, Diogenes pugilafor (after Bloch, 1935). 3 and 4, Before and during sperm release, Eupagurus bernhardus (after Bloch, 1935). 5 , Spermatophore within the vas deferens, Coenohifa rugosus (after Matthews, 1956a). 6, Dardanus usper (after Matthews, 1953). 7, Clibanarius fongifarsus (after Uma and Subramoniam, 1984). 8, Pagurus novae-zeafandiae (after Greenwood, 1972). A: ampoule; CD: connecting cord; P: pedestal; S : stalk; SP: sperm; V: veil; VD: vas deferens.

SPERMATOPIiORES IN MARINE CRUSTACEANS

147

148

T. SUBKAMONIAM

helpful in the liberation of spermatozoa during ovulation, ensuring fertilization of all the eggs within the molluscan shell. Stalked spermatophores are also characteristic of some soil arthropods such as Symphyla and Apterygota (Schaller, 1971). The male symphilids Scutigerella imrnuculutu and Scutigerella silvestrii deposit o n the soil, simple sperm drops o n 1.5 mm long stalks, in the absence of the females. The females bite off the “heads” of the spermatophores and keep them inside the mouth. During ovulation, the eggs are smeared with the sperm stored in the mouth using their mouth parts. In t h e colembolan apterygotes (Orchesetta, Tomoarus) such stalked spermatophores are picked by the vulvae of receptive females. Clearly, the presence of a long stalk is thus helpful in keeping the sperm ball aloft on the soil so that the female can easily pick them up by mouth or vulva. It may be said that the stalked spermatophores of anomurans, though functionally homologous to those of the above soil arthropods, are more protected by definite spermatophore envelopes and accessory mucoid secretion for prolonged protection and storage of sperm on the female body. The difference also lies in the fact that in Anomura the spermatophore is always attached to the feinale directly. Origin of spermatophores: The complex nature of pagurian spermatophore morphology is reflected in the structure of the vas deferens, which produces various secretory materials that comprise the spermatophore proper. However, a basic similarity in the morphology of the vas deferens in the various species of hermit crabs emerges from the pioneering histological work of Mouchet (1931). In all the five forms studied by her, the right and left vas deferens produce the same type of spermatophore that is characteristic of the species, excepting Anapagurus hyndmani where dimorphic spermatophores are produced by the two vas deferens canals. Interestingly, crowding of reproductive organs due to abdominal torsion resulting from lodging of the hermit crabs inside the snail’s shell has no impact on the nature of spermatophore produced by the two male ducts. The vas deferens of the pagurids exhibits several helical twists in the form of the right- and left-handed coils that determine the production of different spermatophoric components inside the lumen of respective regions. Depending upon the complexity of the spermatophore, the number of such morphologically distinguishable regions of a vas deferens may increase. Thus in Durdanus asper, producing a highly complicated spermatophoric ribbon, Matthews (1953) distinguished as many as nine such regions. The vas deferens, in general, is made up of an outer muscular and an inner glandular layer. In Eupagurus hertihurdus, the outer muscular layer is extremely thin and the thicker glandular layer

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varies significantly in thickness in different regions of the duct (Mouchet, 1931). Corresponding to the changes in the thickness of the inner secretory layer, the contour of the lumen also changes. In the proximal spiral, the epithelial cells are cuboidal and line a cylindrical canal. Secretory substance “A” is produced by these cells and used for agglutinating the sperm released from the testis into a sperm mass. The inner shape of the lumen, however, changes into an elliptical condition by the diminution of epithelial cells at the two diametrically opposite points together with the enlargement of the lateral cells. At the opposite points of cellular depletion are found two cavities filled with a new basophilic secretion “B”. This secretion surrounds the inner sperm mass, forming an envelope as well as a stalk that retains connection to the material found in the groove. In the last turn of the first spiral, one of these grooves is well developed, with the gradual obliteration of the other. The substance “B” now accumulates within the groove on one side. The cells at the base of the groove remain small whereas those at the lateral sides grow inwards and restrict the space connecting the axial lumen. Yet another new secretion, emanating from the epithelial cells lining the ventral groove, spreads out to form the basal layer, which is folded laterally on itself. Another mucoid secretion accumulates in the space between the basal layer and the spermatophore ampoule in the upper axial lumen. All these secretions exhibit different tinctorial properties. At the end of the second spiral, the fragmentation of the spermatic cord takes place little by little. The internal lumen becomes enlarged due to thinning of the epithelial cells. The spermatophores take a helical turn to place themselves at 90” to their initial position. In the terminal region, each of the basal layers, which carries many spermatophores, is placed in the transverse plane. In general, different regions of the vas deferens implicated with secretions of various spermatophoric compounds have been found to be remarkably similar in different hermit crabs. Although histological similarities in the secretory epithelial cells of the vas deferens have been recorded in many pagurian species, controversies occur with reference to the fragmentation of discrete spermatophores from the continuous sperm sheath. Mouchet (1931) suggested that the point of inflexion between the opposed coils was the site and cause of the fragmentation of continuous sperm ribbon in the Pagurid species she studied. However, Matthews (1953) and Greenwood (1972) found evidence for the specialized muscular activity as well as the modified lumen shape being the causative agents for the spermatophoric fragmentation from the continuous sperm sheath in D. asper and Pugurus “ovae-zealundiue, respectively. (ii) Sund crabs. Among Anomura, the galatheids, pagurids and porcella-

150

T. SUBRAMONLAM

nids are known to produce typical anomuran spermatophores (described above) in which the continuous, convoluted sperm tube is broken into distinct ampullae, and elevated on peduncles attached to a foot-like base (Mouchet, 1930, 1931; Matthews, 1953; Greenwood, 1972; Uma and Subramoniam, 1984). Conversely, the hippid mole crab, Hippa pacifica, possesses a spermatophore in the form of a continuous, highly convoluted tube raised by a continuous ribbon-like stalk attached to a broad foot (Matthews, 1956a). However, the nature of the deposited spermatophore is not known, as observations on attached spermatophores are lacking. Equally unknown is the mode of sperm release from the spermatophore in this species. As in the pagurids, the changes in the lumen shape (circular, key-hole shape and spindle shape), as well as the differential secretory activity of the glandular epithelial cells lining the vas deferens lumen, bring about spermatophore production in Hippa. Apparently, the vas deferens of N. pacifica differs from that of the pagurids, chiefly in the absence of a region where there is both constriction of the lumen and specific muscular activity that brings about the fragmentation of the continuous sperm sheath into spermatophore units. Further deviation from the basic anomuran plan is found in the spermatophores of yet another hippid mole crab, Emerita asiatica (Subramoniam, 1977, 1984). In this crab, dimorphic spermatophores, one in the form of a truncated cone and the other in the form of a tumbler, are arranged almost alternately in a single file. The lower ends of the spermatophores possess peduncles, which join with a continuous gelatinous ribbon. The whole spermatophoric mass is embedded in a protective jelly-like matrix (Figs 9-15). The extruded spermatophore has a thick, double-layered refractile covering. The spermatozoa are glued together by a viscous fluid and packed closely, irregularly inside the spermatoFic;. 9. Side view of spermatophore ribbon o f Emeriia asiuiica (Subramoniam, 1977). FIGS10 and 11. ‘Truncated cone-shaped spermatophore of Emerita asiatica before and during sperm release (Subramoniam, 1977). FIGS12 and 13. Tumbler-shaped spermatophore of Emerita asiaiica before and during sperm release (Subramoniam, 1977). F I G . 14. Freshly extruded spermatophoric mass of Alhunea symnista showing the loop-like convolution of the spermatophoric tube. (Redrawn from phase contrast micrograph of Subramoniam. 1984.) FIG.15. Diagrammatic representation of the part of posterior vas deferens and distal vas deferens showing the origin and arrangement of the spermatophoric mass. (Redrawn from Suhrainoniam. 1983.) A: ampoule: DVD: distal vas deferens; GC: gelatinous cord: GM: gelatinous matrix: PVD: proximal vas deferens: S: stalk: SP: spermatozoa; SI’: spermatophoric tube.

SPEKMATOPklOKES IN MARINE CRUSTACEANS

G'C

151

152

T. SUBRAMONIAM

phore. In another anomuran sand crab, Albunea symnista, the spermatophore is non-pedunculate and comprises a highly convoluted tube with a firm membrane forming a cord-like mass. This spermatophore ribbon is embedded in a gelatinous matrix, as in Emerita (Subramoniam, 1984).

Origin of spermatophores: The vas deferens in these two sand crabs is simpler than in pagurids. In Albunea syrnnista, the paired vasa deferentia are straight tubes running posteriorly behind the paired testes for a short distance and then turning to the anterior side, forming the descending and ascending limbs. The ascending limb again turns back to open at the base of the fifth walking leg. The entire vas deferens is divisible into a short proximal and a dilated distal portion. The spermatozoa as released from the testis are clumped in the anterior part of the vas deferens. This sperm mass is ensheathed in a thin wall by condensation of the secretory material originating in the vas deferens. This walled sperm mass passes into the enlarged distal portion of the vas deferens. The completed spermatophore is a straight tube without any accessory secretion adhering to it. As the tubular spermatophore enters the first part of the distal vas deferens, it twists and lies opposed to the ventral epithelial wall (Fig. 15). This twisted tube as it extends distally, becomes folded into loops and is set on to a firm membrane. Between the loops the spermatophoric tube tends to constrict towards the ventral region and often forms node-like structures apparently interrupting the continuity of the spermatophoric tube (Fig. 14). This spermatophoric structure is retained even after its extrusion and deposition on the females. Before mating, the ventral thickened gelatinous cord connects the highly convoluted spermatophoric ribbon ventrally. Two typhlosole-like structures made up of epithelial cells in the dorsal region produce a secretion which fills up the rest of the vas deferens lumen, constituting the protective matrix, whereas the secretion of the ventral epithelial cells produces the gelatinous cord. In contrast to A . symnista, a continuous sperm sheath is not evident in the proximal vas deferens of E. asiatica. Here, the rod-shaped mature spermatozoa are agglutinated into many clusters probably similar to a condition described in the spider crab Libinia emarginata (Hinsch and Walker, 1974). The circular lumen changes gradually to an elliptical shape. A thick gelatinous layer formed by the ventral epithelial cells condenses into a thick cord on the ventral luminal surface. This gives off branches linked to individual spermatophore ampoules, which lie distributed on the dorso-lateral periphery of the lumen, providing, also, an outer covering to them. A frothy material produced from the typhlosole of the dorsal epithelial cells constitutes the protective matrix material of the extruded spermatophore.

SPERMATOPHORES IN MARINE CRUSTACEANS

1.53

Recently Hinsch (1991) described the ultrastructural features of spermatophore origin in an oceanic anomuran crab, Pleuroncodes plunipes. The sperm leaving the testis is surrounded by a testicular matrix material, consisting of fine particles and clumped aggregates. In the vas deferens, this matrix material, along with aggregates of sperm, becomes surrounded by an electron-dense homogeneous secretion which forms the individual sperm masses. A stalk-like peduncle, attaching individual sperm masses to the base of the completed spermatophore, also originates from the electron-dense material. In the posterior region of the vas deferens, additional layers of mucopolysaccharides are added onto the spermatophore containing several sperm masses. Within the spermatophore, the nuclear arms of the sperm reside in depressions or indentations of the nuclear membrane. Sperm, when released from the spermatophore, presumably straighten their nuclear arms, which contain microtubules. Similar straightening of the nuclear arms in the released spermatozoa has also been observed in the mud crab, Scylla serratu (personal observation). (b) Chemical composition of the spermatophores Among Anomura, only two sand crabs, Albuneu symnista and Emerita usiaticu, have received attention on the chemical characterization of the spermatophore (Subramoniam, 1984). A detailed histochemical analysis reveals that mucopolysaccharides complexed with proteins form the main components of the spermatophores of A. symnista and E. asiatica (Tables 4 and 5 ) . In A . symnisru, the spermatophore wall is composed mainly of a neutral mucopolysaccharide, and is negative to tests for chitin. The sperm-bearing substance within the spermatophore tube stains metachromatically with toluidine blue, suggesting the presence of strongly acidic groups. These acidic groups are in the form of sulphated polyanions. The sperm cells are PAS-positive suggesting the presence of glycogen. The basal gelatinous cord contains a neutral mucopolysaccharide which is conjugated to a protein rich in basic and aromatic groups. The gelatinous cord also gives a positive reaction for the enzyme phenolase. The protective matrix contains acid mucopolysaccharide rich in carboxylated groups. The protein of the gelatinous matrix is rich in tryptophanyl groups. Histochemically, the spermatophoric mass of E. asiatica is similar to A. symnista, especially in its mucopolysaccharide content (Table 5 ) . Such a mucopolysaccharide heterogeneity in the various spermatophoric components of the two sand crabs may be correlated to their protective and structural functions. This study also indicates that the crustacean spermatophores could serve as a good model system for histochemical investiga-

TABLE> 4.

tlIS1 O C I l b M l C A1 CHARACTERISTICS OF M U C O P O L Y ~ A C C H A R I D CSUBSTANCES OF THE S P h R M A I OPFIOKIC

A LRUNEA Tests

Best’s Carmine (BC) Diastasc

+ BC

Schiff alone Periodic acid Schiff (PAS) Diastase + PAS

+ PAS Delipidation + PAS

Acetylation

Alcian blue - PAS (ABPAS)

MASS OF +, VI P

SYMNIJTA

To i n d i c a t e

S p e r m massa

Spermatophore wall

Gelatinous cord

Gelatinous matrix

R

++ +

+ R +

R

+++ ++

R k

Glycogen and mucopolysaccharides Presence of glycogen

R

R

R

-

-

-

+

++

+++

M Sperm cells

M

M

+ -

+ M + M +++

++ + M ++ M +++

Free aldehydes Glycogen, 1,2 glycol groups, unsaturated fatty acids and acid mucopolysaccharides Removal of glycogen

M

M

Sperm cells -

t

++ M Sperm cells

+

Removal of 1,2 glycol groups

+

+++

Acid and neutral mucopolysaccharides

+

B Sperm mass substance AB-PAS after mild mcthylation

++

+++

+++

+++

M Sperm cells

M

M

B

+

B Sperm mass substance AB-PAS after strong mcthylation

++

+++

+++

+++

M Sperm cells

M

M

B

+

M

w X

Acid mucopol ysaccharides

M B

5

z

Hydrolysis of sulphated groups and esterification of carboxylated groups (Spicer, 1960) Complete hydrolysis of sulphated groups

>

3

Sperm mass substance

+

+++ ++

Aldchyde fuchsin

P

Bracc-Cu rt i

Sulphated and non-sulphated acid muco substances Sulphated groups

P -

BB Toluidine blue at different PH PH 1

+++ +++ V ++ BV ++

+ ++ V ++ V +++

V

pH 3 PH 4

PH 7

0.6M 0.8M

+

++

B k

B

1%) Aqueous alcian blue

B

=

blue; BB

+ ++ B +

benzidine blue; BV

Phosphated mucosubstances

+ +

Strongly sulphated mucosubstances Strongly sulphated mucosubstances Sulphated mucopolysaccharides

B -

Chitin

B

B

-

=

Carboxylated mucosubstances

++ ++

B

Chitosan

Phosphated and carboxylatcd mucosubstances Carboxylated mucosubstances

B

B

1 .OM

Sulphated mucosubstances

BV

B Alcian blue; Critical electrolyte conccntrations of MgClz 0.2M

Sulphated mucosubstances

V

=

bluish violet; M = magenta; P

+ = moderately positive; ++ = positive; +++ = intensely positive.

=

pink; R

=

red; V

=

violet; -

=

negative; k

=

doubtful;

“Sperm mass refers collectively to sperm mass substance, which binds the sperm cells within the spermatophore as well as the sperm cells. When reactions are distinct for sperm cells and sperm mass substance they are indicated accordingly. wl wl

TABLE 5. HISTOCHEMICAL CHARACTERISTICS Tests

Best's Carmine Schiff alone Periodic acid Schiff (PAS) Alcian blue-PAS

Sperm mass"

OF

MUCOPOLYSACCHARIDH SLJBSTANCES EMERITAASIATICA

Spermatophore inneriouter layer

Peduncle/ Gelatinous cord

+

+I+

+ +/+ +

R

WR

R/R

-1-

-1t-+I+ MIM

+++ M Sperm cells

++

M Sperm cells

+ + +/+ MIM ++I+ M/M

+

OF

Gelatinous matrix

+

R

-

+

M

+I+

+++

M/M

B

SPERMATOPHORIC MASS

OF

To indicate

Glycogen and mucopolysaccharides Free aldehydes Glycogen, 1,2 glycols mucopolysaccharide and unsaturated fatty acids Acid and neutral mucopol ysaccharides

+

Bracco-Curti

++

+/-

B

Pi

++

-1-

-I-

++

+I-

+/+

V

€51

-1-

+ P

Sulphated and nonsulphated acid mucosu bstances Sulphated groups

BB Toluidine blue at different pH PH 1

2 icl

>

s 0 5 > 3

B Sperm mass substance Aldehyde fuchsin

i

+ V

Sulphated mucosubstances

PH 3

PH 4 PH 7 Alcian blue: critical electrolyte concentrations of MgC12 0.2M

++I-

+I+

BI

V

BV

Bl

VIV +/+ VIV

++

+++IVI

+/+

+

BIB

V

++

B

0.8M 1.OM 1% Aqueous alcian blue Chitosan

+ + +/-

V

Sulphated mucosubstances Phosphated and carboxylated mucosubstances Carboxylated mucosubstances

w -c

m

w

z

5

: 0

XI

+ + B + + B + B

0.6M

++ +++

++ V

B

-

B/

+I+

+I+ BIB

+/-

-I-

B/ +I+

-/-

-I-

-I-

+/+

+/+

BI -1-

++ ++ B ++ B ++ B ++ B

Carboxylated mucosubstances Phosphated mucosubstances Strongly sulphated mucosubstances Strongly sulphated mucosubstances Sulphated mucosubstances

-

2

5

2 z rn

0 C

?J

? m

B -/-

GI

Chitin

B = blue; BB = benzidine blue; BV = bluish violet; M = magenta; P = pink; R = red; V = violet. "Sperm mass refers to sperm mass substance as well as sperm cells. When reactions are distinct for sperm cells and sperm mass substance they are indicated accordingly.

%

TABLE6. SUMMARY ~~~~

OF T€IE CtIAKAC.~.ERIZAI.ION OF MUCOPOLYSACCHARIIXS OF I H E AL'HUNEA S Y M N I S T A AND EMERITA ASIATIC'A

SPEKMATOPHORIC COMPONENTS

OF

~

Spermatophoric components

Chemical nature

Origin

Albunea symnista 1. Sperm mass substances 2. Spermatophore wall 3 . Gelatinous cord 4. Gelatinous matrix

Sulphated AMPS Neutral MPS Neutral MPS Carboxylated AMPS

PVD PVD DVD, Ventral epithelium DVD, Dorsal epithelium, especially typhlosole

Sulphated AMPS

PVD

Carboxylated AMPS Neutral MPS Neutral MPS Periodate reactive AMPS

PVD DVD, Ventral epithelium DVD, Ventral epithelium DVD, Dorsal epithelium, especially typhlosole

Emerita asiatica 1. Sperm mass substance 2. Spermatophore wall Inner layer Outer layer 3 . Peduncleigelatinous cord 4. Gelatinous matrix

AMPS = acid mucopolysaccharides; MPS From Subramoniam, 1984.

=

mucopolysaccharides; DVD

=

distal vas defercns; PVD

-i

2a:

ti 0

z

5

=

proximal vas deferens

3

SPERMATOPHORES IN MARINE CRUSTACEANS

159

tion of mucopolysaccharides, in view of the occurrence of a variety of mucopolysaccharides in the spermatophore structures (Table 6). (c) Spermatophore transfer and dehiscence In general, anomuran crabs lack intromittent organs for transferring the spermatophores into the female genital tract. The spermatophores are deposited on the ventral sternum of the crab. Therefore, the development of a well-defined peduncle in the anomuran spermatophore is a special adaptation for attachment. The mode of attachment differs markedly among different species of Anomura. In Diogenes pugilutor, only a single spermatophore is attached with its gelatinous base to the sternum of the female (Bloch, 1935). In Pagurus bernhardus, four to five spermatophores are placed on each gelatinous strip and the attachment is only through the stumpy peduncle; the ampoules of the spermatophore are not bound to one another (Jackson, 1913; Bloch, 1935). In the mole crab, Emerita asiatica, spermatophore transfer and mating behaviour have been described by Subramoniam (1977, 1979). As many as five tiny neotenous males deposit their spermatophores in the pleopodal region of one female. In Emerita sp., the spermatophore extrusion occurs through a muscular genital papilla situated at the inner side of the base of the fifth thoracic leg (Wharton, 1942; Snodgrass, 1952; Subramoniam, 1977). In E . asiatica, the spermatophore deposition occurs only in the fresh moult condition; the spermatophore ribbon sticks to the female pleopodal region very firmly. Ovulation occurs only after spermatophore deposition, suggesting that it may stimulate egg laying. The mechanism of sperm release from the spermatophore has long been controversial in anomuran crabs. Hamon (1937), studying this mechanism in Pagurus prideauxi, considered many factors such as mechanical pressure and enzymatic digestion of the spermatophoric envelopes. Oviductal secretions released during egg laying have also been considered responsible for spermatophore dehiscence. The process of sperm release has been recorded in E. asiatica (Subramoniam, 1977). The spermatozoa are released always through a definite spermatophore opening (Figs 11 and 13). In the truncated cone-like spermatophores, the opening is made through the nipple-like projection found at the opposite end of the peduncle. In the other, larger type, the wider region is rimmed by a well-defined lip which is firmly closed before sperm release. Streaming of spermatozoa is first observed in the gaps formed at the corners of the wider end and then in several sites of the centre, resulting in the complete opening of the lips. After extrusion of all spermatozoa, the lips remain completely apart. The fact that the spermatozoa release Occurs only after contact with the eggs suggests that an oviductal secretion

160

T. SUBRAMONIAM

may be responsible for the digestion of the cementing material closing the lip of the spermatophore.

3 . Macrura (a) Lobsters (i) Spermatophore morphology. Lobsters are typical spermatophoreproducing macruran decapods. Their spermatophores are generally complex masses consisting mainly of spermatophoric tubes embedded in a protective gelatinous matrix. However, different families of lobsters produce spermatophores with characteristic differences in their composite structures. Such differences are especially related to the nature and number of layers comprising the protective matrix of the spermatophore (Table 7). Lobster spermatophores have a definite function in the protection and preservation of spermatozoa, pending epizoic fertilization. In spiny lobsters (Palinuridae) and rock lobsters (Scyllaridae) the spermatophore mass is deposited externally on the ventral sternum of the females, whereas in homarid and nephropsid lobsters they are stored within the seminal receptacles (thelycum) of the females. This difference in the mode of spermatophore storage by the females is reflected in the transformation they undergo after deposition in the females. Reports on the spermatophores of lobsters and crayfish go back to 1908 when Dahlgren and Kepner described a fluid which, secreted by their sperm ducts, not only served as a vehicle to carry the mass of sperm but also formed a semi-fluid covering them. Thereafter many reports appeared on the occurrence of spermatophores in lobsters belonging to different species (e.g. Andrews, 1931). However, most of them were casual observations, and it was Matthews (1951, 1954) who, from a series of histological studies of several lobster species, not only traced the origin of spermatophores within the lumen of the vas deferens, but also suggested the possible mechanism of sperm release during fertilization. In the spiny lobster, Panulirus penicillatus, Matthews (1951) found a complex spermatophore consisting of a putty-like matrix surrounding the highly coiled, continuous spermatophoric tube containing the agglutinated sperm cells. He also suggested that spermatophores of Potamobius may be of similar nature. In Panulirus homarus, Berry (1970) recognized three distinct horizontal matrix layers in the deposited spermatophoric mass. They are: (1) an outer crust-like layer, termed the protective matrix; (2) a middle layer, bearing the highly convoluted spermatophoric tube and (3) a basal spongy layer, termed the adhesive matrix. The protective matrix is strongly eosinophilic, containing calcium granules, while the underlying

SPERMATOPHORES I N MARINE CRUSTACEANS

161

spermatophoric matrix differs from the former only in its consistency. The basal adhesive matrix has a striated appearance with strong eosinophilic reaction. The spermatophoric masses of other palinurid lobsters have this basic pattern, although a few deviations, such as the absence of a distinct spermatophoric tube wall, as in Panulirus angulatus and Linuparus trigonus, do occur (Berry and Heydorn, 1970). Nothwithstanding the basic similarities in the spermatophores of five palinurid genera described above, differences in the nature of mucoid substances reflect on the mode of attachment and fertilization process. For example, in Jasus lalandii, the gelatinous matrix disintegrates in sea water, releasing the thread-like spermatophore into the ambient medium, thus necessitating immediate oviposition after mating. In Pulinurus gilchristi, P. angulatus and L . trigonus, the protective matrix is gelatinous with a putty-like consistency, which hardens in the sea water (Table 7). Spermatophore hardening in the palinurid lobsters might have evolved in response to shallow, turbulent water conditions where a soft spermatophoric mass would be liable to be washed off. This condition may imply a trend towards prolonging the interval between mating and oviposition. On the other hand, the unspecialized arrangement of the spermatophoric mass of J . lalandii and its solubility in sea water may be considered as a primitive feature. In the nephropsid lobster, Enoplometopus occidentulis, the tubular spermatophore consists of a highly coiled continuous spermatophoric tube ensheathed by an outer matrix with a narrow sticky region for adhesion to the substratum (Matthews, 1954). An interesting feature of this spermatophore is that it becomes hard when exposed to sea water, although the female receives and stores the spermatophore within the seminal vesicle, which is unconnected to the oviduct internally. In a recent study on the spermatophores of this Hawaiian red lobster, Haley (1984) established a structural homology of the spermatophoric layers ensheathing the inner sperm tube with those described by Kooda-Cisco and Talbot (1982) for Homurus americanus. Nothwithstanding the variations found in the spermatophores of palinurid, homarid and nephropsid lobsters enumerated above, a close examination reveals a basic similarity in the spermatophoric layers. Structural transformation, however, occurs as a result of spermatophore hardening in those species in which the spermatophores are fastened to the ventral sternum of the female. Panulirid spermatophores undergo hardening and blackening after attachment to the female sternum. Martin et al. (1987) recently investigated the structural transformation and chemical changes after spermatophore attachment in Panulirus interruptus. In this species, the spermatophore is composed of a highly coiled sperm tube embedded in an acellular matrix. The extruded spermatophore is white, soft and sticky

Y

cn

h,

TABLE 7. SPERMATOPHORIC COMPOSITION I N LOBSTEKS

Species

Spermatophoric tube

Adhesive matrix

Protective matrix

P. homarus Berry, 1970 Berry and Heydorn, 1970 P. gilchristi Berry and Heydorn, 1970

Highly convoluted cylindrical tube, Spermatophore wall distinct, granular

Present, eosinophilic and globular matrix

Granular with a putty- Becomes hardened and like consistency darkens

P. angulatus Berry and Heydorn, 1970 L. trigonus Berry and Heydorn, 1970

Spermatophore triangular in Globular matrix layer cross-section, wall composed eosinophilic of a homogeneous eosinophilic substance, spermatophoric wall thickness not uniform Globular matrix Consists of large tightly compacted core of spermatozoa embedded in an eosinophilic mucus, but lacks a spermatophore wall Consists of large tightly compacted core of spermatophore embedded in an eosinophilic mucus, but lacks a spermatophore wall

Globular matrix

Gelatinous matrix with dense areas of agglutination

Behaviour in sea water

Remains insoluble in water

Gelatinous but Insoluble in sea water without dense areas of agglutination. Resembles the mucus covering spermatozoa Insoluble in sea water Gelatinous but without dense areas of agglutination. Resembles the mucus covering spermatozoa

9 in

C

m

XI

J . lulundii Fielder. 1964

Spermatophore a thin continuous Absent thread of spermatozoa with no surrounding wall. Convoluted randomly in the matrix P. penicillutus Crystalline granular Not distinguishable Mathews, 1951 spermatophoric wall. Highly convoluted tube Enoplometopus Tubular sperm mass. Possesses A thin outer bounding occidentulis distinct PAS positive wall layer on sides of Mathews, 1954 spermatophore Haley, 1984 adheres to the substratum after ejaculation Panulirus Consists of tightly packed Foot of the interruptus spherical cavities in an spermatophore Martin et ul., 1987 acellular material within which characterized by the sperm lie vertical striations after attaching on the female Thenus Sperm are enclosed in ovalNot distinguishable, spermatophoric orientulis shaped vesicles which are Silas and mass attaches t o the connected t o one another by a Subramoniam gelatinous cord to form a female by any part (unpublished spermatophoric rope. Sperm of the surface observation) capsule wall PAS-positive

Homogeneous gelatinous matrix Present Gelatinous homogeneous matrix, PASnegative

Disintegrates in sea water. It is reasonably fluid and sticky Hardens Not known

Composed of granules The cap of the embedded in a loose spermatophore will weave of filaments harden and darken after exposure to sea water Fibrillar network, contains elastin

Remains insoluble in sea water

164

T. SUBRAMONIAM

on all surfaces. The sperm tube is seen near the surface of the foot of the spermatophore, which is the site of attachment to the female body. The opposite surface, the cap, hardens and darkens after exposure to sea water. In the homarid, H . arnericanus, the spermatophore does not undergo hardening on exposure to sea water and transference to the female, but the stickiness of the freshly extruded spermatophore ensures the successful transfer to the female seminal receptacle (Kooda-Cisco and Talbot, 1982). In this lobster, the inner tubular sperm mass is surrounded by three investment layers: (i) a primary spermatophoric layer, which is amorphous and PAS-positive; (ii) an intermediate layer containing PAS-positive granules and (iii) an outer bounding layer comprising small filaments and a flocculent material. Comparatively, studies on the spermatophores of scyllarid lobsters are meagre. However, as early as 1909, Calman reported that the spermatophores of these lobsters are pedunculate, as in the anomuran crabs. Matthews (1954) studied this pedunculate condition of t h e sperrnatophores in the rock lobster, Parribacus antarcticus. The spermatophoric mass is a continuous, thread-like, highly twisted ribbon from which the pedunculate spermatophores extend. The ribbon as well as the spermatophores are embedded in a gelatinous matrix. On attachment to the female sternum, the outer portion of the matrix becomes hard and dark. Conversely, the inner matrix containing the spermatophoric ampullae, as well as the peduncle, remains soft. Yet another deviation from the macruran pattern of tubular spermatophoric masses is found in another scyllarid sand lobster, Thenus orientalis (Silas, 1991). The electrically extruded spermatophoric mass is pointed at both ends and “bellied” in the centre. The extruded spermatophoric mass is pliable and sticky. It does not harden on exposure to sea water, but becomes flaccid like that of H . arnericanus. It consists of an outer thick protective matrix layer. The interior of this matrix material contains fibrillar structures criss-crossing the entire inner space. Interspersed with these fibrils are the numerous pod-shaped double-walled sperm sacs, containing the inner sperm mass. The sperm sacs are connected together by collagen-like fibrils. The sperm-bearing substances within the sperm sac also contain anastomosing mucoid strands. In the lobsters, fertilization is epizoic or external, even if the spermatophores are stored within the seminal receptacles, the reason being the lack of connection between the seminal receptacles and the oviduct. Waddy and Aiken (1991), however, reported internal fertilization in H . arnericanus, as there is internal continuity between the oviduct and thelycum in this species. This needs verification as the astacoids are known to possess only integumentary thelycum (Hartnoll, 1975). The

SPERMATOPHORES IN MARINE CRUSTACEANS

-

165

evidence is more in favour of external fertilization with the sperm release from the hardened spermatophores being achieved by scratching of the outer hardened layer by the powerful chelae of the lobsters (Matthews, 1954; Berry, 1970). (ii) Origin of spermatophores. Berry and Heydorn (1970) have studied the formation of spermatophores in the vas deferens of five palinurid genera namely, Panulirus homarus, Palinurus gilchristi, Puerulus angulatus, Linuparus trigonus and Jams lalandii. In general, the vas deferens consists of two distinct portions, a proximal portion consisting of a narrow, coiled tubule and a distal enlarged portion. In P . homarus, the proximal vas deferens (PVD) is internally lined by glandular epithelium which is slightly proliferated dorso-laterally. These columnar cells secrete a strongly eosinophilic granular matrix, which surrounds a core of tightly packed sperm and forms a layer over it. This forms the spermatophoric tubule or spermatophore proper. In the distal vas deferens, the dorsal epithelium, by proliferation, has given rise to a typhlosole which runs along the dorsal side up to the terminal end. The typhlosole secretes a less viscous eosinophilic granular matrix, within which the spermatophore remains as a discrete tubule. The spermatophore in the distal region of the vas deferens is arranged in regular convolutions and is pushed to the ventral periphery by the extensive production of matrix material from the typhlosole. The ventral epithelium simultaneously secretes a thin matrix consisting of large strongly eosinophilic granules below the spermatophore; its function is to cement the external spermatophoric mass to the sternum of the females. Due to the same eosinophilic staining, the ventral adhesive matrix has been indistinguishable in species such as P . penicillatus (Matthews, 1951). However, in P. hornarus, the three horizontal layers, namely an outer protective matrix layer, a middle spermatophoric mass layer in which the spermatophores are embedded and a basal adhesive matrix layer which cements the spermatophoric mass to the female’s sternum, are evident even in the externally deposited spermatophoric mass (Berry, 1970). In P. gilchristi, the composition of the spermatophore is similar to that of P . homarus; but the protective matrix is gelatinous and not granular. The typhlosole in the dorsal region of the distal vas deferens consists of a central core of muscle and connective tissue which is merely lined with glandular epithelium. Although the histology of the vas deferens of J . falandii is similar to both P . homarus and P . gilchristi, the composition of spermatophore is different. The lumen of the vas deferens is filled with a gelatinous matrix which is more homogeneous than that of the other two lobsters. Embedded in the matrix is the spermatophore which is a thin continuous thread of

166

T. SUBRAMONIAM

spermatozoa with no surrounding wall. Again, the thread-like spermatophore is not localized ventrally as in the other two lobsters, but is convoluted randomly throughout the matrix. However, a ventral, strongly adhesive eosinophilic globular layer is present. In an ultrastructural study, Kooda-Cisco and Talbot (1986) described the fine structure of the cells of the proximal vas deferens, in relation to their secretory role in producing the spermatophore layers. The wall of the anterior-most part of the vas deferens comprises an outer layer of connective tissue, including several layers of fibroblast-like cells, a thin layer of striated circular muscle and the inner epithelium lining the lumen. The fibroblast-like cells produce electron-dense vesicles, which may be the precursors of collagen that is found as a network in the connective tissue. The inner epithelial cells rest on a thick basal lamina. The basal plasma membrane of the inner epithelium undergoes extensive infoldings and interdigitations with adjacent cells. Microtubules are abundant throughout the cytoplasm. The nucleus is usually located in the basal half of the cell. These cells possess mitochondria, rough endoplasmic reticulum (RER) and Golgi bodies, and the apical region of the cytoplasm contains many electron-dense vesicles which appear to release their contents into the lumen by exocytosis. Within the lumen, the vesicles are supported in a matrix of moderate electron density. Numerous collagenous fibres have also been encountered. Subsequently, the loose spermatozoa are aggregated into a linear mass that extends the entire length of the lumen. The inner epithelium reveals a kind of zonation in ultrastructural features. Multilobed nuclei and mitochondria are positioned in the cell’s basal half; Golgi bodies assume a juxta- or supra-nuclear position. Golgi-derived vesicles migrate mostly towards the apical cell surface. Between the nucleus and the apical surface is the densely packed R E R in the form of parallel and stacked cisternae and vesicles. Near the apex, the vesicular form is more prevalent than the cisternal form. Irregular microvilli cover the apical cell surface. The vesicles of RER in the cell apex are located very close to the plasma membrane and may contribute their contents to the lumen. In addition to the small secretory vesicles originating from R E R , large membrane-bound vesicles form in an area above the nucleus. As these larger vesicles migrate towards the cell apex, they appear to break down into small vesicles which migrate the remaining distance to the apical surface where they appear to fuse with the plasma membrane and release their contents. In this region, the epithelial cells secrete the primary spermatophore layer which contains fibres that can be distinguished from the collagen-like fibres of the sperm-supporting matrix by their smaller size.

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In general, the vas deferens of the lobsters possess an intensely secretory, glandular inner epithelial layer. In the distal vas deferens (DVD), there are specialized typhlosole-like thickenings. In P. homarus. the typhlosole is trilobed with two large foliaceous outer lobes and a small middle lobe in the anterior part of DVD (Figs 16-18) (Radha and Subramoniam, 1985). This configuration gradually changes towards the posterior region, with the trilobed typhlosole changing into a bibbed condition which is finally reduced into a single dorsomedial lobe. In general, the protective gelatinous matrix originates from the typhlosole of the dorsal epithelial cells of the distal vas deferens of the lobster. The distal part of the vas deferens also gets partitioned internally by the

16

17

18

FIGS 16-18, Diagrams representing the typhlosole in the distal vas deferens of PumihruJ homarus. 16, Trilobed typhlosole with two large lateral lobes and a small middle lohc. 17. Bibbed typhlosole. 18, Single lobed typhlosole.

EL: epithelial layer; GM: gelatinous matrix; MC: muscle and connective tissue layer: SP: spermatophoric mass: TY: typhlosole. (Redrawn from Radha and Suhramoniam, 19x5.)

168

T. SUBRAMONIAM

overgrowth of the longitudinal musculature in the rock lobster Enoplometopus occidentulis (Haley, 1984). The glandular epithelial cells of the newly formed tubule secrete a gelatinous, PAS-negative eosinophilic material. This material extrudes from the ductal gland soon after the spermatophore deposition into the female thelycum, thus serving as a plug for the thelycum. A similar process has also been reported for Homurus umericunus (Herrick, 1909). (iii) Chemical composition. Although much information is available on the spermatophore production in lobsters, little is known about their chemical nature. Such knowledge is especially important in view of the fact that they undergo hardening in many species, soon after exposure to sea water. A detailed histochemical study on the spermatophores of the spiny lobster, Punulirus homurus, reveals that the wall of the spermatophore tube consists of neutral mucopolysaccharide, whereas the inner sperm-bearing matrix as well as the gelatinous matrix are rich in acidic mucopolysaccharide (AMPS). The AMPS of the outer gelatinous matrix is rich in both sulphated and carboxylated groups. Further characterization of the isolated AMPS of the lobster spermatophore reveals the occurrence of chondroitin sulphate (Radha and Subramoniam, 1985). The authors suggest that these polyanionic groups of AMPS might bind with inorganic ions such as calcium on exposure to sea water to bring about the so-called hardening of the lobster spermatophore. The predominance of the chondroitin sulphate in the AMPS may have a functional role in providing elasticity as well as resistance to compression, thus keeping the spermatophores from the risk of desiccation. The cementing function of AMPS has been well known in the vertebrate tissues (White et ul., 1978). The spermatophores of lobsters are known to be stored in the female body for a very long time. Judging from the multitude of functional roles of t h e mucopolysaccharides, particularly chondroitin sulphate, it may be suggested that they have a dominant role in spermatophore hardening as well as maintaining a microenvironment within t h e spermatophoric tube for prolonged sperm survival in the spiny lobsters. Martin et ul. (1987) have also made histochemical analysis of the spermatophoric components of Panulirus interruptus and found evidence for tanning and melanization of the outer protective matrix layer. (b) Penueoid shrimps In the penaeoid shrimps, the spermatophores and insemination morphology have been studied with greater interest in recent years in view of their value in establishing phylogenetic relationships and importance for hybridization by artificial insemination. Penaeids show remarkable

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169

variability in the morphology of spermatophores, the complexity being closely related to the nature of thelycum, which is the external modification of the female’s posterior thoracic sternites and/or coxae that are used in sperm receipt and storage (Bauer, 1986). Females with open thelyca generally receive a spermatophore with complicated sperm-free accessory structures such as wings, whereas in the closed thelycal forms the males produce simpler spermatophoric masses (Burkenroad, 1934, 1936; Perez Farfante, 1975; Bauer, 1986). In sicyonid shrimps the “spermatophores” are little more than spermatozoa in a viscous fluid (Perez Farfante, 1985; Bauer, 1991). The open thelycal species deposit a compound spermatophore which is pod-shaped after its assembly from two halves, expelled from the paired terminal ampullae, upon mating (King, 1948). In Penaeus setiferus, the compound spermatophore consists of a main trunk (or germinate body, Perez Farfante, 1975), a pair of wings at the anterior portion and glutinous material clinging to both sides of the trunk (Chow et al., 1991). The paired spermatophores upon extrusion and deposition onto the female sternum are joined along the medial adhesive layer. The germinate body containing the columnar sperm mass is surrounded by thick sheaths of gelatinous materials which undergo hardening. The anterolateral wings, postero-lateral glutinous material as well as the lateral flaps and the dorsal plate serve as the attachment structures of the spermatophores in various Litopenaeus species (Perez Farfante, 1975, Fig. 19). The structural homology in the spermatophores of different species of Litopenaeus is summarized in Table 8. Differences in terminologies of especially the sperm-free mucoid structures of the penaeoid spermatophores used by several workers have been discussed recently by Chow et al. (1991) as well as by Bauer and Cash (1991). The spermatophores are deposited in the sternal plates, XI1 to XIV, posterior to the females’ ovipores. In Penaeus occidentalis the sperm is released through the anterior opening of the sperm sac, whereas in Penaeus stylirostris, Penaeus schrnitti and Penaeus setiferus, the compound spermatophore splits longitudinally into two parts, exposing the inner sperm mass to the surrounding water (Perez Farfante, 1975). In the open thelycan species, the spermatophores are easily dislodged and hence, the time interval between mating and spawning is usually short (Primavera, 1985). In contrast to the highly complicated structures of the open thelycal spermatophore, those of the closed thelycum are less complex, consisting mainly of two basic divisions, namely the main body and the wing, or the appendage (Sasikala and Subramoniam, 1987; Bauer and Cash, 1991). In Penaeus indicus, the main body is composed of a bulky and viscous sperm mass surrounded by a thick envelope, the sperm sac (Sasikala and

170

T. SUBRAMONIAM

FIG. 19. Ventral view of a compound spermatophore of Penaeus (Liropenaeus) setiferus attachcd to female. DP: dorsal plate; F: flap; FG: flange; GB: germinate body with inner sperm mass; GM: glutinous material: GP: gonopore; W: wing. (Redrawn from Perez Farfante, 1975.)

Subramoniam, 1987). The membranous wing, attached to the sperm sac, is slimy and comparatively thinner in texture and more transparent than the opaque sperm sac. In Penaeus kerathurus, the sperm sac contains an inner thick homogeneous layer and a globular outer layer; the inner layer extends freely and serves as anchorage to the membranous wing (Malek and Bawab, 1974b). Further simplification in the spermatophore structure is found in Penaeus monodon (Motoh, 1981) and Metapenaeus monoceros (Sasikala and Subramoniam, 1987). In M . monoceros, the extruded spermatophore has two morphologically distinguishable parts: ( 1 ) a highly viscous sperm mass devoid of sperm sac; and (2) four to five adhesive, milky white, small, grain-shaped crystalline structures. The wing of the closed thelycal species undergoes a characteristic reaction when placed in sea water. I n P. indicus, it is extruded in the form of a cord which unfolds and spreads like an umbrella on contact with sea water, forming a large membranous foliaceous structure (Sasika-

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171

la and Subramoniam, 1987). Similar reactions have also been reported recently in Penaeus aztecus and Penaeus duorarum (Bauer and Cash, 1991). Here, the wing material is composed of delicate anastomosing sheaths that delaminate or unfold upon exposure to sea water. (i) Origin of spermatophores. The development of the spermatophore within the vas deferens of penaeoids, in both open thelycal and closed thelycal species, has received considerable attention (King, 1948; Malek and Bawab, 1974a,b; Champion, 1987; Ro et al., 1990; Chow et al., 1991; Bauer and Cash, 1991). In accordance with the complicated structure of the penaeoid spermatophore, the vas deferens also exhibits certain peculiar features, not found in other decapod crustaceans. In almost all penaeoid species described thus far, the vas deferens lumen is divided by a septum, into two lumina, the larger one containing the sperm mass and the smaller one containing the ‘‘wing’’ materials. Ro et al. (1990) have recently traced the origin of spermatophoric material at ultrastructural level in Penaeus setiferus. The small collecting tubules (segment I), with low columnar epithelial lining, transport sperm from the testis to segment 11. where the sperm pass through a blind pouch, which secretes a small quantity of sperm-bearing material. The blind pouch also functions in aligning sperm in longitudinal columns, in much the same way as the sperm of Macrohrachium orient in the sperm-supporting matrix with the spikes perpendicular to the long axis of the spermatophore (Dougherty, 1987). Distal to the blind pouch, segment IT produces the primary spermatophore layer and other acellular components of the spermatophore that remain separated from the sperm mass till they reach the terminal part of segment TI, where the secondary spermatophore layer forms around the primary layer. Segment 111, a translucent duct, serves as a conduit for transporting the partially formed spermatophore into the last segment, the terminal ampulla, where the spermatophore undergoes final maturation. Chow et al. (1991) and Bauer and Cash (1991) have described the different chambers (up to five in P. setiferus) within the terminal ampulla and the transformation of accessory secretory materials resulting in the hardening of the sheath enveloping the sperm mass. In the penaeid species, the terminal ampulla functions as a site for organizing the components of the spermatophore, as well as deposition of the spermatophore matrices, In the sicyonid shrimps, the seminal plasma is transferred to the females and stored in a pair of bag-like seminal receptacles, concealed under the thelycum (Clark et nl., 1986). Interestingly, the histology of the vas deferens differs significantly from that of other penaeoid shrimps. In Sicyonia ingentis the sperm released from the testis enter into the

172

T. SUBRAMONIAM

TABLE 8. CHARACTERISTICS OF THE SPERMATOPHORES OF FIVESPECIES OF PENAEUS (LITOPENAEUS)

Species

Sperm sac

Wing

Anterior Flange lobe

Blade

P. vannamei

Simple"

Lacking

Lacking Long caudal Present Short, caudal

Lacking

P. occidentafis Simple

Small

P. sty/irostris

Complex" Large

Lacking

P. schmitti

Simple

Moderately Lacking large

P. setiferus

Simple

Moderately Lacking large

Attached at edge extending laterally; broad Attached along midline, Long, extending caudolateral dorsomedially and ventromedially Attached at edge, Long, extending laterally, caudolateral broad anteriorly Attached at edge, Long, extending laterally caudolateral narrow throughout

"Lateral wall continuous with dorsomedial. hLateral wall overlapping dorsomedial wall laterally, forming free shield. From Perez Farfante, 1975.

proximal vas deferens where they are mixed with the secretions emanating from the epithelial cells (Subramoniam, 1992). Two cell types can be distinguished in this region; one bears ultrastructural characteristics of a secretory cell and the other is absorptive in nature. The secretory cells release electron-lucent vesicles and electron-dense granules into the lumen. The sperm found in this region is immature with no spike or spike promordium (Shigekawa and Clark, 1986). The second type has a microvillous brush-border which actively absorbs excessive fluid material secreted into the proximal vas deferens. In the mid vas deferens, the spermatozoa are highly concentrated due to condensation of the spermbearing matrix to form a sperm cord. All the spermatozoa found in this region are in advanced spermiogenic stage with some of them having fully formed spikes. The matrix material seems to condense on the periphery as a membranous layer. Interestingly, the concentrated sperm mass is liquefied in the distal vas deferens, probably by another secretion emanating from the secretory cells lining this region. In S. ingentis and probably in other species of Sicyonia, there is n o compartmentalization of the vas deferens lumen into sperm mass duct and wing duct. Instead, the secretory activity of the proximal vas deferens resembles that of the spider crab, Libinia emarginata (Hinsch and Walker, 1974).

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Table 8 - contd.

Lateral flap

Anterior Dorsal lamina plate

Along sac

Along flange

Broad, fleshy Lacking

Narrow, flexible Broad, highly sclerotized

Lacking Present

Position on female

Long, underlying most of sac and flange Moderately long, underlying blade

Reaching about mid length of sternite XI11 Reaching posterior part of sternite XI1, and embracing gonopore Moderately long, Reaching anterior-most part of sternite XI11 underlying base of sac and flange

Broad, Narrow, membran- flexible ous

Lacking

Narrow, firm

Broad, firm

Lacking

Long, underlying blade and flange

Reaching anteric>r-most part of sternite XI11

Narrow, firm

Broad, firm

Lacking

Moderately long, underlying flange

Reaching anterior-most part of sternite XI11

(c) Caridean shrimps Caridean shrimps produce relatively simple spermatophores, although deposition is external, as in open thelycal penaeoids. Unlike the penaeoid prawns, where the spermatophoric components are formed in a sequential manner from a variety of secretory materials emanating from the glandular epithelial cells within the vas deferens, the spermatophore is not preformed in carideans, but the secretions are poured into the ampullar region. The spermatophore proper is formed upon extrusion. The origin and structure of caridean spermatophores are best known in the freshwater shrimps (Bauer, 1976; Dougherty et al., 1986; Chow, 1982). In the giant freshwater prawn, Mucrobrachium rosenhergii, the pod-shaped spermatophore consists of a lateral sperm mass, a medial mucus mass, and a non-cellular capsule that sticks to the surface of the sternum (Dougherty et a f . , 1986). Homology in the functional morphology of six species of palaemonid shrimps has been revealed in a recent study by Chow et al. (1989). In the freshwater carideans, fertilization occurs externally, by a spermatophoric mass attached to the ventral surface of the female. An interesting deviation from this mode of spermatophore deposition has been described recently in a marine shrimp Crangon crangon (Boddeke et al., 1991). In this species, a tubular

174

T. SUBRAMONIAM

spermatophore is formed within the vas deferens; but during mating, only the sperm mass is injected into the oviduct, leaving behind the spermatophoric covering in the vas deferens, where it subsequently undergoes disintegration, The spermatophore of C. crangon does not possess any of the adhesive structures characteristic of penaeid and other freshwater caridean prawns. In the inseminated females, large numbers of sperm cells can be observed along the inner wall of the oviduct. Unlike the brachyuran crabs, a seminal receptacle in the form of spermatheca has not been reported in C. crangon. It will be interesting to determine whether the internal fertilization of C. crangon is unique o r more widespread among caridean shrimps (Boddeke er al., 1991).

B.

Copepoda

The copepods invariably employ spermatophores in sperm transfer. The first description of spermatophore transfer and mating behaviour in the copepods referred to the cyclopoid, Cyclops castor (Siebold, 1839). Since then, several reports have appeared on spermatophore production and trmisi'er in various copepodan groups (reviewed by Mann, 1984). In copepods, the spermatophore is an alternative to an intromittent organ inasmuch a s it delivers the male gametes directly into the female opening in the absence of copulatory organs. The copepod spermatophore is club-shaped or flask-shaped with its proximal end tapering into a tube-like neck, through which sperm flow into the female opening. An adhesive material deposited around the neck region helps in fastening the sperniatophore onto the female body. Although the general morphology is coinmon in copepod groups, variations d o occur with respect to the sperniatophoric contents causing sperm release and transference to the female gonopore as well as the accessory secretions involved in spermatophore attachment to the female body.

Morphological diversity In the calanoid Euchaera norvegica a simple spermatophore with a slender neck is produced (Fig. 20). The spermatophore proper stores the sperniatozoa and associated seminal secretions. The spermatophore neck, at its opening, contains the adhesive material emanating from the sperniatophore itself. In most of the calanoids, the adhesive material helps in cementing the spermatophore to the genital segment of the female during mating (Hopkins, 1978). A major deviation from the typical calanoidean spermatophore structure is found in two families, viz. Centropagidae and Pontellidae. In the species belonging to these families, (a)

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a special coupling device is found to replace the “adhesive body” in the neck region to effect attachment to the female body. The coupling device consists of a complex group of chitin-like plates, collectively known as coupling apparatus (after “Koppler”, Heberer, 1932). In Centropages typicus, the coupling device is composed of a pale brown posterior coupler which holds the posterior arm of the spermatophore stalk in a right lateral groove and the hyaline, lozenge-shaped. anterior coupler which holds the anterior arm of the spermatophore stalk (Lee, 1972; Fig. 21). On attachment to the female, the posterior coupler is cemented to the ventral and lateral surfaces of the second urosome segment. The anterior hyaline coupler brings the tip of the spermatophore to bear on the attachment point at the tip of the right lateral protrusion of the second urosome segment, which is otherwise called “sub-genital segment” or “ancillary genital segment”. Lee (1972) has described the coupling devices of four other species of Centropages with minor variations. A further interesting variation is found in Centropages furcatus, where the stalk of the spermatophore is short, bell-shaped and continuous with the ventral plate of the coupler. The coupler contains a small sac, called a spermathecal sac, which plays an interesting part in the transfer of spermatozoa to the female (Fig. 22). A granular material is secreted from the neck of the spermatophore which distends the spermathecal sac and forms a plug in the genital opening of the female. The spermatozoa1 material is ejaculated into a thin-walled tube which coils randomly through the granular material terminating at the plug from where the spermatozoa are eventually absorbed into the female (Figs 22 and 23). More interestingly, this species does not have a distinct chitinous spermatheca, as found in Calanus species, and hence, the coupler sac acts as a receptacle for spermatozoa pending their use in egg fertilization. Lee’s description does not, however, explain how the sperm pass into the female opening in other forms, where the tip of the neck is placed in the second urosomal segment, away from the genital opening. (b) Origin of sperrnatophore In a detailed ultrastructural study, Hopkins (1978) traced the origin of the precursor substances in the male reproductive tract and the final assembly of spermatophore proper in the spermatophore sac. The highly glandular proximal part of the vas deferens produces two secretion products, namely the membrane-bound alpha granules and a filamentous matrix material in which the sperm released from the testis and the alpha granules lie. The alpha granules are produced in the vicinity of the well-developed Golgi apparatus and an elaborate system of granular endoplasmic reticulum of the columnar cells lining the proximal vas

176

T. SUBKAMONIAM

deferens. As the materials pass down the posterior region of the vas deferens, the cells lining this lumen secrete the materials responsible for the formation of the wall of the presumptive spermatophore. Closely associated with this wall material are the beta granules, also secreted by the posterior vas deferens. The final position of the materials found in the presumptive spermatophore is evident in this part of the vas deferens. The lumen contains the central core of the alpha granules set in a dense matrix around which is arranged a single layer of sparsely distributed spermatozoa. Outside the zone of the spermatozoa, the beta granules are found closely applied to the inner surface of the presumptive spermatophore wall. The secretions and deposition of the wall material continue in the seminal vesicle, which follows the posterior vas deferens. Furthermore, various components of the spermatophore assume an organized radial symmetry taking up their final position in the formed spermatophore. Such a cylinder of secretions, within the seminal vesicle, is cut off into spermatophores in the short constricted region between the seminal vesicle and dilated spermatophore sac, which bears the completed spermatophore until release. The epithelial cells lining this region produce globular and granular materials which are added on to the spermatophore wall as a new layer on the outside of the spermatophore flask. In the neck region, this secretion becomes moderately thicker. These secretions also function as lubricants, aiding ejaculation of the spermatophore. Yet another secretion consisting of birefringent globules fills the cavity of the spermatophore sac and forms special concentrations on the neck of the spermatophore, as the “adhesive body’?. Blades and Youngbluth (1981) studied the origin of the coupling devices of Lahidocera aestzva, a calanoid belonging to the family Pontillidae. The male reproductive tract is similar to the other calanoids (e.g. Euchaeta, Hopkins, 1978) but for the presence of a well-defined “former”. “Former’‘ is a term coined by Heberer (1932) to represent the

FIG.20. Simple spermatophore of Euchuefa norvegica. AD: attachment disc; SN: spermatophore neck; SPP: spermatophore proper. (Redrawn from a scanning electron micrograph of Blades-Eckelbarger, 1991.) FIG. 21. Spermatophore and two-part coupling device of Centropages typicus (dorsal view). AHC: Anterior hyaline coupler: AP: attachment point; PC: posterior coupler; SPP: spermatophore. (Redrawn from Lee. 1972.) FIGS.22 and 23. Diagrammatic representation of the ejaculation of the spermatophore contents into the spermathecal sac of Crnrropages furcutus. GS: genital segment; PL: plug; SP: spermatozoa; SPS: Spermathecal sac. (Redrawn from Lee, 1972.)

SPERMATOPHORES IN MARINE CRUSTACEANS

20

21

22

177

178

T. SUBKAMONIAM

anterior part of the spermatophoric sac which is morphologically different from the posterior sac proper. The inner wall of the “former” is composed of columnar epithelial cells that release a variety of secretions (up to eight types) into a large, central U-shaped lumen. The infoldings found in the epithelial cells are reminiscent of the typhlosole described for anomurans (Subramoniam, 1984) and macrurans (Berry and Heydorn, 1970; Radha and Subramoniam, 1985). The anterior coupling plate is produced within the first part of the “former” whereas its posterior region produces the posterior coupling plate. (c) Chemical composition of the spermatophore The copepod spermatophores are highly organized, with a well-formed spermatophore layer and a variety of secretory substances that enable spermatophore attachment and sperm expulsion after mating. Raymont et al. (1974) investigated the ultrastructure and histochemical properties of the spermatophoric components in Culanus finmarchicus. The outer covering of the spermatophore consists of about seven concentric layers of amorphous material of medium electron density. Histochemically these layers react positively to tests for proteins, carbohydrates and lipids. The inner core materials consist of (i) large, moderately opaque, rounded masses and (ii) irregularly shaped, smaller, uniformly electron-dense substances. The peripheral ground substance in which the sperm are embedded is generally lighter but both types of inclusions typical of the central core are present. In another calanoid, Euchaeta norvegicu, the wall of the completed spermatophore is about 8-10 p m thick and consists of the inner fibrillar material with an outer coating of electron-dense granules (Hopkins, 1978). At the neck region, the outer coating material is thicker than the ampullar region. The consistency of the spermatophore becomes firmer after its release outside. In the spermatophoric layers of the harpacticoid copepod Tisbe holothuriae, Pochon-Masson and Gharagozlou-van-Ginneken (1977) found a chitin-protein lamellar pattern, as in arthropod cuticle. This cuticular envelope encloses not only the ampullar region but also the thin duct (= neck region of other copepod spermatophores). The ductal region with its chitinous wall continues with a spherule. The spermatophore spherule has an epicuticular material which helps in its adhesion to the segmentary folds of the female genital opening. It may be pointed out in this context that in calanoid copepods, only a sticky adhesive body has been found on the spermatophore neck enabling spermatophore attachment. The neck leads into a tube, through which the sperm passes into the vulva. A unique feature of the spermatophore content of certain calanoid

SPERMATOPHORES IN M A R I N E CRUSTACEANS

179

copepods (Corycacidae; Candacia armata) is the presence of dimorphic spermatozoa (Heberer, 1932). They include the Q-spermatozoa (quell = swell) which provide, by swelling, the propulsive force inside the spermatophore to expel the B (Befruchtungs = fertilization) spermatozoa. With the admission of water, during spermatophore transfer, the Q-sperm swell up and develop a strong pressure inside, causing expulsion of the functional B-sperm. In the absence of Q-sperm, the propulsive force needed to expel the peripherally placed spermatozoa is provided by centrally placed vesicular foam bodies as well as the alpha granules found in E. norvegica (Hopkins, 1978). By virtue of their swelling action on contact with water, these core substances effect the expulsion of the spermatozoa to the exterior. In C. finmarchicus, Raymont et al. (1974) also found such core substances. In the harpacticoid copepod, Diarthrodes cystoecus, Fahrenbach (1962) named the inner vesicular bodies (= core substance) responsible for the ejection of spermatophoric contents Q-bodies by analogy to Heberer’s Q-spermatozoa. In another electron microscopic study, Gharagozlou-van-Ginneken and PochonMasson (1979) described several secretory granules that constitute the “core” of the spermatophore in the harpacticoid, T. holothuriae. Of these, granule A, which is of mucopolysaccharide nature, corresponds to the Q-bodies, inasmuch as they have the capacity to increase in volume by water absorption for the expulsion of the gametes from the spermatophore ampulla. In this species, other secretion granules (B, C, D and D’), mainly proteinaceous and polysaccharidic in nature, may contribute to the attachment of the spermatophore to the female, by forming the spherule lodged in vulva1 folds. (d) Changes in the sperm morphology during their passage through male reproductive duct In general, only mature spermatozoa are discharged from the testis into the vas deferens. However, in a calanoid, Labidocera aestiva, Blades and Youngbluth (1981) have observed a notable change in the morphology of the spermatozoa from spherical to a fusiform shape or somewhat spindle shape inside the seminal vesicle. An extreme case of spermatogenesis being completed within the vas deferens is described in a notodelphid Parasitic copepod Pachypygus gibber (Hipean-Jacquotte and Coste. 1989). During their differentiation inside the vas deferens, the spermatids progressively wrap themselves around a secretion named light vesicle “A”. Further morphological changes in the sperm cells when inside the seminal vesicle include the formation of large vacuoles, making the spermatozoa lighter than the non-modified sperm. These modified Spermatozoa are different from the Q-spermatozoa described by Heberer.

180

T. SUBRAMONIAM

While the calanoid sperm is ovoid, in the harpacticoid copepods, it is fusiform and highly elongated. Interestingly, the transformation of the spheroid spermatid into fusiform spermatozoa occurs in the spermatophoric sac (Pochon-Masson and Gharagozlou-van-Ginneken, 1977). The anterior pointed end contains the acrosome, which is covered by a cell coat, the material for which probably originates from the epithelial secretions. While the anterior end is pointed, the posterior end is very flat. (e) Spermatophore transfer and storage of sperm in the female In copepods, the mechanism of spermatophore transfer and sperm release has received greater attention than any other crustacean group. The flask-like spermatophore is attached to the female genital opening by the neck region. In the calanoid, Acartia tonsa, scanning electron microscope (SEM) studies reveal the correct placement of the spermatophore on to the posterior portion of the female genital opening, with the attachment being effected by an adhesive substance (Hammer, 1978). Attachment is restricted to the spoon-shaped proximal end of the neck, while the cylindrical portion of the neck and remaining distal portion of the spermatophore hang free. The placement of the spermatophore outside the genital field may not permit fertilization of the female. Although, a single, correctly placed spermatophore in the female opening is adequate to fertilize a female, and often to store spermatozoa for subsequent ovulations, multiple placements of spermatophores have been described in various copepods. Hopkins and Machin (1977) have studied in detail the pattern of spermatophore distribution and placement in calanoid species Euchaeta norvegica. Most individual females carry a single, normally positioned spermatophore in the genital opening present in the first urosomal segment. In their analysis of 10,500 females, spermatophores were not found attached to any stage other than the adult female or any region of the female body except the genital segment. Peaks of spermatophore attachment (hence fertilization) occur during February and March, when the number of males in the population also peaked. A strong positive correlation also exists between the mean number of spermatophores per female and proportion of adult males. The number of spermatophores attached on the genital field (vulva) constitutes as much as 70.1%; here the short neck after attachment is found to have gained a long tube through which sperm and other secretions are transmitted. The extended neck is formed by the extrusion of some of the non-spermatozoa1 contents of the spermatophore flask. Since direct placement occurs on the genital cavity, this extension tube merely disappears within the female cavity, but if the attachment is made

SPEKMATOPlfORES IN MARINE CRUSTACEANS

1x1

away from the genital field, the end of the extension tube fows on to the surrounding cuticle to form a circular mass, termed the attachment disc. The majority of the imperfectly positioned spermatophores have no connection with the seminal receptacle and hence are non-viable. A small number, however, have a small “fertilization tube” traversing the cuticle between the attachment disc and seminal receptacle, and are thus considered fully viable. In a parasitic harpacticoid copepod, Lepeophtheirus pectoralis, the placement of spermatophore seems to be more haphazard, but a tube “grows” out of each spermatophore towards the openings of the receptaculum seminis, thus increasing the range of successful sites for spermatophore placement (Anstensrud, 1990). Multiple placement of spermatophores in E. norvegica, as in other copepod forms reported (Hill and Coker, 1930, for Cyclops americanus; Jacobs, 1961, for Pseudodiaptomus coronatus) may be explained by the copulatory competition between several males and an unfertilized female. This especially occurs at high adult male : unmated female ratios, so enhancing the survival of the population. Fleminger (1967) from his studies o n Labidocera jollue, postulated that sexual swarming coupled with a high male:female sex ratio could be the basis for the large number of misplaced spermatophores. Despite multiple placement of spermatophores in copepods, the mating is not indiscriminate and the spermatophore first placed is always found in the female genital pore. In Acartia tonsa, Hammer (1978) reported as many as eight spermatophores precisely placed one on top of the other towards the posterior portion of the genital opening. All this would emphasize the fact that, in spite of the promiscuity of the male copepods, a well-evolved mating system in copepods has developed, probably involving a sex pheromone (Blades-Eckelbarger, 1991). During mating, the adult male must position himself in a specific manner relative to the orientation of the female in order to place a spermatophore correctly to her genital orifice. In E. norvegicu, the placement of the first spermatophore on a female is precise and efficient (Hopkins, 1977). The attachment of additional spermatophores is not functionally important, as the first correctly placed spermatophore is sufficient to fertilize the female. Although many reports have appeared on spermatophore placement in several species, most observations have been made from preserved plankton collections and not from direct observation of mating. Misplacement of spermatophores outside the genital field has been reported for other species also. In Labidocera, Fleminger (1967) grouped spermatophores in two positions: type I position showed the neck of the spermatophore in close proximity to the genital pore and in type I1 the

182

T. SUBKAMONIAM

spermatophore is attached to the left-hand corner of the fifth thoracic segment. Type I1 is considered to be non-functional, due to a lack of connection to the genital pore. In general, type I1 only occurred if a spermatophore was already placed in the type I position. The studies on several species of centropagid calanoid copepods by Lee (1972) has, however, indicated that this is not a general rule. The role of appendages in spermatophore transfer has been suggested by many workers (Blades-Eckelbarger, 1991). However, many conclusions were mainly from t h e morphological features of the appendages and not from direct observation during mating. Establishment of a causal relationship between appendages and spermatophore transfer requires experimental evidence by extirpation of such putative appendages before making direct observations on mating sequences. Similarly, a pheromonal involvement in mating is also speculative. Direct observation of mating in planktonic copepods may necessarily have certain limitations, in that the duration of mating may itself be brief. The presence of special coupling devices in some calanoid families, such as the Centropagidae and Pontellidae, provides an additional mechanism for the exact location of the spermatophore. In these species, copulation refers to the precise act of transfer of the spermatophore and its coupling device from the male and its attachment to the female. The morphology of the coupling plates is unique in each species and corresponds to the external morphology of the conspecific female's urosome and genital region. This is referred to as a "key and lock'' relationship (Fleminger, 1967; Lee, 1972). The female genital opening into which the spermatophore contents are emptied lies on the sternum of the first urosomal segment, called the genital segment. A genital flap, also referred to variously as the operculum. genital plate or genital valve, lies within the genital opening (Hammer, 1978). The genital pore or the oviductal opening lies towards the posterior region of the genital opening. Invariably, the spermatozoa released from the attached spermatophores is taken into the oviduct for internal fertilization. As described in the calanoid species, the oviduct has specialized spermatheca for sperm storage till ovulation occurs (BladesEckelbarger, 1991). In all the free-living male copepods, only one spermatophore is produced at a time. However, in the ascidicolous parasitic cyclopoid copepod Pachypygiis gibber, two spermatophores are extruded through two ventral gonopores, each represented by a slit under the sixth pereiopod of the genital segment (Hipean-Jacquotte and Coste, 1989). By means of antennular hooking and clasping by the fourth pereiopod, the male applies its genital segment strongly to the funnel-shaped female vulva. The pressure thus exerted on the genital segment appears to cause

SPEKMATOPHORES IN MARINE CRUSTACEANS

183

spermatophore extrusion. The spermatophore has the copepodan morphology. The distal end of the neck, after spermatophore extrusion, is fixed to the unpaired copulatory pore situated at the base of the vulva. The spermatozoa as released from the spermatophore penetrates into the two lateral seminal receptacles via a short unpaired duct which originates from the gonopore. From the seminal receptacles, the sperm are passed on to the central antrum for storage until fertilization. Interestingly, the sperm morphology changes significantly inside the antrum with the loss of cell coat, suggesting a process akin to capacitation. Pochon-Masson and Gharagozlou-van-Ginneken (1978) also described a similar phenomenon in Tishe holothuriae.

C. Euphausiids The euphausiids, another group of spermatophore-producing marine Crustacea, resemble mysids and copepods with regard to spermatophore morphology and transport mechanism. As in copepods, the spermatophore consists of a flask-shaped ampoule and a long stalk. The first pair of pleopods, modified into the petasma, is used in some way for positioning the spermatophoral opening at the distal end of the neck, in or over the spermatheca or thelycum (Raab, 1915; Bargmann, 1937; Zimmer, 1913). Alternatively, Brinton (1975) suggested that the hooks and other processes on the petasma are used for preparing or stimulating the female for implantation of the spermatophore. However, in a later analysis of fixed plankton samples of euphausiid species, Brinton (1978) observed the frequent attachment of the spermatophores on the male pleopods of Thysanopoda nqualis. The use of pleopods in the placement of spermatophores on the thelycum has also been indicated in many euphausiid species (Mauchline and Fischer, 1969; Casanova, 1974). The number of spermatophores attached to the thelycum may also vary in different species. In Thysanopoda tricuspidata, only one spermatophore is carried in the thelycum, whereas in Thysanopoda orientalis and Siylocheirori longicorne, two spermatophores are normally seen (Sebastian, 1966). In the majority of the euphausiids, the spermatophores are cemented very firmly to the thelycum by a chitin-like secretion originating from the lateral pockets of the vasa deferentia (Bargmann, 1937). The spermatophore usually remains attached to the thelycum even after discharge of the sperm into the female gonopore and is discarded only during the next moulting. However, in four species of Euphausia, the impregnated females retain the sperm mass, but not complete spermatophores in the

184

T. SUBKAMONIAM

spermatheca (James, 1977). In a recent study on the oceanic species of the genus Stylocheiron, Costanzo and Guglielmo (1980) emphasized the diagnostic value of the thelycum. Surprisingly, nothing is known about the origin and chemical composition of spermatophores in euphausiids. The spermatophore transfer takes place long before the onset of ovarian maturity in the oceanic pelagic forms (Wiborg, 1971; Hollingshead and Corey, 1974). A mating stimulus for the ovarian maturity has also been suggested by Mauchline (1972). D.

Stornatopoda

The Stomatopoda, the only living order of the subclass Hoplocarida, are a small group of carnivorous marine crustaceans with an active predatory life-style. They are highly aggressive, territorial species exhibiting elaborate courtship and maternal behaviour. The stomatopods, unlike several malacostracans, do not package their spermatozoa into spermatophores, but concentrate them into a sperm cord to be transferred into the female during copulation (Komai, 1920). In Squilla holoschista, the paired vasa deferentia carry the sperm forward and embed them in a mucopolysaccharide cement. This forms a sperm cord that is ejaculated into the female during an elaborate mating process (Deecaraman and Subramoniam, 1980). The stomatopod possesses paired intromittent organs which help in the transference of the sperm cord (Fig. 24). In S. holoschista, paired male accessory glands lie in the thorax and open through the gonopods, independently of the vas deferens. The highly proteinaceous secretion of the accessory sex gland appears to have a post-copulatory role in digesting the sperm cord inside the female vaginal pouch 9 liberate active spermatozoa. Deecaraman and Subramoniam (1983) also observed multiple insemination in this stomatopod. Their electrophoretic studies provided evidence that protein from the male accessory glands is taken up by the ovary of the female. Influence of mating on vitellogenesis, by way of contribution from the male accessory gland secretions, has also been reported in an orthopteran insect Melanoplus sanguinipes (Friedel and Gillot, 1976). Clearly, more investigations are needed to provide direct evidence for the influence of the male accessory gland secretions on yolk synthesis in stomatopods.

E. Mysiducea and Other Sperrnatophore-producing Marine Crustaceans Mysids are shrimp-like eumalacostracan Crustacea, having wide distribution in different marine habitats (Mauchline, 1980). Like other peracarid

SPERMATOPHORES I N MARINE CRUSTACEANS

185

AG

ss A0 GO FIG. 24. Intromittent organ of Squillu holoschisru. AGD: accessory gland duct; AO: accessory gland opening; DVD: distal vas deferens; GO: gonopore; SC: sperm cord; SS: spei-m sac. (Redrawn from Deecaraman and Subramoniam, 1080.)

crustaceans, mysids produce filiform spermatozoa which are bundled together by extracellular tubules, secreted by the cells surrounding the developing spermatids (Reger et al., 1970; Reger and Fain-Maurel, 1973). These extracellular tubules may function by cementing the non-motile spermatozoa together into a “spermatophore” for easy sperm transmission. In Praunus flexuosus, Nouvel (1937) observed the implantation of the sperm mass within the female marsupium, using the well-developed penes. Kinne (1955) also reported the possible use of the male pleopod in the transfer of the sperm mass to the marsupium of Neomysis integer. Possible sex pheromone involvement in the attraction of the males to deposit the tube-like spermatophores has also been suggested in the mysidacean, Leptomysis lingvura (Wittmann, 1982). Comparative ultrastructural studies on the spermatophores of other peracarids such as isopods, amphipods and cumaceans reveal striking resemblance with each other in the possession of extracellular tubules and matrix materials which align the long non-motile spermatozoa along their long axis into a sperm bundle (Reger and Fain-Maurel, 1973). In the terrestrial isopod, Armadillidium vulgare, the extracellular tubules are assembled into a cone-shaped structure. Interestingly, the morphology of the matrix material changes gradually before being released for transfer to the female. Reger (1970) reported disaggregation of extracellular tubules at the periphery of the spermatophore at the lower level of vas

1ti6

T. S U B K A M O N I A M

deferens in the mysid, Mysis relicta. In the isopod, Porcellio laevis, Cotelli et al. (1976) could not find the tubules in the female genital tract. Clearly, more studies of the peracarids are needed to determine the fate of extracellular tubules of the spermatophore after transfer to the female genital tract. Brown and Metz (1967), while describing the ultrastructural features of the sperm in a primitive crustacean, Derocheilocaris typicus (Mystacocarida), found bundles of tube-shaped spermatophores within the vas deferens. Each spermatophore contains two spermatozoa which are oriented parallel to each other but face in opposite directions with the anterior (acrosomal) regions at the end of the spermatophore. The thick-walled spermatophoric cylinder has a complex plug at each end. This plug consists of an electron-dense cup with its concave side facing the sperm acrosome. The outer wall of the plug region has a sleeve-like fitting over the wall of the main cylinder of the spermatophore. Interestingly, the mystacocarid spermatozoa are flagellate and motile, but become immobilized within the spermatophore. It will be interesting to know how this structural organization of the spermatophore envelope would facilitate sperm release during fertilization. Remipedia, a new class of Crustacea from a marine cave in the Bahamas (Yager, 1981), produce distinct spermatophores which are surrounded by a moderately electrondense flocculent material. But there is no evidence of an outer spermatophoric wall (Yager, 1989). The two species, namely Speleonectes bevyamini and Speleonectes lucayesis, enclose two to six flagellated sperm within a single spermatophore which is transferred to the female gonopore directly without the help of any copulatory process (Yager, 1991).

111. Spermatophore Hardening In many crustacean species where the spermatophores are externally deposited and carried by the females until fertilization. the spermatophores have been reported to undergo hardening. In the spiny lobster, Panulirus homarus, calcium deposits found in the protective matrix have been suggested to bring about hardening of spermatophore (Berry, 1970). Similarly, in the anomuran crab, Alhunea symnista, the highly acidic groups of the gelatinous matrix mucopolysaccharides might bind calcium ions from the sea water to produce “hardening” of the deposited spermatophore into a putty-like mass (Subramoniam. 1984). Phenolic tanning, as another mode of hardening, has been proposed by Malek and Bawab (1971) from their histochemical studies on the spermatophores of

SPERMATOPIlOKES IN MARINE CRUSTACEANS

187

the penaeid shrimp, Penaeus keruthurus. The spermatophoric layers are lipoproteinaceous in nature, the protein being rich in phenolic groups, owing to the presence of tyrosine. Malek and Bawab (1971) suggested that the aminophenols derived from tyrosyl residues of the protein could form the cross-linking quinones due to oxidation by the enzyme phenolase. Although the tanned layers fail to darken, the layers showed pronounced changes in affinity for stains and in iso-electric point, and they developed resistance to acid treatment. This condition may be comparable to the B-sclerotization involving side-chain linking of the quinones with the tyrosyl residues of the tanning protein, thus conferring softness and resilience to certain insect cuticles (Anderson, 1971). The hardness of the P. kerathurus spermatophore is thus due to enzymatically catalysed chemical transformation, rather than to a mere exposure to sea water (Heldt, 1938). Further evidence for the occurrence of phenolic tanning in spermatophores has been indicated in the sand crab, A . symnista (Subramoniam, 1984) and the lobster Panulirus interruptus (Martin et al., 1987). The latter authors reported bacterial colonization on the hardened spermatophore surface, but bacteria were not found to penetrate into deeper layers of spermatophores, probably because of the antibacterial activity of the phenolic substances present in it. More evidence is necessary to discern the biological significance of phenolic substances in the spermatophores of decapods. Other putative modes of stabilization of crustacean spermatophores include chitinization (King, 1948; Gharagozlou-van-Ginneken and Pochon-Masson, 1979; Uma and Subramoniam, 1979) as well as structural transformation of spermatophore layers (Dudenhausen and Talbot, 1983; Martin et al., 1987). A new type of spermatophore wall stabilization has been reported to involve keratinization in the portunids Portunus sanquinolenies and P. pelagicus (Babu et a l . , 1988; Rao et al., 1989). Recently, Subramoniam (1991) has reviewed the literature on chemical composition of crustacean spermatophores.

IV. Cryopreservation of Spermatophores Chow (1982) first reported the successful preservation of spermatophores of the freshwater shrimp Macrobrachiurn roserzbergii in Ringer solution for a period of 4 days at 2°C. These spermatophores, after their attachment to the female sternum, released viable sperm at the time of ovulation. However, prolonged storage resulted in the degeneration of the protective and adhesive matrices of the spermatophores. Subsequently, Chow e f al. (1985) improved this method by using 10% glycerol as the

P

OF CRYOPRESERVATION OF MALEGAMETES OF AKTHROPODA TABLE9. CONDIT~ONS

SI. Species no.

Cryoprotectant

Temperature Preservation period

Decapod Crustacea Sicyonia DMSO + - 196°C ingentis tre halose 2 S. ingentis Trehalose, - 196°C sucrose proline, glycerol, DMSO 3 Mucrobrachium Glycerol - 196°C rosenbergii 4 Scylla Glycerol, DMSO, -196°C serrata trehalose, - 79°C -4°C DMSO + tre halose 1

Percentage of survival

Method of testing viability

Reference

2 months

6&70%

Acrosome reaction

1 month

56%

Acrosome reaction

Anchordoguy et al., 1987 Anchordoguy et al., 1988

Apis

mellifera

6

Arachnida Limulus polyphemiis

53%

30 days

95% (glycerol, Eosin dye exclusion Jeyalectumie and DMSO + Subramoniam, trehalose) 1989 89% (DMSO, tre halose)

Glycerol, seminal -79°C vesicle fluid, spermathecal fluid

16 days

50%

Motility

Glycerol

50 days

64 96

Eosin dye exclusion Behlmer and Brown, 1984

-74°C

From Jeyalectumie and Subrarnoniam, 19x9.

-i (I:

Fertility

Chow et al., 1985

31 days

Insecta

5

cc, cc,

Sawada and Chang, 1964

C

m

F 3

i

P

SPERMATOPHOKES IN MARINE CRUSTACEANS

189

cryoprotectant and storing the spermatophores in liquid nitrogen at -196°C for a period up to 30 days. These spermatophores were then thawed and fastened to the female sternum using a-cyanoacrylate as the adhesive. A technique for long-term storage of lobster spermatophores has been developed by Ishida et al. (1986) for artificial insemination. This method involves the transference of electrically extruded Homarus spermatophores with a bamboo stick to a plastic test tube containing paraffin oil and storing at 47°C. Sperm stored up to 289 days were morphologically normal and underwent acrosome reaction with calcium ionophore A23187. Although this storage method is simple to execute and inexpensive, degeneration of the sperm occurs when stored longer than 358 days. In addition, this low-temperature storage could not prevent bacterial growth in the sperm mass. Harper and Talbot (1984) suggested that the bacterial epibionts on the lobster body surface could be picked up by the spermatophores at the time of extrusion. Anchordoguy et al. (1988) have successfully frozen the spermatozoa of the penaeoid prawn, Sicyonia ingentis, in liquid nitrogen. In the cryopreservation of sperm of arthropods, several cryoprotectants have been used to reduce the cryoinjuries to the membranes (Table 9). Recently, Jeyalectumie and Subramoniam (1989) have developed methods to preserve the spermatophores together with seminal plasma of the edible crab, Scyfla serruta, at three temperatures (-4"C, -79°C and -196°C). Biochemical alterations of the major substrates such as protein, carbohydrate and lipid, as well as the enzyme lactate dehydrogenase, occurred only at -4"C, reflecting their use in the metabolic activities of the spermatozoa contained in the spermatophores. At -79°C and -196"C, the frozen spermatozoa retain viability but do not exhibit metabolic activity. However, the fertilizing capacity of the spermatozoa could not be tested, as freshly ovulated female crabs (S. serrata) are difficult to obtain. That the sperm frozen in the liquid nitrogen underwent acrosome reaction in the shrimp Sicyonia ingentis is indicative of the fertilizability of the spermatozoa (Anchordoguy et al., 1988). Cryopreservation of crustacean spermlspermatophores certainly needs improvement. However, the available data (Table 9) on a few species of decapod crustaceans are indicative of the possibility that crustacean sperm/ spermatophore banking could be available for artificial insemination as well as for easy transportation.

V. Spermatophores and Artificial Insemination In recent years, with the advent of aquaculture of shrimp and other economically important crustacean species, interest in the methods of

190

T. SUBKAMONIAM

controlling reproduction in them has gained great impetus. Attention has been focused especially on research relating to reproductive engineering to augment seed production under controlled conditions (Subramoniam, 1988). In an attempt to produce culture-suited strains through hybridization in freshwater prawns, Sandifer and Smith (1979) and Sandifer and Lynn (1980) conducted experiments pertaining to artificial insemination, using spermatophores of Macrobrachium and Palaemonetes sp., ejaculated by electrical induction. Chow (1982) used a-cyanoacrylate adhesives to attach these spermatophores to the female sternum. The possibility of extending this technique to marine decapod crustaceans was tried first in the American lobster, Homurus americanus, by Kooda-Cisco and Talbot (1983) and Aiken et al. (1984). Current from a 12 millivolt variable transformer was applied gently near the gonopore at the base of the fifth walking leg to make it extrude a full complement of spermatophore which usually measures 1-2 cm in length. This spermatophore contained normal spermatozoa which underwent acrosome reaction with calcium ionophore A 23187 (Talbot, 1984). Aiken et al. (1984) also achieved artificial insemination in the intermoult H . americanus in a similar way. However, these inseminates yielded only poor results in terms of viable larvae hatched from the eggs. Talbot et al. (1983) improved the method of artificial insemination in the same lobster species. However, they were able to introduce the electro-ejaculated spermatophore into the thelycum only in the post-moult condition, when the thelycal plates were soft and flexible. These authors successfully inseminated lobsters of wild-born H . americanus as well as H . americanus females crossed with H . gammarus or the reciprocal. They also obtained higher survival of the embryos resulting from the hybridization experiments. The hybrid male lobster, however, did not produce sperm. In the laboratory-held lobsters, spermhpermatophore deterioration has been reported to occur with the increase in holding time, together with a decline in male potency (Aiken and Waddy, 1980). Artificial insemination techniques are thus useful in tiding over the difficulty of mating in laboratory conditions as well as selecting desired parentage for the hatchery production. Artificial insemination probably has more relevance to prawn aquaculture than any other crustacean species described. Decline in the reproductive potential of males when kept under captivity has been reported by many workers. Leung-Trujillo and Lawrence (1987) reported significant decline in the spermatophore weight, sperm count and percentage sperm survival, together with sperm abnormalities, when Penaeus setiferus males were kept under laboratory conditions up to a period of 7 weeks. Such a decline in male fertility is also associated with deterioration

SPERMATOPHORES IN MARINE CRUSTACEANS

191

of the spermatophores. Clearly, quality maintenance of spermatophore components is an important prerequisite for effective impregnation of the females. In an attempt to overcome such difficulties, artificial insemination has also been tried in several freshwater and marine prawn species for hybrid production (Table 10). In penaeoid prawns, artificial insemination has been accomplished in both open and closed thelycal species for intra- as well as inter-specific crosses. Bray et al. (1982) removed the sperm mass from the manually extruded spermatophores of males and placed it just anterior to the base of the third pair of periopods near the female gonopores of the open thelycal species, P. setiferus. As the attached spermatophore could easily be dropped by the females, placement of the spermatophore was done just prior to spawning. In the closed thelycum species, spermatophores can be implanted into the thelycum while the female is soft or in the intermoult period (Laubier-Bonichon and Ponticelli, 1981; Lumare, 1981: Lin and Ting, 1986). In the tiger prawn, Penaeus monodon, Muthu and Laxminarayanan (1984) implanted electro-ejaculated spermatophores from the males into the thelycum of newly moulted, eyestalk-ablated females. The implanted spermatophores were retained by the female till the next moult. These authors recommended t h e use of freshly moulted females, in order to rule out the occurrence of any residual sperm remaining inside the seminal receptacle of the thelycum of the intermoult female. Recently, Lin and Hanyu (1990) achieved artificial insemination using fragments of vas deferens removed from the male shrimp and implanting them into the thelycum of pond-cultured P. penicillatus. These authors increased the fertilizability of the females by digesting the spermatophores into a spermatic fluid using trypsin and then depositing the condensed spermatic mass into the thelycum, prior to spawning. In general, the percentage of fertilized eggs produced is much higher in the naturally impregnated females than in the artificially impregnated ones. In the artificially impregnated Penaeus japonicus only 7.5% of fertilized eggs. as against 57.7% to 67.7% from naturally impregnated females, was obtained by Lumare (1981). Furthermore, the hatching rate was 30.9% to 40.1% from natural insemination whereas only 3.3% could be achieved from artificial insemination. It is perhaps important to take into consideration the quality of spermatophores as well as their correct placement in the thelycum, together with the stage of moulting in the recipient female, in order to increase the productivity of seed through artificial insemination. Natural mating between different penaeid species has rarely been reported in the literature. Artificial insemination by way of spermatophore transfer has been reported to be a possible means of interspecific

192

T. SUBKAMONIAM

TABLE10. SPL-CIES IN

No. Species

WHICH

A K IIEICIAL INSFMINATION HAS

BEEN

ACHIEVPD

Thelycum

Insemination

Reference

Open

In terspecific

Bray et al., 1990

-

Open

Interspecific

Lawrence et al., 1984

3 4 5

Penaeoid species P. setiferus x P. schrnitti P. setiferus x P. stylirostris Penaeus sp. Penaeus sp. P. setiferus

Open Open Open

Intraspecific Intraspecific Intraspecific

6

P. japonicus

Closed

Intraspecific

7

P.rnonodon

Closed

Intraspecific

8 9

P. vannarnei P. pencillatus

Closed Closed

Intraspecific Intraspecific

Persyn, 1977 Aquacop, 1983 Bray et al., 1982 Bray and Lawrence, 1984 Laubier-Bonichon and Ponticelli, 1981 Lumare, 1981 Ponticelli, 1981 Muthu and Laxminarayanan, 1984 Goguenheim et al., 1987 Lin and Hanyu, 1991

1

2

1

2

3 4 5

6 7

Macrobrachiurn species Macrobrachiurn Open usperulurn x M. shokitai Palaernonetes Open pugid x P. vulgaris M . nipponense x Open M . forrnosense M . rosenbergii x Open M . acanthurus M . acanthurus x Open M . carcinus M . rosenbergii Open M . acanthus

Open

Lobsters 1 Hornarus aniericanus Closed 2

H . arnericanus x H . gamrnarus

Closed

In terspecific (sterile)

Shokita, 1978

Interspecific

Sandifer and Lynn, 1980; Berg et al., 1986

Interspecific

Uno and Fugita, 1972

Interspecific

Sandifer et al., 1977

Interspecific

Dobkin et al.. 1974

Intraspecific

Sandifer and Smith, 1979; Sandifer and Lynn, 1980 Sandifer et al., 1977

Intraspecific Intraspecific Interspecific

Talbot et al., 1986 Aiken and Waddy. 1980 Hedgecock et al., 1977 Talbot et al.. 1983

SPERMATOPHORES IN MARINE CRUSTACEANS

193

hybridization in several prawn species (Table 10). The first successful hybrid cross of two open thelycal marine prawn species was reported by Lawrence et al. (1984) in reciprocal crosses of P. setiferus and P. stylirostris, through artificial insemination employing manually dissected spermatophores. A minimal hatching rate of 1% was, however, obtained from the eggs of these crosses. More recently, Bray et al. (1990) successfully crossed two species ( P . setiferus and P. schmitti) having close morphological characteristics and geographical range, The egg fertilization rates achieved in these interspecific artificial inseminations were found to be low in comparison with those achieved in intraspecific artificial inseminations, as reported by Persyn (1977), Aquacop (1983) and Bray and Lawrence (1984). Nauplii resulting from 7 of 19 hybrid crosses between P. setiferus and P. schmitti were cultured to adult stage and the hybridization confirmed by isozyme comparisons of parents and offspring (Bray et al., 1990). The hybrid offspring also reached adulthood and produced ova in females and morphologically normal sperm in males, suggesting genomic compatibility in these species. These results also indicate that barriers to interspecific hybridization in penaeids do not appear to be insurmountable. The possibility of artificial insemination in the interspecific crosses has opened up new vistas in selective breeding and genetic manipulation of commercially important prawn species. By this method, genes of specific traits such as disease resistance could be transferred from one species to another. Gene interactions in hybrids sometimes yield heterosis for a commercial trait, in which hybrid performance exceeds that of the selected parents (Bray et al., 1990).

VI. Spermatophore Pathology Chamberlain (1988) noted gonadal swelling and degeneration causing difficulties in spermatophore ejaculation during mating in the prawn Penaeus stylirostris. Chamberlain and Gervais (1984) also reported a low incidence of successful mating in P. stylirostris, due to swollen vas deferens. In another species, Penaeus setiferus, Leung-Trujillo and Lawrence (1987) made detailed observations on the declining sperm quality affecting fertilization success in laboratory-maintained males. During the first 2 weeks, the extruded spermatophores exhibited good morphology and colour; by the third week, external deterioration of the spermatophore was noticed. The wings, outer margins of the germinate body and the flange areas started deteriorating with the wings ultimately falling apart. This was followed by discoloration of the white spermatophore to a dark tan to brownish colour with the germinate body becoming

194

T. SUBKAMONIAM

twisted and hardened. By the sixth and seventh weeks, all spermatophores in the terminal ampoule darkened and appeared as amorphous masses with no distinguishable external features whatsoever. Concurrently, there was a gradual loss of spermatophore weight along with a drastic decline in the sperm count. The sperm also showed deformities such as bent or missing spikes or malformed heads. Brown et al. (1979) observed Vibrio sp. from the terminal ampoules and enclosed spermatophores of P. setiferus, kept under captivity. In addition to the deformed sperm in the spermatophore, the whole reproductive tract and testis were melanized under extreme infection, leading to the failure of spermatophore extrusion. Chamberlain et ul. (1983) isolated melanin-producing bacteria (Pseudornonas sp.) from the infected spermatophores of P. setiferus. lshida et al. (1986) also observed bacterial infiltration into the spermatophores, when stored in paraffin oil for a prolonged period. Possibly, the bacterial epibionts on the lobster body surface (Harper and Talbot, 1984) could have been picked by the spermatophores at the time of extrusion. In the mud crab, Scylla serrata, also, bacterial populations exist in the stored seminal substances in the spermatheca, possibly due to their entry through the female gonopore (Bhavanishankar and Subramoniam, unpublished observations). Ultrastructural studies as well as histochemical observations on the melanized spermatophores of the pond-cultured shrimp Penaeus vannamei have been recently carried out by Dougherty and Dougherty (1989). In the freshwater prawn, Macrobrachium rosenbergii, the melanoid spermatophore was free from bacterial contamination, and the sperm inside the spermatophore were also not affected (Harris and Sandifer, 1986). These authors found a correlation between melanization and the repeated electrical stimulation used in spermatophore extrusion. Possibly, tissue degeneration due to electrical stimulation is a causative factor in melanization. Melanization has also been demonstrated as a woundhealing response in penaeid prawns (Fontaine, 1971; Fontaine and Lightner, 1973) and other decapods (Bazin and Demeusy, 1972; Fontaine et al., 1975). Kooda-Cisco and Talbot (1983) observed necrosis of tissue around the gonopore, during repeated electro-ejaculation in the American lobster, Hornarus americanus. Similar observations have been made in the sand lobster, Thenus orientalis (Silas, 1991). In T. orientalis repeated electrical stimulation in the gonopore region induces tissue necrosis and melanization not only of the spermatophore but also of the entire vas deferens and testis. Histochemical tests revealed the presence of phenol oxidase and phenols in the melanized reproductive tract of T. orientalis. The role of phenol oxidase in melanization and sclerotization, besides showing antibacterial activity, has been well documented in

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insects (Brunet, 1980). Melanization due to phenol oxidase in the spermatophores of lobsters has been observed by Martin et ad. (1987) and the role in spermatophore hardening, discussed by Subramoniam (1991). It thus appears that melanization and bacterial infection of decapod spermatophores are distinct events, the former induced by tissue response to wound healing and electrical stimulus, and the latter caused by bacterial invasion through gonopores from the aquatic environment.

VII. Comparison with Other Spermatophore-producing Marine Invertebrates Mann (1984) has reviewed the occurrence of spermatophores in various animal phyla. Although the reproductively primitive marine invertebrates such as echinoderms and corals practise broadcast fertilization by simply releasing their gametes into the ambient sea water, representatives of several other phyla resort to direct and indirect insemination. Spermatophores predominate in such insemination methods. Their occurrence and mode of transfer to the female seem to be greatly influenced by the life-style of the animal concerned and the environment. Spermatophoreproducing marine invertebrates range from pelagic copepods and chaetognaths to the sessile, tube-dwelling polychaetes. The size and structure of the spermatophores vary from the small, simple vesiculate spermatophores of brachyuran decapods to the large and highly complex spermatophores of cephalopods.

A.

Polychaeta

Although some polychaetes are well known for epitokous spawning and broadcast fertilization (Clark, 1961; Schroeder and Hermans, 1975, for reviews), and others transfer semen by true copulation (Pisonidue and Succocirrida, Gray, 1969; Westheide, 1984) or hypodermic impregnation (Dinophilidue. Protodrilidae and Hesionidae, Ax, 1968; Jouin, 1970; Westheide, 1984, 1988), many sessile benthic families, including burrowing and tube-dwelling forms, practise spermatophore transfer with or without sperm storage in the females (Hsieh and Simon, 1990). For example. in Polydora ligni, the spermatophores are released freely into the sea water, to be picked up by the ciliated palps of females (Rice, 1978). Other spionid polychaetes may deposit the spermatophores in the vicinity of the female's tube (Microspzo mecznikowianus. Cerruti, 1908) or attach them to ;he female (Scolelepis squurnura, Richards, 1970).

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B.

Pogonophora

Pogonophora live in chitinous tubes. In Siboglinum, the cigar-shaped spermatophores bear, at one end, a long but tightly coiled filament, which on release into the sea water uncoils and spreads out. The spermatophore may float near the sea floor until the filaments become entangled with the tentacles of the females and are drawn into their tubes (Ivanov, 1963; Southward, 1975).

C . Chaetognatha All chaetognaths are hermaphrodite, but produce spermatophores for sperm transfer. In the benthic genus Spadella, Ghirardelli (1968) reported that the spermatophore is placed on the neck of the partner and, with the dissolution of the posterior end of the spermatophore, the spermatozoa pass into the seminal receptacles. The sperm further migrate to reach the ovary to fertilize the eggs. In the pelagic Sagirta, Grassi (1883) and Van Oye (1931) described the cross-fertilization process. The spermatophore, when released from the seminal vesicle moves to the caudal fin, which forms a curved repository for it until pairing occurs. The partners are placed head to tail so that spermatophores of one come into contact with the expanded genital orifice of the other. The lateral fins of the partners help in holding the received spermatophore to facilitate sperm penetration into the gonopore. Ghirardelli (1968) described certain serratededged, cap-like structures inside the seminal vesicle. These structures are detached along with the spermatophores during mating and function as copulatory structures by fitting into the female genital orifices of the same species. Laboratory observations on the mating sequences of Sagitta hispida by Reeve and Walter (1972) reveal that the site of spermatophore attachment is always on the lateral trunk wall and the sperm released from the spermatophore begin to stream towards the gonopore on that side. These authors showed that, although this species is capable of self-fertilization, it has rarely been seen.

D. Mollusca Structurally the most complex spermatophores are produced by the cephalopod molluscs. They assume a very large size (up to 1 m in the giant octopus). The spermatophore contains a sperm rope with spermatozoa and a thin portion containing the ejaculatory apparatus. The

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spermatophoral fluid is comparable to the vertebrate semen in its biochemical properties (Mann, 1984). The spermatophoric reaction leading to sperm release is highly complex and characteristic of cephalopod molluscs. The spermatophore transfer is direct and often preceded by an elaborate courtship and mating process. An excellent review of the molluscan spermatophore is given by Mann (1984).

VIII. Conclusion In the Crustacea, the morphology and method of transfer of spermatophores are so varied that it is difficult to draw conclusions about phylogenetic relationships. Decapods, being the ubiquitous users of spermatophores in sperm transfer, exhibit group-specific morphological diversity. True crabs (Brachyura) are comparatively homogeneous, possessing simple, vesiculate spermatophores, bathed in seminal plasma. In Macrura, the tubular spermatophores are enveloped in several acellular accessory mucoid secretions which protect the enclosed sperm cells during their prolonged epizoic storage on the female body. Anomuran crabs, though characteristically possessing a pedunculate type of spermatophore, exhibit an almost species-specific morphological diversity. More interestingly, some of the sand crab species belonging to the family Hippidae, as well as Alhunea symnista, possess a spermatophoric ribbon which is more characteristic of the macruran species such as spiny lobsters. Subramoniam (1984) found evidence for the possession of peduncles in the dimorphic spermatophores of the hippid mole crab, Emerita asiatica. The intermediary conditions in the evolution of a pedunculate type of spermatophore from the tubular macruran type of spermatophore is further evidenced in the spermatophores of A . symnista, in which a tubular spermatophore shows node-like constrictions leading to internal discontinuities. Such a breaking up of a continuous spermatophoric tube by constrictions (Albunea) and distinct spermatophoric ampullae with drawn-out peduncles set on a basal filamentous pedestal (Emerita) suggest that these anomuran sand crabs may be mid-way forms in the evolution of discrete pedunculate spermatophores of the anomurans from t h e tubular spermatophores of Macrura. Based on the occurrence of spermatophores in different arthropods, no possible phylogenetic relationship between Crustacea, Antennata and Chelicerata could be established (Schaller, 1980). However, a distinction could be drawn between the utilization pattern of spermatophores in the Crustacea and other arthropods. Whereas the spermatophores of Crustacea are mainly used in marine environmental conditions, the other

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arthropod groups employ them as a means to transfer semen in terrestrial habitats. Crustacean spermatophores are always transmitted directly to the females whereas in the majority of the stalked spermatophoreproducing terrestrial arthropods, there is indirect transfer. Comparatively, the spermatophores of Crustacea are simpler than those found in Chelicerata, but the structure still varies within Crustacea according to the method of sperm transfer. When the spermatophores are transmitted to the females for storage within the internal seminal receptacles for internal fertilization (Brachyura), the spermatophores are simple and degenerate structures. Conversely, when the spermatophores are deposited outside the female body, they assume structural complexity which could be attributed to the presence of multilayered mucoidal secretions. A careful perusal of spermatophoric structures and their behaviour in sea water may provide insights into their adaptive value to suit the environmental conditions in which the male has to transfer the spermatophore to the female for varying periods of storage, pending fertilization. The best example is provided in the spiny lobsters, where the spermatophoric mass quickly adheres to the female body on deposition, and undergoes hardening to protect the internal delicate spermatozoa from environmental hazards. Another interesting example to explain the adaptive value of spermatophores is found in the two sand crabs, Emerita asiatica and Albunea symnista. In E. asiatica, spawning rapidly follows spermatophore deposition and hence the spermatophoric ribbon remains as a jelly. Conversely in A . symnista, there seems to be a long period between mating and spawning so it is necessary for the spermatophore to undergo hardening. However, in both species, the spermatophoric ribbon is sticky, and can be deposited quickly and firmly, since both inhabit shifting sands of the surf zone (Subramoniam, 1984). Crustaceans also differ from other arthropods in the manner in which the spermatophores are produced. In insects and arachnids, there are well-defined male accessory glands to secrete various components of the spermatophore and assemble them in a common chamber (Schaller, 1980). On the contrary, in Crustacea, the entire spermatophore is made from secretions of the glandular epithelial cells lining the vas deferens. Surprisingly, endocrine control of the secretory activity of the vas deferens is hardly known for any crustacean species. Evolution of reproductive processes, especially involving spermatophoric sperm transfer mechanisms, seems to have occurred independently among arthropod groups. Schaller (1980) contends that the ability to produce spermatophores was a form of preadaptive differentiation reached in all annelid-like arthropod ancestors long before their coloniza-

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tion of land. In the terrestrial forms the distribution of spermatophores seems to be predominant in the primitive forms, with more emphasis on the indirect, contact-free transfer of spermatophores. As an example, the primitive apterygotes employ spermatophores for indirect transmission; in older orders of flying insects such as Orthoptera, spermatophores arc produced, but they are transmitted directly to the females. In the more advanced insects, there is a tendency for spermatophores to be replaced by liquid semen transferred by well-formed penes (Alexander, 1964). In contrast to insects, advanced marine crustaceans such as decapods always use spermatophores for sperm transmission. Even the brachyurans, with direct transfer into the female seminal receptacle, enclose sperm in discrete spermatophores. Conversely, the primitive orders included in the Euphyllopoda (Anostraca, Cladocera, Notostraca, Ostracoda) do not produce spermatophores at all; the amoeboid but motile sperm cells are transmitted in fluid semen by penes into the female reproductive tract for internal fertilization. It is notable, however, that most of the euphyllopods are freshwater forms (exception: brine shrimp) and have R-strategic reproductive patterns. Land crustaceans such as terrestrial isopods also have true copulation, but many of them use B primitive type of “spermatophore”. Clearly, spermatophore production is a speciality of the marine crustaceans to transfer semen. Biochemical studies o n the spermatophores of a variety of marine crustaceans indicated the predominant mucoid nature, which helps in the quick fastening to, and long storage in, the female body exposed to a marine medium. Furthermore, the acidic mucopolysaccharide components of the spermatophore also are responsible for maintaining a microenvironment for sperm survival within the stored spermatophore in species such a s lobsters (see Radha and Subramoniam, 1985). The adhesive nature of the crustacean spermatophore is also helpful in its transfer in planktonic forms such as copepods. All this evidence is in favour of the idea t h a t there is a parallel evolution in the use of spermatophores in marine crustaceans, together with their counterpart arthropods on land. The nonmotile nature of most crustacean spermatozoa obviously necessitates the use of spermatophores in sperm transfer. The widespread occurrence of spermatophores in other marine invertebrates such as molluscs, annelids, chaetognaths, etc., lends further evidence to the suitability of spermatophores in sperm transfer (both direct and indirect) in sea water. Commensurate with a wide use of spermatophores, the crustaceans have also developed various sperm-storing devices in the females, pending fertilization. In many, the spermatophores may be stored externally on the female bodv or internally in specialized spermathecal

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sacs. Brachyuran decapods have specialized various internal spermstoring mechanisms. As discussed earlier, Hartnoll (1979) has classified brachyuran crabs from the position of the spermatheca in the ventral sternum of the females. When the spermatheca forms the dilated proximal region of the oviduct, the fertilization is internal, whereas when the spermatheca is an epidermal invagination, unconnected to the oviduct, the fertilization is perforce external. In most crustaceans, however, spermatophore deposition is external and the oviduct is short and unspecialized for sperm storage. Extensive histochemical and a few biochemical studies, carried out on these spermatophores, emphasize the role of mucopolysaccharides in spermatophore hardening and sperm protection. In a few instances, phenolic tanning of the protein associated with the mucus as well as melanization have also been reported. In other cases (e.g. copepods), the spermatophore may be placed precisely on the female genital apparatus with the sperm mass streaming into the oviduct through the neck or a fertilization tube formed from neck materials. In such instances, the mechanism of sperm release parallels the conditions obtained in the spermatophores of cephalopod molluscs. Evidently, spermatophores have long been utilized by marine crustaceans and the structure and mechanism of dehiscence have been modified in tune with the life-style and reproductive adaptation to the environment. Apparently, analogies found in the spermatophore morphology of unrelated crustacean orders, as well as other invertebrate forms, only reflect parallelism in sperm-transporting mechanisms and do not show any phylogenetic relationship between the groups.

IX. Acknowledgements This review is dedicated to Mme Jaquiline Pochon-Masson, Laboratory of Evolution, University of Paris. I am grateful to Ms Elanor Ulinger, Librarian, Bodega Marine Laboratory, University of California at Davis, for helping me to obtain many papers on crustacean spermatophores. I am beholden to Drs C . T. Indra and N. Munuswamy for critically reading through the manuscript. I thank Dr Jeyadev Babu for helping me with the line diagrams. I am also grateful to my students Jose Philip, Bhavanishankar, Vidya Jayasankar, Gunasekaran, Abdul Nazar, Senthil Murugan and Suresh for their help in the preparation of the manuscript. Studies on Scyllu serrutu reported here were financially supported by the Department of Science and Technology, Government of India.

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X. References (References marked with an asterisk have not been checked by the author and are included here for completeness of the bibliography.) Adams, A . E. and Paul, A . S. (1983). Male parent size, sperm storage and egg production in the crab Chionoecetes bairdi (Decapoda, Majidae). International Journal of Invertebrute Reproduction, 6, 181-187. Aiken, D. and Waddy, S. (1980). Reproductive biology. In “The Biology and Management of the American Lobster” (S. Cobb and B. Phillips, eds), Vol. 1, pp. 215-276. Academic Press, New York. Aiken, D., Waddy, S. L., Moreland, t i . and Polar, S. M. (1984). Electrically induced ejaculation of the American lobster Homarus umericanus. Journal of Crustacean Biology, 4, pp. 519-527. Alexander, R . D. (1964). The evolution of mating behaviour in Arthropods. In “Insect reproduction” (ti. Highnam, ed.), pp. 78-94. Royal Entomological Society of London. Anchordoguy, T., Crowe, J . H., Clark, W. H . Jr and Griffin, F. J. (1987). Cryopreservation of sperm from the penaeid shrimp Sicyonia ingentis. World Ayuuculture Society, 18th Annual Meeting, Ecuador (Abstract). Anchordoguy, T., Crowe, J. H . , Griffin, F. J. and Clark, W. H. Jr (1988). Cryopreservation of sperm from the marine shrimp Sicyonia ingentis. Cryobiology, 25, 23g-243. Anderson, S. 0. (1971). Phenolic compounds isolated from insect hard cuticle and their relationship to the sclerotization process. Znsect Biochemistry, I , 157-170. Anderson, W. A . and Ellis, R . A . (1967). Cytodifferentiation of the crayfish spermatozoan: acrosome formation, transformation of mitochondria and the development of microtubules. Zeitschrift fur Zellforschung und Mikroskopische Anatomic, 77, 8 C 9 4 . Andrews, E. A . (1931). Spermatophores of an Oregon crayfish. Americun Naturalist, 45, 277-280. Anilkurnar, G. and Adiyodi, t i . G. (1977). Spermatheca of the freshwater crab Paratelphusu hydrodromous (Herbst) in relation to the ovarian cycle. In “Advances in Invertebrate Reproduction” (ti. G. Adiyodi and R . G. Adiyodi, eds), pp. 269-274. Peralam - tiemoth, tiarivellur, India. Anstensrud, M. (1990). Male reproductive characteristics of two parasitic copepods Lernaeoceru branchiulis (L.) (Pennellidae) and Lepeophtheirus pectoralis (Muller) (Caligidae). Journal of Crustacean Biology, 10, 627-638. Aquacop (1983). Contribution of broodstock, maturation, spawning and hatching systems for penaeid shrimps in the Centre Oceanologique du Pacifique. In “CRC Handbook of Mariculture” Vol. 1, “Crustacean Aquaculture” (James P. McVey, ed.), pp. 105-121. CRC Press, Boca Raton, Florida. A X , P. (1968). Das Fortpflanzungsverhalten von Trilobodrilus (Archiannelida, Dinophilidae). Marine Biology, 1, 33C335. Babu. B. T., Shyamasundari. K . and Hanumantharao, K. (1988). Cytochemical nature of spermatophore layers in the edible crab Portunics sanitinolentus (Herbst) (Portunidae). Proceedings of Indian National Science Academy, Part B (Biological Sciences). 54, 129-132.

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Southward, E. C. (1975). Fine structure and phylogeny of the Pogonophora. Symposia of the Zoological Society of London, 36, 235-251. Spalding, J. F. (1942). The nature and formation of the spermatophore and sperm plug in Carcinus maenas. Quarterly Journal of Microscopic Science, 83, 399422. Spicer, S. S. (1960). A correlative study of the histochemical properties of rodent acid mucopolysaccharides, J. Histochem. Cytochem., 8, 18-35. Stevenson, J . R. (1961). Polyphenol oxidase in the tegumental glands in relation to the molting cycle of the isopod crustacean Armadillidium vulgare. Biological Bulletin, Marine Biological Laboratory, Woods Hole, 121, 554560. Subramoniam, T. (1977). Aspects of sexual biology of the anomuran crab, Emerita asiatica. Marine Biology, 43, 369-377. Subramoniam, T. (1979). Some aspects of reproductive ecology of a mole crab Emerita asiatica (Milne Edwards). Journal of Experimental Biology and Ecology, 36, 259-268. Subramoniam, T. (1981). Sexual and reproductive endocrinology of Crustacea. Journal of Scientific and Industrial Research, 40, 39W03. Subramoniam, T . (1984). Spermatophore formation in two intertidal anomuran crabs, Emerita asiatica and Albunea symnista (Decapoda: Anomura). Biological Bulletin, Marine Biological Laboratory, Wood’s Hole, 166, 78-95. Subramoniam. T. (1988). Reproductive engineering in Crustacean aquaculture. Journal of Indian Fisheries Association, 18, 27-37. Subramoniam, T. (199 1). Chemical composition of spermatophores in decapod Crustaceans. In “Crustacean Sexual Biology” (R. T. Bauer and J. W. Martin, eds), pp. 308-321. Columbia University Press, New York. Subramoniam, T. (1993). Ultrastructural studies on the vas deferens of the penaeid shrimp, Sicyonia ingentis. Journal of Biosciences (submitted). Talbot, P. (1984). Problems and progress in controlling reproductive biology in the American Lobster (Homarus). In “Advances in Invertebrate Reproduction” (W. Engles, W. H. Clark, Jr, A . Fischer, P. J. W. Olive and D. F. Went, eds), Vol. 3, pp. 473480. Elsevier, Amsterdam. Talbot, P. and Zao, P. (1991). Secretion at molting by the pleopod tegumental glands of the lobster Homarus americanus (Milne Edwards). Journal of Crustacean Biology, 1 I , 1-93. Talbot, P., Hedgecock. D., Borgeson, W.. Wilson, P. and Thaler, C . (1983). Examination of spermatophore production by laboratory-maintained lobsters (Homarus). Journal of the World Mariculture Society, 14, 271-278. Talbot, P . , Thaler, C . and Wilson, P. (1986). Artificial insemination of the American lobster (Homarus). Gamete Research, 14, 25-31. Tudge, C. C. and Jamieson, B. G. M. (1991). Ultrastructure of the mature spermatozoon of the coconut crab Birgus latro (Coenobitidae: Paguroidea: Decapoda). Marine Biology, 108. 395402. Uma. K. (1978). Studies on comparative sperm morphology and spermatophores of Crustaceans. MPhil Dissertation, University of Madras. Uma, K. and Subramoniam, T. (1979). Histochemical characteristics of spermatophore layers of Scylla serratu (Forskal) (Decapoda: Portunidae). fnternarional Journal of Invertebrare Reproduction, 1. 3 1 4 0 . Uma. K. and Subramoniam, T. (1983). A comparative study on the spermatophore formation in Scyllu serrata (Forskal) (Decapoda: Brachyura) and Clibanarius longitarsus (De Haan) (Decapoda: Anomura). Journal of the

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Murine Biological Association of India, 26, 103-108. Uno, Y. and Fugita, M. (1972). Studies on the experimental hybridization of fresh water shrimp Macrobrachiurn nipponense and M . formosense. Second International Ocean Development Conference, 5-7 October, Tokyo, Japan (abstract). ‘;Van Oye, P. (1931). La fecondation chez les chetognathes. Bulletin Musee Royale d’Histoire Naturelle de Belgique, 7 (7), 1-7. Varadarajan, M. (1982). Studies on the reproductive tract and accessory sex organs of the ghost crab, Ocypoda platytasis Milne Edwards (Crustacea: Brachyura). MPhil Dissertation, University of Madras. Waddy. S. L. and Aiken, D. E. (1991). Mating and insemination in the American Lobster, Homurus americunus. I n “Crustacean Sexual Biology” (R. T. Bauer and J. W. Martin, eds), pp. 290-307. Columbia University Press, New York. Westheide, W . (1984). The concept of reproduction in polychaetes with small body size: Adaptations in interstitial species. In “Polychaete Reproduction” ( A . Fischer and H. D. Pfannenstuhl, eds), Vol. 29, pp. 267-287. Westheide, W . (1988). The ultrastructure of Polychaeta. In “Microfauna Marine” (W. Westheide and C . 0. Hermans, eds), Vol. 4, pp. 263-279. Gustav Fischer Verlag, Stuttgart, New York. Wharton, G . W. (1942). A typical sand beach animal the mole crab, Emerita tulpoida (Say). Ecological Monographs, 12, 137-181. White, A , . Handler, P., Smith, E. L., Hill, R. L. and Lehman, R . (1978). “Principles of Biochemistry”. p. 1155, Academic Press, New York. Wiborg. K. F. (1971). Investigations on Euphausiids in some fjords on the West Coast of Norway in 19661969. Fiskeridirektoratets Skrifter, serie Huvunder.rvkelser, 16, l(L35. ’Wittrnann. K. J. (1982). Untersuchungen zur Sexualbiologie einer inediterranen Mysidacen: Leptomysis lingvura G. 0 . Sars. Zoologischer Anzeiger, 209, 362-375. Yager, J. (1981). Remipedia, a new class of Crustacea from a marine cave in Bahamas. Journul of Crustacean Biology, 1, 328-333. Yager, J. (1989). The male reproductive system, sperm and spermatophores of the primitive hermaphrodite remipede crustacean, Speleonectes benjammi. Invertebrate Reproduction and Development, 15, 75-84. Yager, J. (1991). The reproductive biology of two species of Remipedes. In “Crustacean Sexual Biology” (R. T. Bauer and J. W . Martin, eds), pp. 271-289. Columbia University Press, New York. -‘Ziinmer, C. (1913). Untersuchungen uber den inneren Bau von Euphausia sriperbu Dana. Zoofogica Stuttgart, H. 67 (Bd. 26), 65-128.

The Bristol Channel Sole (Solea sslea (L.)): A Fisheries Case Study J. Horwood Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Lowestoft, Suffolk NR33 OHT, UK

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Introduction .. .. .. .. .. A . Classification and identification .. .. B. Description and related genera .. .. Distribution and Movements .. .. A. Physical characteristics of the region .. B. Eggs and larvae .. .. .. C. Juveniles . . .. .. .. .. .. .. D. Adults .. E. The Bristol Channel “stock” .. .. Feeding, Size and Growth .. .. .. .. .. .. A. Feeding .. .. B. Size and growth: general aspects C. Length at age .. .. .. .. .. .. D. Weight at age .. .. .. .. .. Reproduction .. .. .. A. Spawning behaviour .. B. Seasonal development and time of spawning C. Distribution, size and age with maturity . . .. .. D . Fecundity .. .. .. .. Natural Mortality Rates .. .. .. A. Eggs and larvae .. .. .. .. B. Juveniles .. .. .. .. C. Adults .. .. .. .. D. Comments .. .. .. .. Harvesting Options .. .. .. A . Yield per recruit ..

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ADVANCES IN MARINE BIOLOGY VOLUME 29 ISBN 0-12-026129-4

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B . Absolute yields .. .. C . Spawning stock biomass per recruit D. The stock and recruitment relationship E. Bioeconomics and dynamics .. F. Appropriate fishery targets .. VII. Exploitation of the Bristol Channel Sole A . Early fisheries .. .. .. B. Early trawl fisheries .. .. C . Early quantitative information .. D. Catches from 1903 .. .. E. Evolution to the modern fishery , , VIII. Status of the Stock .. .. A . ICES assessments .. .. B . Egg-production based assessments C . Comparison of assessment methods D. Mark-recapture estimates .. E. Simulation of population trajectories F. Concluding remarks .. .. IX. Some Final Comments .. .. X. Acknowledgements .. .. .. XI. References .. .. .. ..

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1. Introduction The common or Dover sole (Solea solea (Linnaeus, 1758)) is a “prime” species of marine fish, commanding a price of f5000lt at auction in 1990. Its firm white flesh, impressive appearance on the plate, easy culinary dissection and delicate taste mean that it is seldom absent from the menus of good restaurants. The sole is a flatfish, named from its similarity in shape to a sandal, dark to black on its upper side and creamy-white underneath. Both eyes are found on the upper, right side of the fish. The fish seldom exceeds S0cm in length. The sole is nocturnal in habit and buries deep in sand during the day. Sole taken with the conventional otter-trawl were mainly caught during the night and catches were low until the modern beam-trawl fishery started in the 1960s. The powerful beam-trawlers can dig-up the sole from the sand, and fish effectively throughout the day, so that fishing mortality on all European stocks of sole increased over the past two decades. Even so, nearly 100 million soles were sold annually at Billingsgate in the mid nineteenth century (Couch, 1864). An adequate supply of sole is necessary for the survival of the international. high-cost, beam-trawl fleets as other fish either command too low a price to cover costs (e.g. plaice, Pleuronectes platessa

THE BRISTOL CHANNEL SOLE ( S O L E A SOLEA

(L.))

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Linnaeus, 1758), or else are too few in numbers to yield a profit (e.g. turbot, Scophthalmus maximus (Linnaeus, 1758)). The sole is restricted to the shallower waters of the eastern North Atlantic and Mediterranean. Its distribution extends southwards along the west coast of Africa as far as Senegal (12-16"N) (Whitehead el al., 1986). Couch (1864) noted its presence off the Cape of Good Hope, but this was probably Austroglossus, a species which has supported a trawl fishery (Smith, 1949). The sole is frequently referred to as a warm-water fish, but it has bred successfully in areas experiencing water temperatures from 4 to 30°C. In any large numbers, the northern extent of the sole's range is about 55"N. Commercial records of England and Wales vessels show occasional catches off north and northwest Shetland (61Y242"N), and there is only one record, in forty years, from north of 62"N. Holt and Byrne (1905) reported it to be rare at the Faeroe Islands but there are recordings of catches off Trondheim (63!h0N) on the Norwegian coast (Anon., 1964a). The sole is also found in the western Baltic Sea. This review describes work on the sole of the Bristol Channel, mainly undertaken since 1987. Four related, specific programmes are involved: (i) the annual assessment work undertaken within the framework of the International Council for Exploration of the Sea (ICES), which involves determination of distribution, recruitment and abundance; (ii) juvenile tagging programmes to examine the dispersion of young sole; (iii) a programme to investigate the dispersion of sole eggs and larvae and their route to nursery grounds and (iv) the determination of the abundance and relative status of the sole independent from the ICES assessments. These studies have allowed a presentation of the population biology of the sole in the Bristol Channel and northern Celtic Sea, approximately between 5CL52"N. This comprises ICES Divisions VIIf-g and, although the boundary of the stock is not precisely determined, it is shown that sole in this region are relatively isolated, and that they can be regarded as comprising a population in that birth and death rates dominate those of immigration and emigration. This allows a comparison between the ICES and alternative assessment that is not possible for many "stocks" assessed by ICES. In describing the population biology of the Bristol Channel sole it is natural to relate it to that from other populations, and biological Parameters from other populations have had to be used in some evaluations of the Bristol Channel sole. Cunningham (1890) described the comparative anatomy and morphology of the sole, and related species. However, there is no general review of the population biology of the sole, and much of the information on it is available only through unpublished

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manuscripts. Consequently, this study details the population biology of the Bristol Channel sole relating it, as appropriate, to the population biology of the species; it is an exercise in fisheries biology (Cushing, 1981). Details in this section cover the classification and identification of the sole and related genera. In Section I1 the physical and hydrodynamic characteristics of the Bristol Channel and northern Celtic Sea are described, with new information presented on residual currents, salinity, temperature and density gradients. New information is also presented on the spatial and temporal distributions of eggs and larvae, showing the isolated nature of the spawning and drift in the plankton. The distributions of juvenile and adult sole are described from a variety of sources including new data from mark-recapture programmes, and research and commercial catches. The isolation of t h e Bristol Channel population is argued. In Section I11 limited new information on feeding is given. Growth in length and weight in the population are inferred and temporal increases in average weight at each age identified. In Section IV the spawning behaviour of the sole is described, and new data presented on the seasonal development of the gonads, time of spawning, size and age at maturity, determinacy of fecundity and absolute fecundity. In Section V the natural mortality rates of eggs, larvae, juveniles and adults are estimated and reviewed. The above biological information allows an evaluation of the potential yields and status of the Bristol Channel sole, which has given catches of about 1000 t/year over the last decade. This evaluation is undertaken in Section VI, along with the development of a new sized-based model to predict yields given the selection characteristics of meshes used in the fisheries; it is a complement to the age-based, yield-per-recruit analysis of Beverton and Holt (1957). The population has been fished since the Middle Ages and the fishery and its catches are described in Section VII. In Section VIII the current status of the stock is briefly reviewed from the latest ICES assessment. Research has allowed an independent eggproduction based estimate of the female spawning biomass. This is the first time that the main ICES assessment methodology - tuned virtual population analysis (VPA) - has been subjected to a rigorous independent evaluation. Previous egg-production based assessments have been conducted for stocks for which there was no independent or reliable VPA (western mackerel, Californian anchovy, North Sea sole) or where the VPA did not correspond to a biological population (North Sea sole and plaice). A large discrepancy between the two methods is demonstrated. The population size is simulated from 1820, using all the above information, and the relative status of the population is discussed.

T H E BRlSTOL C H A N N E L SOLE (SOLEA S O L E A ( L . ) )

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219

Classification and Identification

The following classification is based upon that of Ahlstrom et al. (1984) and Hensley and Ahlstrom (1984), which is generally similar to those offered by Norman (1934) and Whitehead et al. (1986). The nomenclature for the sole (S. solea) is taken from Wheeler (1988). The flatfish comprise the order Pleuronectiformes (Heterosomata of Norman), and have been taxonomically separated from other fish since 1871. Pleuronectiformes are recognized by having a flat, laterally compressed body, both eyes on the same side, a short precaudal region, long anal and dorsal fins, 17 or fewer principal caudal fin rays, pelvic fins with six rays or less and various cranial characteristics; 520 species have been recognized. Ahlstrom et al. (1984) divided the order into three sub-orders and eight families, and these are given below along with examples of the genera or species. Sub-order: Psettodoidei Family: Psettoidae (single, tropical genus: Psetrodes) Sub-order: Pleuronectoidei Family: Citharidae (single genus in NE Atlantic: Citharus) Scophthalmidae (4 genera: brill, megrim, turbot, topknot) Paralichthyidae (16 genera, not found in E or NE North Atlantic) Bothidae (many genera, 2 in N E Atlantic: scaldfish) Pleuronectidae (many genera, 9 in NE Atlantic: flounder, plaice, dab, lemon sole, plaice, halibut) Sub-order: Soleoidei Family: Soleidae (33(?) genera, 7 in N E Atlantic: see later) Cynoglossidae (3 genera, single specimen from NE Atlantic: tonguesoles) In the Soleoidei the hind margin of the preoperculum is hidden below the skin, whereas in the Pleuronectoidei the margin is free and clearly visible. Within the Soleoidei the true soles, Soleidae, have both eyes on the right side and the caudal fin is separated from the elongated dorsal and anal fins. The lateral line is well developed on the eyed side and runs in a relatively straight line along the body but changes shape and distinctness as the lateral line passes over the head. Scales are ctenoid. The Cynoglossidae are much more narrow in shape, with the caudal fin confluent with dorsal and anal fins, both eyes are on the left side and, depending upon species, the lateral line is either absent on both sides or else well developed o n the eyed side only. In the eastern North Atlantic

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they are found off west Africa and in the Mediterranean, although a single specimen of Cynogfossus browni Chabanaud, 1949, was found off the Netherlands coast (Nijssen, 1966). Five species of symphurine Cynoglossidae are recognized from the western Atlantic (Munroe, 1991).

B.

Description and Related Genera

Whitehead et al. (1986) described seven genera of Soleidae from the northeastern North Atlantic, comprising 17 species. They give a key and illustrations to distinguish the species. Plates and descriptions are also given by Couch (1864) and Cunningham (1890). Of these species, Solea sofea has the widest distribution and is the most abundant of the larger soles. The taxonomy of the Atlanto-Mediterranean soles was further reviewed by Ben-Tuvia (1990). The following gives a brief description of these genera and species, and notes the features that allow them to be readily distinguished from S. sofeu. Solea: the North Atlantic species are described later. Pectoral fins are developed on both sides with at least seven fin rays; the supra-temporal branch of the lateral line is distinct and smoothly rounded; on the scales the intercanicular striae are not strongly curved and are longer than the width of the intercanicular zone. Bathysolea: comprising a single species ( B . profundicufa (Vaillant, 1888), the deep-water sole), which inhabits waters of over 200m depth from southwest Ireland southwards, the pectoral fin is on the eyed side and is small with four to five fin rays, the supra-temporal branch of the lateral line is indistinct, and the adult length is uncertain, but can exceed 21 cm. Buglossidium: comprised of a single species ( B . futeum (Risso, ISlO), the solenette), which is common on the European shelf; it is sandy coloured and small, growing to 15 cm, and the supra-temporal branch of the lateral line is indistinct. Dicofogoglossa: has two species (the wedge sole and six-eyed sole); the supra-temporal branch of the lateral line has a pronounced “S” shape, the pectoral fin has an angular shape and the intercanicular striae are strongly curved and are shorter than the width of the intercanicular zone. D. cuneata (Moreau, 1881) is of a similar colour to S. sofea and grows up to 30cm; D. hexophthalmus (Bennett, 1831) is lighter in colour, has six pronounced spots, is smaller and has a more southern distribution. Microchirus: has four species in the northeastern North Atlantic ( M . azevia (Capelio, 1867), the bastard sole, M. boscanion (Chabanaud, 1926), M . oceffatus (Linnaeus, 1758), the four-eyed sole, M . vuriegatus (Donovan, 1802), the thickback sole. The pectoral fin on the eyed side is small

TFIE BRISTOL CHANNEL SOLE ( S O I , E A SOLEA (L.))

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and on the blind side is much reduced, the supra-temporal branch of the lateral line is indistinct and "S" shaped, and the intercanicular striae are strongly curved and shorter in width than the intercanicular zone. Monochirus: comprising a single species ( M . hispidus Rafinesque, 1814, the whiskered sole), it has no pectoral fin on the blind side and is mainly found around the margins of the Mediterranean. Synaptura: has one species in the northeastern North Atlantic (S. lusitanica (Capello, 1868), the Portuguese sole), which has the dorsal and anal fins joined to the caudal fin; it grows to about 35 cm and is found off the southern coast of Spain and off north-west Africa. Whitehead et al. (1986) described seven species of Solea, but this was reduced to four by Ben-Tuvia (1990) and his interpretation is given below. The main changes were that the two species S. impar (the Adriatic sole) and S. nasuta (the snouted sole) were not recognized as separate taxa and were synonymized under S. lascaris (Risso, 1810) (the sand sole), and similarly S. aegyptiaca was synonymized under S. solea. There was some electrophoretic evidence to support a separation of S. solea and S. aegyptiaca, but this was not sustained by morphometric criteria, and it was implied that the study was further complicated by a possibility of hybridization with S. senegatensis Kaup, 1858. The characteristics of the four species of Mediterranean and northeastern North Atlantic Solea are given in Table 1; the length given for S. solea is generous (Section II1.B). The species fall into two groups, each of two species. Those with the anterior nostril on the blind side much enlarged (S. lascaris and S. kleini Bonaparte, 1833), and those with it not enlarged (S. solea and S. senegalensis). In the latter group, S. senegalensis is rare north of about 45"N. In the waters of, and near to, the Bristol Channel the only other species likely to be encountered is the sand sole (S. lascaris), which is not uncommon. This is a much browner and fatter fish which has an enlarged, rosette-shaped nostril on the blind side. For all the species, Ben-Tuvia (1990) demonstrated the large effect that geographic position, or temperature, can have on the number of vertebrae and fin rays. Amongst the four species the numbers overlap and provide no basis for differentiation unless the position of sampling is known.

11.

Distribution and Movements

A. Physical Characteristics of the Region The area under consideration is the Bristol Channel and northern Celtic Sea, approximately from 5&52"N and west of 9"W to the coast of

TABLE1.

CHARACTERISTICS OF THE

FOURNORTHEASTERN NORTH

Colour

A T 1 ANTIC' SPbClES OF

Distribution in N E Atlantic and Mediterranean

Common name

Blind side anterior nostril

S. solea

Common sole

S. senegulensis

Senegal sole

S. kleini

Klein's sole

S. 1uscari.s

Sand sole

Not enlarged, without Black or dark on eyed Widespread in fringes, diameter side, white under, shallow seas of about size of scales black tip to eyed side Europe and pectoral North Africa, south of about 64"N Not enlarged, without Eyed side brownigrey Biscay coast south fringes, diameter and pectoral fin of about 4 T N , about size of scales mainly black NW Africa and west Mediterranean Enlarged, cupula Eyed side light brown Mediterranean and shaped, fringed, with light spots, NW Africa not radiated and pectoral fin has black separated from spot in middle posterior nostril Enlarged, fringed, Light to dark brown West Scotland, rosette shaped and with spotted Ireland, English near posterior appearance, pectoral Channel, Mediterrancan nostril fin has small central black spot and NW Africa

After Ben-Tuvia, 1990, supplemented by Whitehead er al., 1986.

SO LA^

Adult size (cm)

Dorsal fin rays

Anal fin rays

Vertebrae

<70

65-97

53-79

41-52

<60

72-95

61-75

4>46

<40

72-91

57-74

42-48

~ 4 0 65-90

4G75

42-47

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England and Wales. It will be shown that this region contains the adult and juvenile sole and the spawning grounds of the population of interest. This region corresponds largely to that of ICES Divisions VIIf-g. 1. Depth and geomorphology The waters of the Bristol Channel and northern Celtic Sea have provided a hospitable environment for the sole for only about the last 3000 years or 500 generations. During the Ice Ages the ice sheets extended to approximately 50"N, and some 20,000 years ago the water depths in the region were 100-150m less than today (Carter, 1988; Evans, 1990; Hardisty, 1990). The exposed and semi-exposed sea bed was subjected to great erosion which resulted in a now submerged peneplain. These flattish areas house the traditional fishing grounds of the Smalls, in the east, and the Nymphe Bank and the Saltees, Mine Head, Ballycottin and Kinsale Head grounds in the west (Fig. 1). In the northeast the area is dominated by the Severn Estuary.

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FIG. 1. Bathymetry and geography of the Bristol Channel and northern Celtic Sea; depth in m.

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A broad central channel, 120m deep, narrows and shallows from the southwest to a depth of 100-110m, but then deepens to 120m to the south of the St George's Channel. From both east and west coasts the water shoals rapidly to 2&40 m. In a broad band 20-30 miles wide, from the mouth of the Severn Estuary and along the coast of Wales to the Saltees, the bottom is covered with sand ridges whose long axes run north-south. In the south the major southwest-northeast running Banks of Labadie, Jones, Cockburn and the Great Sole Bank are sand ridges which were formed by the tides when the sea was shallower (Pantin and Evans, 1984; Hardisty, 1990). The substratum is predominantly of sand or sandy-gravel. West of Cardiff to about 4"W strong currents have scoured the sea bed to expose the bottom rock and there are no bottom sediments. Fringing the rock, and in a band parallel to the north Cornwall coast, there are large areas of gravelly-sand. Local areas of mud can be found (British Geological Survey, 1987).

2. Temperature The monthly surface water temperatures in the Celtic Sea and adjacent areas, averaged over 1905-1954, are illustrated in Anon. (1962). Minimum temperatures occur in February and March when most of the region is between 7 and 9°C. By August, much of the region is 15-16°C. Seawater temperatures at Newlyn (used in Section 1V.B) are about one degree warmer than those at the St Gowan Light Vessel (51'30" 05"OO'W) for the first 6 months of the year. Cold winters affect the behaviour of sole and fifty years of data from Newlyn show that the lowest average monthly temperature was in February at 8.4"C, and that in two years, 1947 and 1963, the mean February temperature was 3 4 c " below the long-term average, and in 1986 it was 2.5c" below. The highest mean temperature for that month was in 1943 and was 1.55c" above average. Thermal stratification is first established in early April to the southeast of Ireland associated with the weak tidal currents. Throughout April the stratified region extends to the north and east to about 5"W (Pingree, 1975, 1980). Simpson ef af. (1977) predicted a front from Dunmore to Lundy Island and to the south of Hartland Point, and this is seen in the satellite image shown by Pingree (1980). This feature appears to form the northern boundary of the sole spawning. However, the frontal region is very dynamic and can move tens of miles within a day (Simpson, 1981). Stratification in the eastern Celtic Sea and Bristol Channel in 1990 followed the general predictions outlined above, but, as described in

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T H E BRISTOL C H A N N E L SOLE ( S O L E A S O L E A ( L . ) )

detail later, local haloclinic stratification was sometimes observed before heating dominated the system. In 1990, average temperatures in the water column were sampled during collections of plankton, from February to June (Fig. 2). They display the general features described above, with strong horizontal thermal gradients established from May, but local structures are seen in March and April (see Section II.B.2). 4"

7"

6"

.

.

I

5"

4"

. . . . .

F K . 2. Distribution of temperatures ("C) averaged over the water column in 1990: a) 15-22 February. b) 9-13 March, c ) 1-6 April. d) 17-24 April, e ) 18-23 May, f) 2 S 2 9 June.

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3 . Salinity

The seasonal surface salinity, averaged over 1905-1954, is illustrated in Anon. (1962). In most of the region salinity is about 34.5-35.5%0. The two main features are the influence of the River Severn, giving less saline water to the Bristol Channel, and the inflow of more saline water from the south or southwest. Fig. 3 illustrates the surface salinity found on the

FIG.3 . Distribution of surfacc salinities (%); a-f as Fig, 2

T l f E BKlSTOL CHANNEL SOLE ( S O L E A S O L E A (L.))

227

1990 plankton surveys; however, differences were detected in the water column as described in Section II.B.2. 4. Currents (a) Tidal streams At the time of high water at Dover tidal southwesterly currents are removing water from the Bristol Channel. The tide turns 2 h later and water is drawn in from the Irish Sea and the southwest. Six hours after high water at Dover the Irish Sea tide turns and all flows are to the northeast. The outward flow of water starts at 4 h before the Dover high water. Tidal speeds vary considerably over the region with the maximum spring tide in the Bristol Channel, near Cardiff, being over 2 m/s. In the central deeper waters the maximum tides are about 0.3 m/s. Spring tidal range at Cardiff is 11.3 m and at Milford Haven and Padstow it is 6.5 m. (b) Residuul velocities Residual current velocities were estimated from lunar tide (M2) driven, vertically integrated, hydrodynamic models. For the Bristol Channel, east of 6"W, the results are given in Fig. 4 (results were provided by E. Jones of the Proudman Oceanographic Laboratory, Birkenhead). Weak residuals of <1 cm/s occur away from the coasts. A small residual current flows north along the coasts of Cornwall and Devon with some backcirculation in the bays. Flow north is arrested at Lundy Island (51'10") except for a near-coastal current, between Lundy and the mainland, of 10-20cm/s. Mean flow eddies within the Severn Estuary and water is taken around Carmarthen and Swansea Bays at residual velocities of about 5 cm/s. Owen (1980) calculated residual currents from models with different spatial resolution, east of 5"W and 4"W. His results give a greater detail within the Severn Estuary but are similar in magnitude and direction to those of Fig. 4, except that some greater velocities are predicted about Lundy Island. The effect of steady-state winds was modelled by Pingree and Griffiths (1980) from M2 tidal flows plus wind effects. Results were given for steady SW and SE winds and they show that the residual flows of Fig. 4 were enhanced for these winds, and were increased by about the same magnitude for winds of 1&20 mis. A three-dimensional wind-driven model of the circulation in the Celtic Sea has also been recently developed by Davies and Jones (1992).

( c ) Current meter resuits In 1989 current meters were positioned about 15 miles off the north Cornwall coast from 51'35' to 51"55'N, near the centres of sole spawning.

228

J . HORWOOD

FIG.4. Direction and magnitude of modelled tidal residual currents; the arrow shows a magnitude of 10 cm/s.

Four gave records of between 19 and 34 days. From a range of depths within the water column they all demonstrated a similar residual current of 2.G3.9 km/d (2.3-4.5 cm/s) towards 006453"N. Over this time the average wind was of 2.7m/s from the SW to WSW. These results are consistent with the modelled tidal residual currents assisted by a small Sw wind. In August to October 1971 meters were positioned at four stations in the St George's Channel (Howarth, 1971). They indicated a complex flow pattern. To the west, currents followed the Irish coast southwest, whilst to the east they flowed south and east. The central station showed variable directions and speeds.

T I E BKISTOL C € I A N N E L S O L E ( S O L E A SOLEA (L.))

229

(d) Drifter returns Sea-bed and near-surface Woodhead drifters were released in 1989 and 1990. Near-surface drifters were released in Carmarthen Bay and off the Gower in May 1989 and most recoveries were from the northern coasts of Devon and Cornwall 1-3 months later. Similar drifters were released 40-60km northwest of Trevose Head in April 1990. Recoveries were made from the north Devon or south Wales coast (Jennings and Pawson, 1992); however, recovery times were large (7G180 days) in relation to the duration time of sole egg and larvae. Near-surface drifters are affected by the tide and wind (Hill and Horwood, 1974), but recovery time is always likely to exaggerate the passage time because of the difficulty of the drifters actually getting ashore and the need for them then to be discovered. Sea-bed drifters were released at ten stations throughout the plankton grid in 1989 (described later). These drifters ride into the water column when the tide flows (Dickson, 1976) and in this locality of high tidal streams are likely to capture the movement of a large part of the water column. Recaptures were reported from the coasts of north Devon and south Wales but the large majority were found over 150 days after release. A few drifters were returned outside ICES Divisions VIIf-g and many were recaptured by trawlers at sea. It is difficult to reconcile the previous interpretation of residual flow with the pattern of drifter returns.

B.

Eggs and Larvae 1. Description

Fertilized eggs of members of the family Soleidae are spherical and contain several oil globules. The yolk is segmented around the perimeter and there is a small perivitelline space. Eggs of the sole are of 0.95-1.58mm diameter and the oil globules occur in a few large groups around the surface of the yolk (Holt, 1899; Nichols, 1976; Russell, 1976). Care is required to separate sole eggs from those of the solenette ( B . luteurn), the sand sole (S. lascaris) and the thickback sole ( M . variegatus) all of which are found in the Bristol Channel and northern Celtic Sea. Sole eggs have been classified into four developmental groups by Riley (1974), following the scheme for plaice of Simpson (1959), and ten phases of development are illustrated in his Fig. 3. Stage IA is the blastula phase from fertilization to when the blastoderm diameter is 80% of the egg diameter. Stage IB is the gastrula phase up to the appearance of the Primitive streak. Stage I1 eggs are still in the gastrula stage but are from

230

J . HORWOOD

the formation of the primitive streak to when the blastopore closes. Stage 111 eggs undergo organogenesis with growth of the tail, free from yolk-sac attachment, extending to the equator of the egg, and with the body axis running from pole to pole. At Stage IV the tip of the tail grows through the equator to 60-40" from the nose; hatching then occurs. Temperature-dependent stage-duration times of sole eggs have been measured in several experiments (Riley, 1974; Fliichter, 1970; Irvin, 1974; Fonds, 1979; Devauchelle et a l . , 1987); such information is necessary in the determination of egg mortality and production rates. The natural logarithm (In) of the stage duration ( d ) in days is given for temperature ( t ) in "C modified from Riley (1974): Stage Stage Stage Stage

I: 11:

Ill: IV:

ln(d) In(d) In(d) ln(d)

= = = =

2.0193-0.1227t 1.49414.1530t 2.5075-0.1509t 1.410W.0687t

Viability of eggs is low below 7°C but salinities of 2CL35700 do not appear to affect egg viability and development. Yolk-sac stage, larval soleids are characterized by the many oil globules present in the segmented yolk-sac. The frontal region is prominent, and the mid-brain is well developed with vesicular expansion of the embryonic dorsal fin above the mid-brain. On hatching the eyes are not pigmented. Sole larvae hatch at about 3 m m , they have a few large groups of oil globules in the yolk-sac and large stellate chromatophores in the marginal fins. The development of sole larvae was described by Fabre-Domergue and BiCtrix (1905) and sole larvae at different sizes are illustrated by Nichols (1976). In the yolk-sac stage the larvae may be confused with those of the solenette, although the latter are usually much smaller and of a different shape. After the yolk-sac stage, but before the head becomes asymmetric, the sole is identified by the large stellate chromatophores and myotome number of 45-50; the solenette has many fewer myotomes. The yolk-sac is exhausted after 5-9 days (Lagardkre, 1989). Asymmetry of the head is observed at about 8-9mm. The sole is then distinguished from the solenette by its greater number of vertebrae (48-52, cf. 36-38), and from the sand sole by its more distinct swim bladder, pattern of the chromatophores and more protruding lower jaw. Pelagic life ceases at about 12 mm. Fabre-Domergue and Bietrix (1905) described metamorphosis starting at 30-33 days after hatching and which was completed by 4&52 days; however, growth rates are sensitive to temperature and feeding (Section 1II.C).

T1IE BKISTOL CHANNEL SOLE ( S O L E A .SOI-EA ( L . ) )

23 1

2. Distribution

In 1989 and 1990 plankton surveys were carried out in the outer Bristol Channel to sample sole eggs and larvae. Information from these exercises is used to demonstrate the location of spawning and the dispersion of eggs and larvae and, later, to determine the abundance o f eggs and adult sole. The 1990 surveys spanned the onset and completion of spawning, from February t o June, and are described first. Six surveys were conducted and the plankton grids, and egg and larval distributions are shown in Figs 5 and 6 and physical parameters are illustrated in Figs 2, 3 and 7. At each station samples were collected with a single haul of a high-speed sampler, which was towed at 5 knots and traversed from the surface to a s near the sea bed as practical (usually within 1-2m). Depth, temperature and conductivity were continuously monitored, and internal and external flow meters on the sampler allowed a calibrated calculation of water sampled (Milligan and Riches, 1983; Arnold et ul., 1990). The sampler was fitted with a conical nose cone of 40cm opening, and a nylon net of 270pm aperture. Approximately 400 000 litres were filtered o n a typical station o f 6 0 m depth. Samples were fixed in 4% buffered formalin a n d t h e n transferred to propan-1-2 diol and propylene phenoxetol. In the laboratory all sole eggs and larvae were extracted from the samples. Fig. 5 illustrates the distribution of Stage I eggs o n each survey. These are on average 1-day-old and reflect the location of spawning. The March and early April surveys show the main spawning activity which is largely contained coastwards of 6"W and 51"N. Peak spawning activity is between 5-6"W from just north of Padstow to south of Newquay, with a second, slightly smaller centre, north of Padstow to the latitude of Hartland Point. The centres of production are generally offshore and most of t h e near-shore stations had either none or few eggs. Spawnings outside the main spawning area are small. Fig. 6 shows the distribution of total numbers/m2 of the pelagic larvae and maximum densities were found on the survey covering 17-23 April. The distribution of the larvae is similar to that of the eggs, although the centre of the distributions is about 20 miles to the north, consistent with the flow of the residual currents. However, very few larvae are found to the north of Lundy Island and t h e implications of this are discussed later. The evolution of stratification over the course of the surveys is illustrated in Fig. 7 as plots of Simpson's (1981) integrated, potential energy anomaly 4 (the work that would be required to mix the water column); values above 5-10 J/m' indicate a degree of stratification. N o stratification is seen in Februarv but it is atmarent in March and Atxil.

232

J . IIOKWOOD

7" 0)

6" 0

-

.

.

.

.

'

5"

.

.

4"

.p

.

.

.

D

.

O

.

.

. . .. . . . . .

FIG.5. The distribution of Stage I eggs in 1990 (nosh'): March, c) 1-6 April, d) 17-24 April, e) 18-23 May.

a) 15-22 February, b) 9-13

This early stratification (Figs 7 b d ) is caused by cooler and less saline water, apparently of estuarine origin, overlying warmer and more saline water, although top-to-bottom differences are less than 0.5c". True thermal stratification is seen in Figs 7e-f. Average temperature and surface salinity distributions are shown in Figs 2 and 3. The 1989 surveys were designed to examine the dispersal of eggs and larvae and so only the second half of the 1989 egg production was

THE BRISTOL C H A N N E L

_ * _ . . . 51"

+ +

' .

233

. . . ^ _ I

o d

SOLE ( S O L E A S O L E A (L.))

. .

o .

, l

. . .

"

50"

. .

51".

.

.

-

0

O -

o . j. ,.

.;

:J--

*

. . . . . . . * . . 50"

FIG.6. The distribution o f pelagic larvae in 1990 (nodrn'); a) 15-22 February, b) 9-13 March, c) 1-6 April, d ) 17-24 April, e) 18-23 May. Only two stations had larvae over 23-29 June.

monitored. The dates of sampling were: 5-9 April, 13-16 April, 23-25 May, 6-10 June and 2-3 July and sampling methodology was as described above. The first survey encountered eggs in comparable abundance to those found at the peak of the 1990s surveys (see Figs 16 and 17), and thus probably identified the main spawning locations in 1989 (Fig. 8). The Spatial distribution is very similar to that of 1990. In addition, a few eggs

234

J . HOKWOOD

FIG.7. Distribution of Simpson's stratification parameter (Jim'); a) 15-22 February, b) %13 March. c) 1-6 April, d ) 17-24 April, e ) 18-23 May, f) 23-29 June.

are found west of 6"W and north of Hartland Point, and some sole eggs were also found on the northeasten Nymphe Bank, along with an equal number of eggs of the thickback sole. The location of peak spawning and the extent of spawning, in the Bristol Channel, are similar to those described by Riley et af. (1986), from sampling in February, April and June 1975. They are also consistent with limited unpublished data from March 1953 and 1971.

235

TIIE BRISTOL CHANNEL SOLE ( S O L E A S O L E A ( L . ) )

6"

7"

4"

6"

7"

5"

4"

51" .

l

.

.

O

O

*

.

O

.

f

I

"

r

.

.

0

.

I

D

.

I

.

o

0

0

.

. o o

,

o

.

.

.'.

g

50"

Eggs m-*

0

1

0 10

0 2 0

FIG.8. The distribution of Stage I eggs in 1989 (nosh') from surveys on 5-9 April (a) and 1.1-16 April (b).

It might be anticipated that sole spawn along the southeast coast of Ireland. Sole are caught there, and Riley et al. (1986) and Anon. (1986a) show sole spawning, in varying concentrations, from the southern North Sea, in the Channel, along the west coasts of England and Wales and in the Irish Sea. Walshe (1980) described results from 22 Irish plankton surveys on the Nymphe Bank that spanned from 18 September 1978 to 10 April 1980. The maximum geographical extent covered was of 6-1O"W and 5l0l0'-52"15'N, which was sampled in April and hence was appropriate for finding sole eggs and larvae. The abundance of fish larvae (but not eggs) was given and from all surveys only three sole larvae were found. On the first 1990 survey in the present study (15-22 February) an excursion was made to sample on the Irish side of the St George's Channel but no eggs were found, possibly because of the early date. Sole eggs and larvae are never very common in the plankton, but this paucity indicates that the southeast coast of Ireland does not harbour any major spawning. It is concluded that the spawning of the Bristol Channel sole is discrete and occurs in a consistent location, described above. The centres of spawning are clearly offshore. Those stations that had over 10 Stage I eggsim2, were found in depths of 40-74 m, the highest abundance being in a depth of 50 m. This is contrary to that found for many spawnings in the North Sea, which are close inshore and frequently in the estuaries. However, the Irish Sea spawnings are offshore (Rogers and Symonds, in preparation), those in the Bay of Biscay are over 50 km offshore in waters

236

J . HORWOOD

of 5(L70m depth (Arbault and Boutin, 1968; Arbault et ul., 1986; Koutsikopoulos et al., 1989) and in the Mediterranean, Gulf of Lyons, the sole spawns in waters of 5C100m depth (Farrugio, 1991). The location of the Bristol Channel sole spawning is therefore more typical of t h e species than those of the North Sea. The main spawning areas were predominantly over a sea bed of sand with some gravel (5-30% of gravel grains >200pm, the remainder sand of 62.5-200p.m) or else of gravel with some sand (>30% of grains over 200 pm). Apart from allowing the sole to burrow during the daytime it is not evident that the character of the substratum is relevant to spawning since mating is in mid-water. The area of the sole spawning is common to many other fish species, and it can be assumed that the area is relatively productive in food for larvae. Of particular interest is the problem of how the sole larvae, or metamorphosed sole, reach the nursery grounds. Those of the South Wales coast appear to be the most important locations (see below). The distribution of Stage I eggs, Fig. 5 , is similar to that for Stages 11-IV. The time to the beginning of metamorphosis is about 20-40 days and by that time the sole would need to be near the nursery grounds if the mechanism for transport was passive. The residual currents are described in Section II.A.4, and away from the slightly stronger coastal current the typical M2 tide and wind-driven current is estimated to be 2-4 km/d. The South Wales nursery grounds are some 110 km distant. Such currents may be just adequate to take the pre-metamorphosed sole to near the nursery grounds, but the implication is that the numbers arriving depend upon the contribution of the wind-driven circulation. However, the stronger coastal currents, typically over 4 km/d, appear always likely to transport into the Estuary that proportion of the population that encounters them. Notwithstanding the above, the distribution of larvae (Fig. 6 ) shows that the northern transport in 1990 was not strong, and that very few larvae were found to the north of Lundy Island. The arrest of a northern flow, to the west of Lundy Island, is predicted from the calculated tidal residual currents (Fig. 4). Further investigation is still needed into the diffusion and advection of the sole eggs and larvae, under different wind regimes, to determine more precisely the significance of different winds over the post-spawning period. An initial interpretation is that it would be imprudent of the sole to rely upon the tidal or wind-driven residual currents to transport the premetamorphosed larvae to the major nursery grounds. A similar sentiment was expressed by Koutsikopoulos et ul. (1989, 1991) about the sole of the northern Bay of Biscay; the implications are discussed below (Section 11.C.4).

THE BRISTOL CHANNEL SOLE ( S O L E A S O L E A (L.))

237

C. Juveniles The juveniles are defined as metamorphosed sole, over about 12 mm or 3%50 days, up to the onset of maturity at about 3 or 4 years. 1. Distribution of 0-group sole The 0-group sole are defined as metamorphosed sole that have not experienced a new calendar year; on the first January 1 they become 1-groups, etc. The offshore distribution of immediately post-metamorphosis sole is not known. Sole larvae are uncommon in the plankton and metamorphosed forms even more so. However, the latter are found in high densities on suitable near-shore nursery grounds. The distribution of 0-group sole around the British coasts was determined by Riley et al. (1981, 1986). The fish were sampled with either a 2 m beam-trawl, worked from a small boat, or with a 1.5 m push net worked from the shore; the meshes were of 0.6-1.0cm. Estimates of 0-group abundance were obtained from sample densities averaged by depth band and multiplied by the areas of that depth in the locality. A moderate abundance of 0-groups was found in Barnstaple Bay, Swansea Bay and Carmarthen Bay and a greater abundance was found in the mouth of the Severn Estuary. The relatively high abundances estimated for the Severn Estuary may be exaggerated by the inclusion of areas within depth bands that were unsuitable for the young sole, some regions being tidally scoured to bare rock. Densities were generally lower than on nursery areas of south-east England and in the Irish Sea. The presence of very small sole in the coastal waters of the Severn Estuary and Bristol Channel has been known for many years, from the large catches in the fixed net fisheries and especially in the weirs (Section VI1.A). The presence of 0-group sole, in the upper and lower reaches of the River Severn and the Estuary, is demonstrated by fish trapped on the screens of water inflow pipes to power stations, at Hinkley Point and Uskmouth, and above the Severn Bridge, at Oldbury and Berkeley (Claridge and Potter, 1987; Henderson and Holmes, 1991). Riley et al. (1981) found that 0-group sole were at maximum densities in depths of CL5 m. The densities (1000/m2) by depth band were: 0-6m, 3.85; &lorn, 2.08; 10-20m, 1.19; 2 W O m 0.70. The relative densities with depth vary amongst the flatfish species that inhabit the nursery grounds. For example. the plaice is concentrated even more in the shallow waters, whereas the dab ( L i m a n d a limanda (Linnaeus, 1758)) and t h e solenette have peak densities at 5-10 m depths. Depth per se may be an important factor influencing distribution of young flatfish; 0-group

238

J . 110RWOOD

plaice transported offshore into deeper waters quickly returned to their original depths of capture (Riley, 1973). Depth, however, may simply influence the distribution of essential prey species. Dorel et al. (1991) described the distribution of 0- to 2-group sole in the Bay of Vilaine. Recently recruited O-group sole, in July and September, were strongly concentrated in waters less than 5 m deep, although they were found in waters of 1&40 m in December. Beam-trawl surveys were undertaken for young sole in the Bristol Channel, and although they focused on older age groups some O-groups were caught. The stations fished are shown in Fig. 9 and Table 2 gives the relative density of O-group sole per depth band, averaged over surveys in September 1988 and 1989. Very shallow waters were not trawled and so the highest densities of O-groups were missed. Nevertheless, the results show that the sampled stations in the depth strata &20 and 21-40m housed similar densities of O-groups in September and that these sole are still found in waters of over 40 m depth. Based upon the data of Riley et al. (1986) it is estimated that 2.8 times as many O-group sole are found in depths of &12 m than in 12-20 m. However, in the Bristol Channel there are large areas deeper than 20 m, and if they contain densities similar to those found in 12-20m, as the trawling data suggest, then it is possible that the greatest abundance of O-group sole are to be found in waters over 20 m. At present the data are inadequate to resolve this question. The distribution of O-group sole is associated with low salinities and river estuaries (e.g. Ehrenbaum, 1908; Christensen, 1960; Riley et al., 1981), but the relationship is not precise and sole are found mainly in salinities of 1&33?400. Nursery grounds of sole and plaice were recognized by Riley et al. (1981) as being shallow, with salinities less than 33'Yoo and having a relatively stable sea bed. The substratum was of sand to mud, of particle sizes 4 to 500pm, and the nursery grounds had at least some protection from wave and tide action. However, juvenile sole can be found outside this salinity range and in distinctly muddy environments (Coggan and Dando, 1988; Marchand, 1988). Catches of O-group sole at Hinkley Point power station are from intertidal mud flats. The significance of the substratum for 0- and l-group sole was examined in more detail by Rogers (1992) for Irish Sea nursery grounds. Within a recognized nursery ground on the north Wales coast he found that high densities of sole were associated with a fine-sand of grain diameters Iess than 200pm. High catch rates were not found in areas of coarser sediments, although some areas of apparently suitable sediment size did not yield high catch rates. The high densities were also associated with a high organic content of the substratum. Rogers speculated that the preferred substratum is associated with an acceptable abundance of prey

T H E BRISTOL CIIANNEL SOLE (SOLLA S O L E A ( L . ) )

239

40

20'

51"

FIG.9. Distribution of research beam-trawl stations and o f 1-group sole. Stations shown by dots and densities of sole indicated at t 1 0 caughti30min. (blank), 1&30/30min. (hatched) and >30/30 min. (cross-hatched). a-b: April 1989 and 1990; e-e: September 1988-00.

species and the ability to bury in the sand to avoid predation, but that the Occurrence of apparently suitable sites and low catches indicates that we still lack information on this topic. Dorel er al. (1991) claimed a similar result, with the summer distribution of 0-group on fine and medium muddy sand, but in winter on soft mud.

240

J.

nouwooD

TABLE2. AVERAGERELATIV~: DENSITIES OF CL3 GROUPSOLE, CAUGHTI N BEAM-TRAWL SLJKVEYS I N APRIL A N D SWTEMBER, n Y DEPTHBAND

April

September

Age

@20 m

2140 m

41+ m

1 2 3 0 1 2 3

1.OO 1 .OO 0.48 0.79 1.oo 1 .oo 0.31

0.79 0.32 1.00 0.79 0.73 0.71 0.92

0.05 0.13 0.51 0.30 0.06 0.46 0.71

2. Distribution of l-group sole Catches from the power station inflows at Hinkley Point and Oldbury and from stake nets on Steart Flats in Bridgewater Bay confirm the presence of 1-group sole in the River Severn. A description of the more widespread distribution relies on trawl catches; these cannot be taken very near to the shore and push-net catches may underestimate the abundance of active 1-group sole; consequently the distribution of l-group close inshore is not well defined. The distributions of 1-group sole caught in research beam-trawl surveys in April 1988-89 and September 1988-90 are shown in Fig. 9, and relative densities by depth band are given in Table 2. The overall distributions show a variability of pattern and abundance amongst the surveys, although Carmarthen Bay appears consistently to house a concentration of l-group sole. Within a depth band, there is a similarity between the April and September results, with peak densities in &20 m, a reduction to 80% of the peak in 21-40 m, and to a much reduced density in 41+ m. As described above, the shallower waters within the CL20 m depth band are poorly sampled. Symonds and Rogers (pers. comm.) give additional details. N o 1-group sole were caught on the spawning grounds off Trevose Head in February to April 1990. Dorel et al. (1991) show that, in summer, 1- and 2-group sole in the Bay of Vilaine are in deeper water than the O-group. Reviews of the distribution of young sole in the North Sea, English Channel and Irish Sea are given by Rijnsdorp et al. (in press) and Symonds and Rogers (pers. comm.)

24 1

THE BRISTOL> C H A N N E L S O L E ( S O L E A S 0 I . W (L.))

3. Distribution of 2- and 3-group sole

The research beam-trawl catch data described above revealed a pronounced segregation of young immature sole from the mature sole at spawning time. Fig. 10 shows the distributions of 2-group sole near the nursery areas and away from the spawning ground. Highest densities of 2-group sole in April are still in waters of 0-20m but in September they are more widespread. The 3-group sole, near the nursery areas, are found 5"

4"

5"

4"

40'

20'

51'

40

20

51

Flc;. 10. Distribution of research beam-trawl stations and of 2-group sole. Stations shown by dots and densities o f sole indicated at <10 caughti30 min. (blank). 1(&3O/Xlmin. (hatched) and >30/30 min. (cross-hatched). a-b: April 1989 and 1990: c-e: September 1 98x-90.

242

J . HOKWOOD

in deeper water but are distributed similarly in April and September. On the spawning ground, from February to April 1990, no 2-group females were found and < I % of the males were aged 2. Of 3-group sole, <1% of females were of this age but 29% of males were of age three. Near the nursery areas only a few 3- and 4-group males remained, but 4-group females were only 28% less than 3-group females. Although differences may be masked by variations in year-class size, the relative numbers do not imply a marked developmental migration of 3- and 4-group females, and peak catches of females on the spawning ground were of age 6. The results indicate that at spawning time only mature sole are found on the spawning ground, and that the immature females, generally age 3 4 and less, and immature males, age 2 and less, are elsewhere; details are given in Section 1V.C. Sampling of all juvenile stages has been limited and 0- and l-group distributions are based on sampling in locations where experience has indicated that these reside. As the Welsh coast trawling data show, the l-group distribution may be quite broad and much of ICES Divisions VIIf-g has not been sampled with fine mesh nets. In particular the Nymphe Bank and southeastern Irish coasts have not been fully surveyed. 4. Movements of juveniles Studies on metamorphosing plaice entering the Wadden Sea suggest that they may move to the nursery grounds by selective use of the tidal currents (Creutzberg et al., 1978; Rijnsdorp et aZ., 1985) and it is possible that sole behave in a similar way. It was suggested earlier that a passive transport with the residual currents may be insufficient to carry the pre-metamorphosed sole to the major nursery areas of the South Wales coast. Koutsikopoulos et al. (1989, 1991) also found that a passive drift of eggs and larvae, from the sole spawning in the northern Bay of Biscay, would not take the sole onto the nursery areas of Vilaine and Loire, although once in the Bay of Vilaine onshore currents will help the O-groups reach the coast (Marchand and Masson, 1989). The information from these two spawning groups suggests an active movement by the youngest O-group sole in order to find and remain on the nursery areas. For the Bristol Channel in particular, and for sole more generally, the mechanisms of movement from metamorphosis to appearance on the known nursery grounds are unknown, and this part of the life history may be crucial in determining relative year-class strengths within a population. Creutzberg et al. (1978) discovered that lack of food stimulated pelagic swimming in young 0-group plaice, whereas in regions with food they were benthic and less active. Champalbert and Castelbon (1989) and

THE BKISTOL CHANNEL SOLE ( S O L E A .S O f .EA (L.))

243

Marchand and Masson (1989) confirmed a nocturnal activity in 0-group sole which was triggered by light levels. Macquart-Moulin el al. (in press) and B. Howell (pers. comm.) reported this behaviour pattern in the smallest 0-group sole, but they also found that nocturnal activity was dramatically suppressed by the presence of food. The implication is that the 0-group sole remain in the benthopelagic environment until suitable feeding areas are encountered. Such a mechanism would explain the retention of 0-group sole in nursery areas. It also provides a mechanism for “transport” to the nursery areas as an apparent random walk with a sink. That is, movement may be random but once a location is encountered movement stops; such a scheme would ultimately end with all sole in the one location. This mechanism would not, however, help in circumstances where the advective and random eddy processes were too weak to bring the sole near the nursery grounds in a reasonable time - as may be in the cases discussed above. A combination of selective transport with food acting as a final stimulus to retain sole on the nursery grounds may be speculated as the most plausible mechanism. As described above, the highest densities of 0-group sole are relatively near-shore although the greater abundance may be in deeper water. So far unexamined is the distribution of suitable feeding grounds throughout the outer Bristol Channel, and the distribution of young 0-group sole in relation to potential feeding sites. The timing of recruitment into the nursery areas has not been explored for the South Wales and north Devon coasts; the 0-group surveys of Riley et al. (1981, 1986) were late for this purpose, being in September and October. More detailed data are available from the Severn Estuary. Sole caught on the power station inflows were sampled from 1972 to 1977 by Claridge and Potter (1987). They found low or zero catches in November-February, an increase in March and peak numbers in July-September. The 0-group sole first appear at Hinkley Point in June. Henderson and Holmes (1991) described sole caught at Hinkley Point from 1981 to 1990. They reported 0-group sole from late July with maximum numbers in September. The standard length of the sole in August was 71 mm, and, since fish much smaller would have caught on the screens, the authors concluded that the catches were associated with an immigration from Bridgwater Bay or a movement from the littoral zone. Henderson and Holmes found a reduction in the catches of 0-group during winter and an increase in catches of 1-group in March. They ascribed this to a migration of late 0-group out of the Estuary to return as I-group. From the Wadden Sea, Creutzberg and Fonds (1971) described a seasonal pattern in the density of 0- and 1-group sole in catches. The @group sole migrated out of the Wadden Sea in October, even when

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temperatures were as high as 8"C, to return as 1-group in summer. The 1-group left the Wadden Sea earlier than the 0-group. In the Bay of Vilaine, Dorel et al. (1991) demonstrated the winter offshore movements of 0- and 2-group sole. The results complement those of Marchand (1988), Koutsikopoulos et al. (1989) and Marchand (1991). Such migrations are consistent with the variation in catch rates in the River Severn and the Estuary which experience cool winter temperatures below 7°C. In contrast, a different description of juvenile sole migration was given for the nearby Tamar Estuary, which opens to the western English Channel (Coggan and Dando, 1988). The 0-group sole are found in the Tamar from their smallest size and from April onwards. There does not appear to be the delay experienced at Hinkley Point, although the upstream migration is later as in the upper Severn Estuary. Detailed movements were described through the branding of individual 0- and 1-group sole and trawling was conducted throughout the year. Coggan and Dando found that 0-group sole avoided the mud flats whereas the 1and 2-group sole moved onto the mud flats at high tide to feed; in the Bristol Channel 0-group are taken on the power station screen near mud flats. The main difference, however, is the absence of a migration of 0-groups out of the Tamar Estuary, although they do leave as 1-group in October-November. Generally there was very little movement of tagged sole around the Estuary and between mud flats. Rogers (1992) also reported Iittle movement after settlement on the nursery grounds off north Wales. Further details on the behaviour of individual tagged juvenile sole were given by Lagardere and Sureau (1989) and Sureau and Lagardere (1991). Tagged sole of 19.5-24.5cm were very inactive at 2-5°C but activity increased ten-fold by G9.5"C. This suggests that the late appearance of sole at Hinkley is not due to inactive sole being present (but not caught) since the river temperatures are in excess of 10°C from May. The reasons are unclear for the apparent differences in behaviour amongst the sole from the various locations, but they may be associated with this species' dislike of cold conditions, and to the local environmental variations experienced in the different regions. As yet, however, the seasonal distribution of juvenile sole from the Bristol Channel is inadequately known. The present description of spatial distribution is not precise, sampling is from very few locations and times and detailed information gained from individual sole is sparse.

5 . Juvenile tagging studies A mark-recapture experiment was conducted to examine the dispersion of young sole from the nursery grounds. In June 1988, 1023 sole were

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245

tagged in Swansea Bay with Petersen discs; they were of 15-26 cm length with a modal length of 19cm. In July, a further 826 were tagged in Carmarthen Bay of 14-28 cm length and with a modal length of 21 cm. Subsequent returns showed that most sole were tagged at ages 2 and 3 years, with a few of age 1 and age 4, To June 1991, there had been 127 returns, of which information on dates and positions of recapture were available for 92. The numbers of returns by ICES rectangle are illustrated in Fig. 11. Within the region, the geographical pattern of returns is similar to the distribution of effort and catch rates, described below (Fig. 12), and it particularly reflects the concentration of effort onto the spawning grounds off Trevose Head. Of the 92 returns, 82% were recovered in Divisions V1If-g, and 93% in or near to VIIf-g. Of the returns from outside the area, illustrated in Fig. 11, only one of these is thought to be reliable, and hence the percentages could be higher. Further details are given by Symonds and Rogers (in preparation). Recoveries of 87 sole by age show, for males and females combined, a few caught at age 3, and a major increase at age 4. The more limited data by sex do not show any difference in the pattern of recaptures by sex, and the results suggest a recruitment to the fishery at age 4 years. This complements information on catches from the nursery and spawning

FIG. 11. Marking positions (hatched) and numbers returned by ICES rectangles of sole marked it1 mainly 2-3 years. Arrows indicate six returned from outside thc illustrated region.

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grounds (Section 1V.C) which indicates that females recruit to the spawning fishery at ages 4-5 years, and males 1 year earlier. Large numbers of juvenile sole have been tagged on nursery areas in the North Sea and English Channel (Anon., 1989a; Riley, 1991). Results were analysed from those sole that were of lengths 1&24cm (mainly 2-group) when tagged and that were returned within 4% years after the end of the year of release. Two important conclusions were reached from 3000 recoveries. The first was that recoveries of sole of age 3 or over (assumed to be mature in the study) on the spawning grounds were predominantly from locally tagged juveniles. Generally sole in the nursery area recruited to the local spawning. The second was that the young sole exhibited an onshore-offshore seasonal migration, with offshore movement between mid-August and mid-November and a return in March and April. It was claimed that the typical minimum migration distance was 77 miles. However, analysis is possibly confounded by the seasonal distribution of fishing effort and this could not be corrected for in the study. Less pronounced seasonal movements were found in the fish tagged in the English Channel compared with the North Sea, and this was explained by the warmer waters not forcing a migration due to cold winter conditions. The results from the Bristol Channel also show that the nursery grounds of the South Wales coast supply fish to the local spawning population. Very few migrate large distances, and none were found on other spawning grounds. Only two of 80 sole, tagged as juveniles in the Irish Sea, were returned from the fisheries in VIIf-g (Rogers, pers. comm.), and there have been no returns from sole tagged in other regions.

D. Adults 1. Distribution Close’s Fishermen’s Chart of the English Channel identifies sole grounds between Trevose and Hartland Point, and to the southwest, on the Great Sole Bank and the south of the Cockburn Bank. The sole is buried during the day and it is associated with a sandy substratum. In fact, much of ICES Divisions VIIf-g has a sandy substratum (British Geological Survey, 1987). Sole is a most valuable fish and where it is present in any moderate density the fishing fleet will attempt to catch it. The beam-trawl fleet targets sole but its methods are expensive and without a significant catch

247

T H E BKISTOL CIIANNEL SOLE ( S O L E A S O L E A ( L . ) ) 9" I

8"

7"

6"

5"

4'

3"

9"

8"

7"

6"

5"

4"

3"

52"

5 1"

50"

FIG.12. Catch per hours fishing (kgih) of England and Wales beam-trawlers 1981-85 by quarter of the year. Blank: n o fishing, dash: C-10. hatch: 1C20, cross-hatch: 20+ kg/h.

of sole the beam-trawlers could not profitably operate. Consequently the fishing effort of beam-trawlers reflects the distribution of high densities of the sole. A better measure is the catch per effort expended. Catch per hours fishing by England and Wales beam-trawlers is illustrated in Fig. 12, by quarter of the year, averaged over 1981-85. In all quarters there was virtually no fishing in the southwest, around the Labadie, West and North West Banks. This does not mean that sole are absent there, but catch rates in adjacent localities are lower than those in the north and west. High catch rates, of over 20 kg/h, are found in the east of the region and in October to December, although the highest local catch rates are found on the spawning grounds off Trevose Head in the second quarter. 2. Seasonal patterns The catch rate data show a weak seasonal pattern; in quarters 1-3 catch rates are much higher in the east with a particular high at spawning time, whereas in quarter 4 the high catches are more widespread.

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Fish tagging studies in the North Sea (Anon., 1965; de Veen, 1970) show a rapid movement of sole from feeding and over-wintering grounds to the spawning grounds at spawning time. The aggregation of sole, off Trevose, is thought to be relatively rapid in late spring, but data are not available to quantify or confirm this. The North Sea studies showed that those sole migrate to the shallow coastal waters to spawn and return to deeper waters in summer and autumn. Some southern, North Sea groupings of sole appeared less migratory, and this was ascribed to them living in waters that rarely went below 5”C, and hence did not necessitate a migration. Subsequent tagging studies found, however, that the southern North Sea sole also moved into deeper, northern waters later in the year (Wallace, 1977; Anon., 1989a). A migration into deeper water in winter has also been ascribed to Irish Sea sole (Anon., 1964b). Tagging of Bristol Channel sole has been limited and seasonal migrations cannot be identified from the few returns which are described below. However, the very concentrated spawning location and the extensive distribution of catches of adult sole imply that an annual migration must occur in the Bristol Channel and Celtic Sea, but it may not be related to seasonal changes in temperature.

3 . Mechanisms of migration The ICES working group on sole (Anon., 1965) noted that tagging data showed that sole could move up to 4 miles in 24 h, and remarked “this seems rather much for a fish which is generally regarded as sluggish”. Subsequently it was discovered that several species, including sole, use the tides selectively to aid migrations, a mechanism which has been termed “selective tidal transport” (Greer Walker et al., 1978, 1980; Arnold and Cook, 1984). Research fishing has confirmed that this movement is general for a population. De Veen (1967) also reported extensive observations by fishermen of sole near to the surface of the water and he associated this with a migration. Greer Walker and Emerson (1990) found that in December in the Straits of Dover during night-time, three times as many sole were caught on southerly tides than on northerly tides, whereas in March five times as many were caught on northerly tides; they interpreted this as showing a pre-spawning migration into the North Sea after wintering in the Channel. N o similar work has been undertaken in the Bristol Channel, but such movements can be assumed to occur because of the ubiquitous nature of the mechanism. Nevertheless, the weak tidal currents in the centre and west of the region may pose some interesting problems - can tidal transport be used here?

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249

4. Effects of cold winters Cold winters have a significant effect on adult sole in the North Sea. Wood (1911) described myriads of sole driven into the deep water of the Silver Pits in the winter of 1834, while extensive natural mortality and high catch rates of sole occurred in the cold winters of 1928/29, 1946/47 and 1962/63 (Woodhead, 1964a-c). Woodhead found that during the prolonged cold spell of 1962/63 the diurnal behaviour pattern of the sole changed and h e ascribed this to their torpid state. It appears that if temperatures drop below 3 4 ° C then the sole experiences physiological problems, and temperatures below 2°C cause massive mortalities (Section V). It was reported that the late winter fishery of 1962/63 failed to develop in the Bristol Channel, but that catch rates recovered later in 1963 (Anon., 1964b). The implication was that the winter temperatures had retarded the seasonal migration. In February and March 1963 the water temperatures at Newlyn were 3 4 ° C lower than usual and may well have been below 5°C in some regions, and catches in those months were only 1&20% of the catches in 19.5942. However, the relatively low catches in these months continued for the rest of the decade, and one must consider catch rates rather than absolute catches. In January to March of 1963, catch rates were 7096, 62% and 32% of the averages of the previous 5 years. However, they were 8 4 1 2 0 % of those in the following 5 years. Spring catch rates were slowly falling over the 1950s, and it is not possible to recognize that the spring fishery of 1963 was anything other than part of this decline (Section VII1.E). Catches and catch rates were disappointing compared with those of the previous decade, but there is no evidence to suggest that the sole in the Bristol Channel were affected by the relatively low temperatures. In support of this, it was reported that the sole fishery off Brixham, in 1963, was normal (Anon., 1964b). It is possible, but not established for the Bristol Channel, that the water temperature may affect t h e timing of the spawning migration (Section IV.B), but the monthly catch data are too coarse to detect any such changes.

E. The Bristol Channel “Stock” Ideally a “stock” or population is genetically isolated with no immigration to, or emigration from, its range. This is rarely, if ever, found in marine fish and to a marine fisheries biologist a stock is a grouping of fish that is sufficiently isolated for t h e practical purposes to hand; generally the birth

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and death rates should be much larger than those of immigration or emigration rates. We examine here the validity of this concept to the Bristol Channel sole. The subject was reviewed by ICES, in the context of considering whether the present assessment boundaries (ICES Divisions VIIf-g) were appropriate for the assessment and management of sole in the region (Anon., 1989b, 1991b). No investigation of the genetics of the Bristol Channel sole has been conducted. A comparison of allozyme frequencies of sole from the eastern English Channel and the central Irish Sea showed no heterogeneity between samples at six polymorphic loci (D. Thompson, pers. comm.). No differences were detected by Dando (pers. comm.) in a comparison of enzymes in sole caught from the western English Channel and off the Dutch coast. It is probable that the Bristol Channel sole, located amidst these sampling locations, will not reveal any protein differences at this level of resolution. Movements of adults can be inferred from tagging, and in April 1959, 608 sole were tagged on the spawning grounds off Trevose (Williams, 1963, 1965). In the following 9 years 72 tags were returned, some 68 of these from the Bristol Channel (Horwood and Nicholson, 1991). The locations of tagging and recaptures are given in Fig. 13. Of the other four, two were returned from immediately south of Land’s End, just out of VIIf-g; one off the Irish coast just north of VIIf-g, and the remaining return (from the North Sea) may have been misreported. Although the interpretation of returns is complicated by an uneven distribution of fishing effort, they d o indicate that once adults have recruited to an area they will largely remain there. The dispersion of the 71 fish over 9 years is remarkably small. This relatively small dispersion is thought to be general for adult sole once recruited to a spawning area. The results show a dispersion similar to that of the sole tagged as juveniles (Fig. 11). The tagging data do show a possibility that sole can be recruited from other areas, and that small numbers of sole from the Bristol Channel may immigrate. In order to determine whether the immigration and migration was of any significance for management purposes (rather than having genetical implications), VPA-estimated recruitments (Section VI1I.A) from adjacent management areas of the Irish Sea and western English Channel were inter-correlated (Anon., 1991b). Correlation coefficients (3)were all less than 0.05, indicating that estimated recruitment within each region is largely independent. In conclusion it can be seen that the spawning grounds of the sole in the Bristol Channel and northern Celtic Sea are well defined and discrete. Exclusive nursery grounds exist. A large proportion of sole from the nursery grounds examined, and probably from those not examined,

THE BRISTOL CHANNEL SOLE

( S O L E A SOLEA ( L . ) )

25 1

FIG.13. Positions of 608 adult sole tagged on spawning ground (circled), and of the 72 recaptures. two from outside of the area shown.

recruit to the adult spawning stock of the Bristol Channel. A large proportion of the adult sole, once recruited to the Bristol Channel stock, remain in the region. Few sole appear to recruit into the region. Consequently the sole of the Bristol Channel can be regarded as a valid population for studies of dynamic population demography and for management purposes. However, the extent of the migration range is unclear, and the tagging results indicate that the boundaries of the population may be a little more extensive than those of ICES Divisions VIIf-g.

111. Feeding, Size and Growth Feeding is included with size and growth because the two are obviously related, but more pragmatically because there is material from the Bristol Channel to add to a short A. Feeding

d

Sole larvae feed during the day, finding their food by sight (Blaxter. 1972). They begin to feed well before the end of the yolk-sac stage. Sole larvae reared in natural sea water from the North Sea preferred

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polychaete larvae, copepods and cladocerans (Fonds, 1979). The lengths of prey were typically 10% of the larval length. Last (1978) examined the stomach (not gut) contents of preserved larvae from the Southern Bight of the North Sea and the eastern English Channel. He found that dinoflagellates were important for all sizes of larvae, but before the yolk-sac was absorbed the nauplii of small copepods and lamellibranch larvae were also important. At a larger size, polychaete larvae became the dominant food. Fonds (1979) questioned Last’s identification of the sole larvae, since he had measured larvae less than 2 m m , which is well below the typical length at hatching of 2.5-3.0 mm;