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Potential for mass culture of the estuarine amphipod Eogammarus confervicolus Sharp, Joan Catherine 1980

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POTENTIAL FOR MASS CULTURE OF THE ESTUARINE AMPHIPOD EOGAMMARUS CONFERVICOLUS by JOAN CATHERINE SHARP B.A., M c G i l l U n i v e r s i t y , Montreal, Quebec, 1972 B . S c , M c G i l l U n i v e r s i t y , Montreal, Quebec, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1980 (c) Joan Catherine Sharp, 1980 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f i nanc ia l gain sha l l not be allowed without my writ ten permission. Department of "2-OOLOQ The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS Date P \ r w \ \ 3 Q , R ^ Q i i ABSTRACT The gammarid amphipod Eogammarus confervicolus (Stimpson) was investigated as a p o t e n t i a l mass culture organism, with u t i l i t y as a diet supplement for a r t i f i c i a l l y reared f i s h . Suitable conditions for large-scale culture were determined i n a s e r i e s of experiments. E_. confervicolus demonstrated wide s a l i n i t y and temperature tolerances, with best s u r v i v a l at low s a l i n i t i e s (5 - 10^/00) and temperatures (5 - 10 C). Populati on densities greater than 2 mg/1 reduced amphipod growth and s u r v i v a l , although densities may be increased with a flow-through system. .E. confervicolus showed good growth and s u r v i v a l on a v a r i e t y of algae and associated epiphytes, demonstrating the broad d i e t of the species. Clumping diatoms or phytodetritus were suggested as s u i t a b l e foods for mass culture. Maintenance of populations over three generations showed the f e a s i b i l i t y of long term culture of t h i s amphipod. Short term growth rates of j u v e n i l e coho at 12°C were s i m i l a r on l i v e amphipods (3.2%/day), freeze-dried amphipods (2.4%/day) and Oregon Moist P e l l e t s (3.1%/day). Protein analysis showed E_. confervicolus to have a well-balanced amino acid spectrum, and proximate analysis indicated that the amphipod was a n u t r i t i o n a l l y s a t i s f a c t o r y component of f i s h d i e t s . A L e s l i e matrix model was developed from information about growth, mortality and fecundity of Eogammarus confervicolus under optimal conditions, and was used to test various harvest s t r a t e g i e s . Highest y i e l d of the strategies examined was produced by a weekly 41% harvest applied to amphipods between 0.6 and 2.2 mg dry weight. Further experiments testing the predictions of the L e s l i e matrix model were recommended. i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF FIGURES v LIST OF TABLES v i ACKNOWLEDGEMENTS v i i 1. INTRODUCTION 1 2. DESCRIPTION OF EOGAMMARUS CONFERVICOLUS 5 3. FIELD COLLECTION AND MAINTENANCE 8 3.1 Description of c o l l e c t i o n s i t e 8 3.2 C o l l e c t i o n methods 10 3.3 Maintenance 10 4. REARING CONDITIONS • H 4.1 Introduction 11 4.2 Materials and Methods 11 4.21 Tolerance Experiments 11 4.22 Feeding Experiments 12 4.23 Density Experiments 13 4.24 Long Term Culture 13 4.3 Results 14 4.31 Tolerance Experiments 14 4.32 Feeding Experiments 16 4.33 Density Experiments 22 4.34 Long Term Culture 2 7 4.4 Discussion 31 i v 5. EOGAMMARUS CONFERVICOLUS AS A FOOD FOR FISH 40 5.1 Introduction 40 5.2 Materials and Methods 40 5.21 Chemical Analyses 40 5.22 F i s h Feeding T r i a l s 41 5.3 Results 42 5.31 Chemical Analyses 42 5.32 F i s h Feeding T r i a l s 42 5.4 Discussion 47 6. EOGAMMARUS CONFERVICOLUS HARVEST MODEL 51 6.1 Introduction 51 6.2 Development of Model 51 6.3 Results and Discussion 58 7. RESULTS AND CONCLUSIONS 67 8. LITERATURE CITED 69 V LIST OF FIGURES Page 1. Diagram of adult Eogammarus confervicolus 6 2. Major features of the Squamish estuary 9 3. M o r t a l i t y at various s a l i n i t i e s 15 4. M o r t a l i t y at various temperatures 17 5. Relationship between head length and dry weight 18 6. Growth i n weight and s u r v i v a l on various d i e t s 19 7. Growth i n weight at f i v e experimental de n s i t i e s 24 8. Changes i n numbers of E_. confervicolus i n r e p l i c a t e cultures 29 9. F i r s t generation mortality and changes i n biomass i n r e p l i c a t e cultures 30 10. Frequency d i s t r i b u t i o n s of wet weight of coho before and a f t e r feeding t r i a l s 46 11. Relationship between brood s i z e and dry weight 56 12. Predicted age d i s t r i b u t i o n of harvested f r a c t i o n with 18% harvest applied to a l l age classes 61 13. Predicted age d i s t r i b u t i o n of harvested f r a c t i o n with 40% harvest applied to age classes 0 to 8 63 14. Predicted age d i s t r i b u t i o n of harvested f r a c t i o n with 41% harvest applied to age classes 9 to 17 64 15. Predicted age d i s t r i b u t i o n of harvested f r a c t i o n with 90% harvest applied to age classes >17; 15% harvest applied to age classes 0 to 17 65 v i LIST OF TABLES Page I. Growth rates and mortality c o e f f i c i e n t s f o r E_. confervicolus reared on various diets 20 I I . S ignificance of differences i n pairwise comparisons of mean growth rates on s i x diets 23 I I I . Growth rates and mor t a l i t y c o e f f i c i e n t s f o r E_. confervicolus reared at f i v e experimental densities 25 IV. Significance of differences i n pairwise comparisons of mean growth rates at f i v e d e n s i t i e s 26 V. Growth and mortality c o e f f i c i e n t s and f i n a l d ensities f o r three experimental cultures 28 VI. Proximate composition of amphipod sample 43 VII. Amino acid composition of amphipod sample 44 VIII. Comparison of growth rates and i n i t i a l and f i n a l wet weights and forklengths of coho of three d i e t groups 45 IX. Summary of ANOVA on f i n a l wet weights and forklengths and growth rates of coho on three test d i e t s 48 X. L e s l i e matrix elements 57 XI. Predicted weekly y i e l d from harvest strategies 59 v i i ACKNOWLEDGEMENTS Many people have contributed to the development of t h i s t h e s i s . I am g r a t e f u l to my thesis supervisor, Dr. T. R. Parsons, for h i s encouragement, advice and patience throughout the study. I thank my fellow graduate students, Alan Carruthers and Brenda Harrison, for t h e i r very h e l p f u l comments on the th e s i s . E r i c Woodsworth and Pam Mace provided cheerful assistance i n f i e l d c o l l e c t i o n s , f o r which I am gr a t e f u l . Thanks are also due to J u l i e O l i v i e r a , f or her i d e n t i f i c a t i o n of a l g a l species. I appreciate the assistance provided by researchers at the West Vancouver Laboratory, West Vancouver. Dr. C. D. Levings was very h e l p f u l throughout the project, providing information, equipment and advice. B. Dosanjh gave generous assistance i n laboratory analyses. I would l i k e to thank my thesis committee members, Drs. C. D. Levings and D. McPhail for t h e i r advice and suggestions i n improving the manuscript. F i n a l l y , I acknowledge the f i n a n c i a l assistance of the National Research Council of Canada. 1 1. INTRODUCTION C u l t i v a t i o n of marine or brackish water invertebrates i n large numbers under controlled conditions i s of i n t e r e s t both f o r b i o l o g i c a l research and for a r t i f i c i a l rearing of f i s h . Many researchers have established breeding populations of crustaceans, e i t h e r as a preliminary to commercial mariculture or to provide a continuous supply of animals of known p h y s i o l o g i c a l status f o r further experimentation. Fewer than 2% of marine organisms can be reared through t h e i r e n t i r e l i f e cycle under co n t r o l l e d laboratory conditions (Kinne, 1970). Ryther and Bardach (1968) have summarized the b i o l o g i c a l c h a r a c t e r i s t i c s of organisms lending themselves to intensive c u l t u r e . They should reproduce r e a d i l y i n c a p t i v i t y , and the eggs and juve n i l e s should be hardy and capable of hatching and developing under c o n t r o l l e d conditions. They should demonstrate good growth on a wide v a r i e t y of inexpensive and abundantly a v a i l a b l e foods, and should be tolerant of high density conditions. Further desirable a t t r i b u t e s include wide s a l i n i t y and temperature tolerances and year-round reproduction with high fecundity (Chang and Parsons, 1975). Several steps must be taken p r i o r to establishment of a large scale culture. Foods of sui t a b l e q u a l i t y to allow reproduction and development of the culture organism must be i d e n t i f i e d (Nassogne, 1970). The temperatures and s a l i n i t i e s allowing best s u r v i v a l must be determined (Sastry, 1970). S e n s i t i v i t y of the species to high density, and i t s e f f e c t s on feeding, growth and behaviour should be assessed (Ryther and Bardach, 1968). There i s a need f o r a sui t a b l e organism to be c u l t i v a t e d to serve as a di e t supplement f o r a r t i f i c i a l l y reared f i s h . The brine shrimp, 2 Artemia s a l i n a , i s a v a i l a b l e for t h i s purpose, but at a very high p r i c e . Young hatchery-reared f i s h frequently need l i v e or fresh-frozen invertebrate preparations to stimulate t h e i r appetite before they w i l l accept commercial foods (Walker, i n Fulton, 1976). With d i e t supplements of invertebrate preparations added to the standard hatchery d i e t , Oncorhynchus species have been observed to feed more voraciously and grow more quickly (Brett, 1974). In western Norway, an experimental fi s h e r y has been established f o r the copepod Calanus finmarchicus. Calanus i s used as a d i e t supplement i n salmonid rearing, to increase c o l o r a t i o n of f i s h f l e s h (Heath, 1977). The p o t e n t i a l of' the gammarid amphipod Eogammarus confervicolus (Stimpson) f o r mass culture and i t s u t i l i t y as a d i e t supplement for a r t i f i c i a l l y reared f i s h was explored i n t h i s study. The u t i l i t y of amphipods as stocking organisms i n impoundments and r e s e r v o i r s has been investigated by several researchers. I o f f e (1972) introduced 49 invertebrate species, including 18 amphipod species, into man-made reservoirs and storage lakes i n an attempt to enrich the food supply f o r commercial f i s h . The amphipods established themselves su c c e s s f u l l y and were widely u t i l i z e d by demersal f i s h . Fish production estimates for the Tsimlianskoe storage lake before and a f t e r introduction of invertebrates indicated that increased growth rate and improved production of commercial f i s h species resulted from these introductions. Ivanova and Abrosimova (1975) were able to increase carp production at decreased cost per kilogram of product by stocking f i s h fattening ponds with mysids and gammarid amphipods. Eogammarus confervicolus served as the primary food source f o r coho salmon (Oncorhynchus kisutch) 3 reared i n n a t u r a l enclosures (Powers, 1973). Powers a l s o modelled the growth and s u r v i v a l of the amphipod to assess s u i t a b l e c o n d i t i o n s f o r maximum p r o d u c t i v i t y . The p o s s i b i l i t y of m a r i c u l t u r e of amphipods ha* been discussed by Zebchenko (1975), who recommended the development of methods f o r the mass c u l t u r e of Gammarus b a l c a n i c u s , described as a high q u a l i t y food f o r domestic animals and f i s h . Tenore, Browne and Chesney (1974) set up a polyspecies a q u a c u l t u r e system i n which a Corophium species formed part of a s u c c e s s f u l d e t r i t u s feeding component, u t i l i z i n g pseudo-feces from oyster c u l t u r e . They proposed t h i s amphipodas a food source i n the production of f i s h species such as winter flounder. The present study proposes Eogammarus c o n f e r v i c o l u s as a s u i t a b l e organism f o r mass c u l t u r e , possessing the d e s i r a b l e b i o l o g i c a l c h a r a c t e r i s t i c s o u t l i n e d e a r l i e r and having p o t e n t i a l as a d i e t supplement f o r young f i s h . To assess the s u i t a b i l i t y of the amphipod as a m a r i c u l t u r e organism, i t s s a l i n i t y and temperature tolerances were explored. Amphipods were reared on v a r i o u s foods and food combinations, and the r e s u l t i n g growth and s u r v i v a l were compared. E f f e c t s of d e n s i t y on growth r a t e , m o r t a l i t y and f e c u n d i t y were i n v e s t i g a t e d i n cohorts reared at f i v e experimental d e n s i t i e s . The causes of reduced growth at h i g h density were b r i e f l y explored. R e p l i c a t e c u l t u r e s were maintained f o r three generations ( c l o s e to one year) and demonstrate-the f e a s i b i l i t y of long term c u l t u r e . To assess the p o t e n t i a l u t i l i t y of E l . c o n f e r v i c o l u s as a d i e t supplement f o r f i s h , chemical analyses were performed to determine the composition of amphipod t i s s u e . The short term growth response of 4 j u v e n i l e coho to amphipod preparations was assessed i n feeding t r i a l s . F i n a l l y , i n f o r m a t i o n about growth r a t e , m o r t a l i t y and f e c u n d i t y of amphipods reared w i t h excess food at low de n s i t y was used to construct a L e s l i e matrix model of the amphipod's p o p u l a t i o n dynamics. Various harvest r a t e s and p o l i c i e s were modelled to p r e d i c t the r e s u l t i n g p o p u l a t i o n age s t r u c t u r e and weekly y i e l d . 5 2. DESCRIPTION OF EOGAMMARUS CONFERVICOLUS Eogammarus confervicolus (Stimpson), the most abundant and widely d i s t r i b u t e d amphipod on the North American P a c i f i c coast, i s a member of the newly defined family Anisogammaridae (Bousfield, 1979). The family can be recognized by the peglike spines on the gnathopod palms and the spines on the postero-dorsal surface of the urosome segments. Eogammarus confervicolus, known as Anisogammarus confervicolus before i t s recent r e c l a s s i f i c a t i o n by Bousfield (1979), i s described by Barnard (1954) and Bousfield (1958, 1979). An adult male i s diagrammed i n Figure 1. The head bears two p a i r s of antennae, the f i r s t with an accessory flagellum. The mandibular molars are strong and well-developed, with a cutting edge with two to three teeth. Gnathopods on the f i r s t two pereon segments function i n food handling and as precopulatory grasping appendages. Pereopods 3 to 7 are used for walking and c l i n g i n g . Coxal g i l l s are found on pereon segments 2 to 7. The female marsupium i s formed by brood plates (oostegites) attached to coxal margins 2 to 5. The three pleon segments bear pleopods, used for swimming and g i l l v e n t i l a t i o n . Uropods on the three urosome segments are also used for swimming. Males of E_. conf ervicolus can be recognized by t h e i r larger gnathopods, mature females by the presence of oostegites. Mating i n t h i s species i s t y p i c a l of gammarid amphipods, as described by Hynes (1955). Mating pa i r s are formed following a random encounter between a male and a receptive female (Holmes, 1903), the male using h i s gnathopods to grasp the female's thoracic segments. Amplexus may l a s t f o r up to a week. Copulation follows the female moult, then the male and female separate. 6 Figure 1. Diagram of adu l t Eogammarus c o n f e r v i c o l u s (male) (from B o u s f i e l d , 1979). A, antennae (2 p a i r s ) ; AF, accessory f l a g e l l u m (on f i r s t antennae); G, gnathopods (2 p a i r s ) ; HL, head l e n g t h ; P, pereopods (5 p a i r s ) ; PL, pleopods (3 p a i r s ) ; U, uropods (3 p a i r s ) ; T, t e l s o n . u 7 The female ovulates, releasing her eggs i n t o the marsupium, where they are f e r t i l i z e d and develop u n t i l hatching. The marsupium i s v e n t i l a t e d by the beating of the female's pleopods. Young are released before or during the female's next moult. The female can reproduce at each moult. The newly released young have a dry weight of ca. .02 mg. Sexual dimorphism i s not expressed u n t i l maturity, reached at a dry weight of between 1.1 and 1.2 mg. Common i n B r i t i s h Columbia estuaries and coastal areas with f r e s h -water influence, E. confervicolus i s adapted to low s a l i n i t y habitats, but can survive over a wide range of s a l i n i t i e s (Levings at a l . , 1976). Waldichuk (1969) notes that _E. confervicolus can t o l e r a t e low dissolved oxygen l e v e l s (1.92 mg/1) and can survive for several days at temperatures as high as 20°C. This amphipod i s abundant within the zone of influence of the Port Mellon pulp m i l l , suggesting a high tolerance f o r toxicants from bleached k r a f t m i l l e f f l u e n t (Harger and Nassichuk, 1974). _E. confervicolus i s found i n a s s o c i a t i o n with vascular plants, benthic algae and d e t r i t u s , with f i e l d abundance d i r e c t l y correlated with the presence of a sedge rhizome habitat (Hoos and Void, 1975). Benthic algae and d e t r i t u s are u t i l i z e d as food (Pomeroy, 1977). E-. conf ervicolus i s capable of rapid c o l o n i z a t i o n of new habitats, and i s widely dispersed by the spring freshet. In the c e n t r a l d e l t a of Squamish estuary,where Eogammarus confervicolus i s present i n high abundance, amphipod biomass i s greatest i n A p r i l and May (Levy and Levings, 1976). Juveniles and ovigerous females are present i n the population year round, with ju v e n i l e s predominating i n the early winter months (September to January). 8 3. FIELD COLLECTION AND MAINTENANCE 3.1 D e s c r i p t i o n of c o l l e c t i o n s i t e Amphipods were c o l l e c t e d from the t i d a l f l a t s of the c e n t r a l b a s i n of Squamish estuary (49°41'N, 123°10'W) at the head of Howe Sound, a l a r g e f j o r d i n southern B r i t i s h Columbia (Figure 2). The c e n t r a l b a s i n has r e c e i v e d no d i r e c t freshwater input s i n c e dyke c o n s t r u c t i o n i n 1972 r e s t r i c t e d the flow of the Squamish r i v e r to the west s i d e of the c e n t r a l d e l t a . Water movements i n the c e n t r a l b a s i n are due to t i d a l flows. Surface temperatures i n the c e n t r a l b a s i n ranged from 4.4°C (winter) to 14.7°C i n l a t e summer. Surface s a l i n i t i e s ranged from 30^/00 (winter) to 3.4^/00 ( e a r l y summer) (Levings, 1976B). The middle and upper i n t e r t i d a l zones i n the c e n t r a l d e l t a (1.6 to 4.0 m O.D.) are covered by sedge meadows (Carex l y n g b y e i ) . Benthic algae, i n c l u d i n g diatoms and attached seaweeds ( p r i m a r i l y Enteromorpha and Fucus), grow on l o g s , p i l i n g s , exposed sediment and sedges. The most abundant benthic i n v e r t e b r a t e s i n the i n t e r t i d a l are the amphipod Eogammarus c o n f e r v i c o l u s and the isopod Exosphaeroma oregonensis. E. c o n f e r v i c o l u s i s found p r i m a r i l y i n the middle i n t e r t i d a l zone of the c e n t r a l d e l t a . I t s abundance i s greatest i n sedge rhizomes, which form a refuge at low t i d e (Levings, 1976B). The biomass of E_. c o n f e r v i c o l u s f l u c t u a t e s over the year, w i t h maximum values o c c u r r i n g i n m i d - A p r i l (Levings and Levy, 1976). J u v e n i l e s and mating p a i r s are found throughout the year. Other amphipod species recorded i n the Squamish estuary i n c l u d e Ramellogammarus ramellus (formerly Anisogammarus r a m e l l u s ) , Locustogammarus l o c u s t o i d e s (formerly A. l o c u s t o i d e s ) , Anisogammarus  puge t t e n s i s , Lagunogammarus setosus (formerly Gammarus setosus) and 9 Figure 2. Major fe a t u r e s of the Squamish estuary (courtesy of Dr. C. D. Levings, 1973). 10 Corophium spinicorne, a tube-dwelling amphipod (Levings, 1973). Eogammarus confervicolus i s the major food organism for j u v e n i l e salmonids i n the estuary, and also serves as prey f o r herring, s c u l p i n and flounder (Goodman and Vroom, 1972). 3.2 C o l l e c t i o n methods Amphipods were c o l l e c t e d by h o r i z o n t a l plankton tows at high t i d e . 2 A 0.25 m , 350 ym SCOR/UNESCO net was towed j u s t over the sedge mat. Two other methods of c o l l e c t i n g amphipods were t r i e d . Nylon net bags were f i l l e d with Fucus and suspended from p i l i n g s at the 2.5 m ti d e l e v e l , as described by Levings (1976A). Amphipods swimming i n the water column accumulated i n the bags at high t i d e . A f t e r two to three weeks, bags were c o l l e c t e d and amphipods rinsed from the Fucus. Individual amphipods were c o l l e c t e d by dip net from t i d e pools and by hand from sedge rhizomes and from under debris at low t i d e . These methods proved le s s successful than net tows at providing large numbers of _E. confervicolus. 3.3 Maintenance Amphipods were maintained i n f u l l y aerated f i v e and ten gallon aquaria p r i o r to use. Nylon mesh was provided for cover, and animals were fed excess Enteromorpha p r o l i f e r a . Rhizoclonium served as the maintenance food source during density experiments. Before tolerance experiments, animals were maintained at 10^/00 and 5°C. For a l l other experiments, s a l i n i t y was kept at 7^/00 and temperature at 10°C. The maintenance l i g h t regime was 12:12. 11 4. REARING CONDITIONS 4.1 I n t r o d u c t i o n A major aim of t h i s study was to determine s u i t a b l e c o n d i t i o n s f o r mass c u l t u r e of E_. c o n f e r v i c o l u s . S a l i n i t y and temperature tolerances and growth at d i f f e r e n t d e n s i t i e s were i n v e s t i g a t e d . Growth and s u r v i v a l of amphipods reared on v a r i o u s foods and food combinations were compared. Populations were maintained over three generations to demonstrate the f e a s i b i l i t y of long-term c u l t u r e . 4.2 M a t e r i a l s and Methods 4.21 Tolerance Experiments S a l i n i t y and temperature tolerances of E. c o n f e r v i c o l u s were evaluated i n l a b o r a t o r y experiments. Animals were c o l l e c t e d from the f i e l d i n June and J u l y , 1977 and maintained at 10^/00 and 5°C i n the seven days before experiments were i n i t i a t e d . Healthy j u v e n i l e amphipods of roughly equal s i z e (0.6 to 0.8 mg dry weight) were placed i n i n d i v i d u a l compartments ( 5 x 5 x 5 cm) of a covered p l e x i g l a s s box. Animals were provided w i t h 100 mis of water and excess food (Enteromorpha p r o l i f e r a ) . Photoperiod was 12:12. T r i a l s a l i n i t i e s and temperatures were s e l e c t e d to i n c l u d e the range of values experienced by animals i n the f i e l d . For the s a l i n i t y s e r i e s , f i f t e e n animals were kept i n a 10°C coldroom at each of s i x experimental s a l i n i t i e s : 0, 5, 10, 15, 20 and 25^/00. For the temperature s e r i e s , s a l i n i t y was maintained at 10^/00 f o r each group of f i f t e e n animals. Animals were placed i n c o l d rooms kept at 5°-and 10°C, and i n incubators at.18° and.22°C. , E f f e c t s of i n t e r a c t i o n s between temperature and 12 s a l i n i t y were not i n v e s t i g a t e d . Each week, the amphipods were t r a n s f e r r e d to clean water and s u p p l i e d w i t h f r e s h excess food. Weekly m o r t a l i t i e s were noted. An amphipod was considered dead when no movement was observed d e s p i t e prodding w i t h a glass rod. Experiments were terminated a f t e r four weeks. 4.22 Feeding Experiments To determine d i e t s s u i t a b l e f o r r e a r i n g of Eogammarus c o n f e r v i c o l u s , newly released j u v e n i l e s (.016 mg dry weight) were r a i s e d on s i x foods or food combinations. One food o f f e r e d was Amphiprora paludosa, a benthic diatom forming clumps that can be e a s i l y manipulated by the amphipod. Three a l g a l species abundant i n the Squamish estuary were s e l e c t e d : two green algae, Enteromorpha p r o l i f e r a and Rhizoclonium, and the brown a l g a Fucus, w i t h associated epiphytes. H e r r i n g meal and a combination d i e t of Fucus, _E. p r o l i f e r a and h e r r i n g meal were a l s o t e s t e d . J u v e n i l e s were placed i n i n d i v i d u a l p l e x i g l a s s containers c o n t a i n i n g 100 mis of 7^/00 water, maintained at 10°C w i t h a photoperiod of 12:12. Ten to twelve amphipods were s t a r t e d on each food type. Head le n g t h was measured at weekly i n t e r v a l s and converted to dry weight by the e m p i r i c a l r e l a t i o n s h i p : l o g (dry weight) = 0.2125 + 3.4783 l o g (head length) Animals were returned to c l e a n water w i t h f r e s h excess food f o l l o w i n g measurement. Growth and s u r v i v a l were monitored f o r ten to eleven weeks, when mean s i z e of amphipods reared on three of the d i e t s exceeded minimum adult s i z e (1.15 mg dry weight). 13 4.23 Density Experiments The e f f e c t s of den s i t y on growth, m o r t a l i t y and f e c u n d i t y i n Eogammarus c o n f e r v i c o l u s was explored to determine s u i t a b l e d e n s i t i e s f o r mass c u l t u r e . The amphipods used were newly hatched j u v e n i l e s of known s i z e , released from f i e l d c o l l e c t e d ovigerous females. Cultures were e s t a b l i s h e d at f i v e experimental d e n s i t i e s - 1., 2., 5., 7.5 and 10. mg dry weight/1. The lowest d e n s i t y c u l t u r e was e s t a b l i s h e d w i t h twenty j u v e n i l e s , w h i l e s i x t y j u v e n i l e s were s t a r t e d at each of the other four d e n s i t i e s . Amphipods were maintained at 10°C, wi t h 7^/00 seawater and excess Rhizoclonium. Weekly head length measurements were made of a subsample of ten amphipods from each c u l t u r e . Amphipods were t r a n s f e r r e d to a f r e s h container i n order of s i z e , w i t h i n d i v i d u a l s being s e l e c t e d at reg u l a r i n t e r v a l s f o r measurement of head length. Head length measure-ments were converted to dry weight. The number of amphipods s u r v i v i n g and presence of mating p a i r s and ovigerous females were a l s o noted weekly. Females bearing young were removed from the c u l t u r e and the number of young rel e a s e d was determined. Animals were replaced i n c l e a n water. Water volume was adjusted f o r changes i n t o t a l dry weight of each c u l t u r e , to maintain constant d e n s i t i e s (mg/1). I n i t i a l l y , c u l t u r e s were grown i n gl a s s s t a c k i n g dishes h o l d i n g a maximum volume of 350 mis. As the animals grew, c u l t u r e s were t r a n s f e r r e d to l a r g e g l a s s s t a c k i n g dishes (maximum volume 1800 m i s ) , s m a l l p l a s t i c p a i l s (4 l i t e r s ) and la r g e p l a s t i c p a i l s (10 l i t e r s ) . 4.24 Long Term Cult u r e D u p l i c a t e E. c o n f e r v i c o l u s c u l t u r e s were e s t a b l i s h e d , and growth, 14 mortality and reproduction of the amphipod populations were followed f o r one year. Each culture was kept i n a 2.5 1 cylinder made of p l e x i g l a s s , (R) with open ends covered with 35 ym N i t e x ^ mesh. The cylinders were suspended i n an InstantOcean ® aquarium f i l l e d with 7^/00 seawater, maintained at 10°C. R e c i r c u l a t i o n of water within the aquarium kept water flowing through the cyl i n d e r s . F i f t y newly released E. confervicolus were placed i n each c y l i n d e r and supplied with twisted nylon net for cover. Each cohort was fed d a i l y with equal amounts of the benthic diatom Amphiprora paludosa. This diatom forms clumps that can be e a s i l y manipulated by the amphipod. Approximately 18.5 mg C were supplied to each c y l i n d e r d a i l y . The Amphiprora was grown as a u n i a l g a l culture i n one l i t e r f l a s k s i n a cold room maintained at 15°C. Each day, 325 mis were removed from each f l a s k , and the culture r e d i l u t e d to 650 mis with Enriched Seawater (McLachlan, 1973). Weekly observations were made to note the presence of mating pa i r s or the release of young i n the cyl i n d e r s . Each month, amphipods i n each cylinder were measured and sexed. Cultures were followed f o r one year, allowing time f o r three separate generations. 4.3 RESULTS 4.31 Tolerance Experiments i . S a l i n i t y Tolerance M o r t a l i t y of Eogammarus confervicolus at various s a l i n i t i e s i s i l l u s t r a t e d i n Figure 3. _E. confervicolus tolerates a wide range of s a l i n i t i e s (from 5 to 25^/00) with maximum s u r v i v a l at 5^/00 and 10^/00. 15 Figure 3. M o r t a l i t y i n E. c o n f e r v i c o l u s at v a r i o u s s a l i n i t i e s . 0°/oa 2 WEEKS 20% 25°/oO " • 15°/oo • 1 6 The species shows poor s u r v i v a l i n f r e s h water, i i . Temperature Tolerance Low temperatures ( 5 ° - 1 0 ° C ) proved optimal f o r s u r v i v a l of E. c o n f e r v i c o l u s (Figure 4). Although short term temperature e l e v a t i o n s could be t o l e r a t e d without high m o r t a l i t y , low temperatures would be most s u i t a b l e f o r long term maintenance. 4.32 Feeding Experiments A l i n e a r r e l a t i o n s h i p i s demonstrated i n the l o g a r i t h m i c p l o t of head length vs dry weight f o r Eogammarus c o n f e r v i c o l u s , shown i n Figure 5. (Data from Dr. C. D. Levings, West Vancouver Laboratory, West Vancouver). Growth and s u r v i v a l of E. c o n f e r v i c o l u s v a r i e d w i t h food type. Weekly values f o r mean s i z e and number s u r v i v i n g f o r each d i e t are given i n Figure 6. Since 1 0 0 % m o r t a l i t y ended the h e r r i n g meal experiment a f t e r ten weeks, only the f i r s t ten weeks were used to c a l c u l a t e the growth rates and m o r t a l i t y c o e f f i c i e n t s l i s t e d i n Table I. Growth c o e f f i c i e n t s (%/day) were c a l c u l a t e d as the c o e f f i c i e n t s of d a i l y exponential growth k according to the equation: wt = w/fc ( M u l l i n and Brooks, 1 9 6 7 ) , where W^  = dry weight at time t WQ = i n i t i a l dry weight ( . 0 1 6 mg) t = time i n days Li n e a r r e g r e s s i o n of l o g e dry weight on time was c a l c u l a t e d f o r each food type, to o b t a i n the values f o r the growth c o e f f i c i e n t k. Growth r a t e s (mg/wk) were c a l c u l a t e d by d i v i d i n g the increase i n dry 17 Figure 4. M o r t a l i t y i n E. c o n f e r v i c o l u s at v a r i o u s temperatures. 17a 18 Figure 5. R e l a t i o n s h i p between head length and dry weight f o r Eogammarus conf e r v i c o l u s (Dr. CD. L e v i n g s ) . (Base 10 logarithms) 19 Figure 6. Growth i n weight and s u r v i v a l of E. confervicolus raised on various d i e t s . Mean dry weight, 95% confidence l i m i t s and number surviving given for each week. W E E K S 1-40-1-00-B. Herr ing M e a l E •60-1 UJ >-Q • 20-0 12 10 9 — i 1— 4 5 W E E K i — 6 i 3 T 3 ,3 1 8 9 10 11 C. Enteromorphg p r o l i f e r g 1-4Ch 1-OCH 0 1 2 3 4 5 6 7 8 9 10 11 W E E K S F. Rhizoclonium 1 - 4 0 -1 - 2 0 -" T r 1 1 1 1 1 1 1 i i 1 0 1 2 3 4 5 6 7 8 9 1 0 11 1 2 W E E K S Table I. Growth rates and mo r t a l i t y c o e f f i c i e n t s f o r Eogammarus confervicolus reared on various d i e t s . A. Amphiprora paludosa B. Herring meal C. Enteromorpha p r o l i f e r a D. Fucus E. Choice F. Rhizoclonium Days (to adult size) 75 93 75 80 88 Growth c o e f f i c i e n t (k) Growth rate Mortality c o e f f i c i e n t ( k f ) %/day %/day .063 .025 .051 .063 .059 .054 mg/wk .082 .005 .040 .079 .076 .056 0. .001 .035 .001 .008 21 weight of i n d i v i d u a l amphipods over ten weeks by the number of weeks. Mean values for growth on each food type are presented i n Table I. M o r t a l i t y c o e f f i c i e n t s (%/day) were calculated as k' from the expression: -k 't N F C = N^e , where = number surviving at week ten NQ = i n i t i a l number t = 70 days Exponential growth c o e f f i c i e n t s k were used to predict days to adult s i z e - 1.15 mg dry weight, the weight of the smallest ovigerous female observed. Diets A - Amphiprora paludosa - and D - Fucus and associated epiphytes - provided good growth (k = .063 %/day) and low mortality (k' between 0. and 0.001 %/day). Amphipods reared on Rhizoclonium (Diet F) and Enteromorpha (Diet C) grew more slowly {k = .054 (F); .051 (C)}, but s t i l l showed low m o r t a l i t y . Herring meal (Diet B) was an unsatisfactory food. Although amphipods did feed on the clumped meal, growth was very poor (k = .025 %/day) and the meal did not support growth to adulthood. High m o r t a l i t i e s were noted with diets B - herring meal alone, (k' = 0.035 %/day) - and E - choice of Fucus, Enteromorpha p r o l i f e r a and herring meal, (k' = .008 %/day). This suggests that i n c l u s i o n of herring meal i n diet E increased the mortality of animals otherwise provided with a s a t i s f a c t o r y d i e t allowing good growth (k = .059%/day). The assumption of homoscedasticity of growth rates (mg/wk) of the experimental d i e t groups (calculated to week 9) was evaluated with the F test (Sokal and Rohlf, 1969). As variances of the s i x groups were max 22 not s i g n i f i c a n t l y d i f f e r e n t , an analysis of variance was performed using i n d i v i d u a l growth rates. The Student-Neuman-Keuls procedure (Sokal and Rohlf, 1969) was used to evaluate s i g n i f i c a n c e of differences between means. Results of pairwise comparisons of mean growth rates are summarized i n Table H> showing l e v e l of s i g n i f i c a n c e of differences between each pair of means. 4.33 Density Experiments Figure 7 shows weekly values for mean s i z e of E_. conf ervicolus reared at f i v e d e n s i t i e s - 1.0, 2.0, 5.0, 7.5 and 10.0 mg dry weight/1. Amphipod growth rates decreased with increasing density, with good growth at the two lowest densities - 1.0 and 2.0 mg/1. Only the f i r s t twenty weeks were used to c a l c u l a t e the growth rates and mortality c o e f f i c i e n t s l i s t e d i n TableIII,due to the removal of large numbers of ovigerous females from low density cultures i n subsequent weeks. Growth (k) and mortality ( k ' ) c o e f f i c i e n t s and mean growth rates (mg/wk) were calculated as previously described (Section 4.32). The time taken by each cohort to reach a mean s i z e of 1.15 mg i s given as 'days to adult s i z e ' . Growth of E_. conf ervicolus decreases with increasing density, while m o r t a l i t i e s increase. After homogeneity of variances was established (Section 4.32), an ANOVA was performed on the growth rates (mg/wk), using the SNK procedure to evaluate s i g n i f i c a n c e of differences between means. Results of pairwise comparisons of mean growth rates i n Table IV show the l e v e l of s i g n i f i c a n c e of differences between each p a i r of means. Growth at 1.0 and 2.0 mg/1 were not s i g n i f i c a n t l y d i f f e r e n t . However, amphipods showed s i g n i f i c a n t l y 23 Table I I . S i g n i f i c a n c e of d i f f e r e n c e s i n p a i r w i s e comparisons of mean growth ra t e s of Eogammarus c o n f e r v i c o l u s on s i x d i e t s . H e rring meal Enteromorpha Rhizoclbnium Choice Fucus  Enteromorpha * Rhizoclonium * ns Choice * ns ns Fucus ** * ns ns Amphiprora ** ** * ns ns * P < . 0 5 * * P < . 0 1 24 Figure 7. Growth i n weight of Eogammarus c o n f e r v i c o l u s reared at f i v e experimental d e n s i t i e s . Mean dry weight and 95% confidence l i m i t s given f o r each week. 24a 24c E - 10.0 mg I WEEKS IS Table III. Growth rates and m o r t a l i t y c o e f f i c i e n t s of Eogammarus confervicolus reared at f i v e experimental d e n s i t i e s . Density p ays Growth coefficient ( k ) Growth rate M o r t a l i t y c o e f f i c i e n t ^ ' ) (to adult size) %/day mg/wk %/day 1.0 mg/1 86 .034 .19 .003 2.0 mg/1 84 .031 .17 .004 5.0 mg/1 122 .028 .09 .007 7.5 mg/1 123 .027 .08 .007 10.0 mg/1 125 .026 .06 .009 26 Table IV. Significance of differences i n pairwise comparisons of mean growth rates of Eogammarus confervicolus at f i v e experimental d e n s i t i e s . 2.0 mg/1 5.0 mg/1 7.5 mg/1 10.0 mg/1 1.0 mg/1 2.0 mg/1 5.0 mg/1 7.5 mg/1 ns •k* ns ns ns * P < .05 ** P < .01 27 reduced growth at the three higher d e n s i t i e s - 5.0, 7.5 and 10.0 mg/1. The factors causing reduced growth and increased mortality of E_. confervicolus at high d e n s i t i e s were b r i e f l y investigated i n three cultures, each started with 48 newly released j u v e n i l e s . Two cultures -A and B - were set up i n 2.5 1 p l e x i g l a s s cylinders, suspended i n an accumulation of and excretory products, and depletion of 0^. Culture A was provided with excess food (Rhizoclonium). Twisted nylon net was supplied f o r cover, reducing i n t e r a c t i o n s between amphipods. Culture B was supplied with excess Rhizoclonium only. Culture C was set up i n a p l a s t i c p a i l , also with 2.5 1 of water. Excess food was supplied, and water was changed every two weeks. No attempt was made to maintain constant density during the experiment. Cultures were maintained f o r 95 days. Growth and mo r t a l i t y c o e f f i c i e n t s (%/day) and f i n a l d e n s i t i e s are given i n Table V. T tests (Sokal and Rohlf, 1969) were used to assess s i g n i f i c a n c e of differences between mean growth c o e f f i c i e n t s (k) i n the three cultures. Amphipods i n container C showed slowest growth and highest mortality; reduction of growth was highly s i g n i f i c a n t (P < .01) i n comparisons with growth i n cultures A and B. Growth of amphipods i n cultures A and B was not s i g n i f i c a n t l y d i f f e r e n t , but mortality was increased i n container B. Mean dry weights of amphipods a f t e r 95 days - 1.26 mg (A) and 1.17 mg (B) - were very close to those of amphipods reared at low dens i t i e s - 1.27 mg at 1.0 mg/1; 1.32 mg at 2.0 mg/1. 4.34 Long Term Cultures Data are presented i n Figures 8 and 9 for the two r e p l i c a t e amphipod aquarium, with r e c i r c u l a t i n g water flow preventing 28 Table V. Growth and m o r t a l i t y c o e f f i c i e n t s and f i n a l d e n s i t i e s f o r Eogammarus c o n f e r v i c o l u s reared i n three experimental c u l t u r e s . C u l t u r e Growth c o e f f i c i e n t (fc) %/day M o r t a l i t y c o e f f i c i e n t ) %/day F i n a l d e n s i t y mg/1 .044 .006 .13.7 .043 .008 10.5 .032 .012 2.6 29 Figure 8. Changes i n numbers of Eogammarus confervicolus i n r e p l i c a t e cultures. 30 Figure 9. F i r s t generation mortality and changes i n biomass i n r e p l i c a t e Eogammarus confervicolus cultures. 31 populations i n which growth, mortality and reproduction were monitored for 340 days. Three separate generations of Eogammarus confervicolus were followed over t h i s period. Figure 8 shows the changes i n amphipod numbers i n the two cylinders over the year. The f i r s t appearance of mating pa i r s and the f i r s t release of young i s noted for each generation. A l l adults of the previous generation died before t h e i r o f f s p r i n g reached maturity, so no interbreeding between generations occurred. Generation time i n the populations was approximately 150 days. Figure 9 shows the changes i n t o t a l biomass i n the two cylinders over the course of the experiment. M o r t a l i t y of the f i r s t generation amphipods i s also i l l u s t r a t e d . Biomass of the amphipod population reaches a maximum as the f i r s t and second generation amphipods reach maturity. The maximum biomass f o r the f i r s t generation coincides with the minimum mortality values f o r t h i s generation. Highest m o r t a l i t i e s are seen as ju v e n i l e amphipods compete for a l i m i t e d food resource, with, m o r t a l i t y values decreasing as amphipods grow to maturity. A second increase i n mortality i s seen a f t e r mating and release of young, as large males cannibalize moulting females and compete with abundant juv e n i l e s f o r food. 4.4 DISCUSSION Eogammarus confervicolus has wide s a l i n i t y and temperature tolerances, with best s u r v i v a l at low s a l i n i t i e s (5 - 10^/00) and temperatures (5 - 10°C). This species would be w e l l able to t o l e r a t e f l u c t u a t i o n s i n s a l i n i t y ( e s p e c i a l l y increased s a l i n i t y ) and temperature 32 in culture. Levings et a l . (1976), investigating survival of E_. conf ervicolus in seawater and bleached kraft m i l l effluent at various temperatures and s a l i n i t i e s , found maximum juvenile survival at 8^/00, with adults surviving best at 16^/00. He also noted that f i e l d observations show E. confervicolus i s adapted to low s a l i n i t i e s , and i s more abundant and widespread in the brackish Squamish estuary than in the more marine Cowichan estuary (Levings, 1976A). Pomeroy and Levings (1980) reared Eogammarus confervicolus at various temperatures and s a l i n i t i e s , and found survival and growth was best at 15^/00. A salinity of 10^/00 would be most suitable for large scale culture of this amphipod. Moderate fluctuations about this value would have l i t t l e effect on survival. Temperature plays an important role in determining growth rate as well as survival of E . confervicolus. Temperature tolerances reported here agree with those of Levings et a l . (1976), who found that low temperatures (3° - 10°C) provided optimum conditions for survival of E. confervicolus. Again, this corresponds with the f i e l d distribution of the species. Surface temperatures in the Squamish estuary range from 4.4* to 14.7°C (Levings 1976B) . Pomeroy and Levings report best growth of E_. confervicolus at 10°C. Thus 10°C is the most suitable temperature for culturing Eogammarus confervicolus, combining high survival with good growth. In feeding experiments, _E. confervicolus showed good growth and low mortality with the clumping benthic diatom Amphiprora and with partially decayed Fucus. With the green algae Rhizoclonium and Enteromorpha, 33 growth was slower but mortality remained low. Herring meal was an unsatisfactory food for _E. confervicolus, despite i t s high n u t r i t i v e value f o r f i s h . Amphipods ate i t , but demonstrated poor growth and high mort a l i t y . Martin (1966) reports that Marinogammarus species r e a d i l y ingested undigestible materials, and concludes that n u t r i t i v e value of foods seems of l i t t l e importance i n food s e l e c t i o n . Results of the feeding experiments demonstrate the wide d i e t of Eogammarus confervicolus. A number of a l g a l species appear s u i t a b l e for rearing t h i s amphipod. C h a r a c t e r i s t i c b a c t e r i a , micro-algae and protozoans associated with each a l g a l species are also used as a food source by the amphipod, and may be important i n determining growth. Pomeroy and Levings (1980) reared _E. confervicolus on a number of d i e t s , and mentioned epiphytes as a major food source. The high growth rate demonstrated with Fucus may be l a r g e l y due to epiphytic growth. Watanabe (pers. comm.) reported that Fucus provides shelter for Lagunogammarus setosus but i s not used f o r food. Epiphytes serve as the main food source f o r many amphipod species. Hargrave (1970A) reported that growth of H y a l e l l a azteca was proportional to the amount of m i c r o f l o r a i n the d i e t . He also reported high (> 50%) a s s i m i l a t i o n e f f i c i e n c y of n a t u r a l l y occurring epiphytes on elm leaves, but poor digestion of the leaves themselves (Hargrave, 1970B). Gammarus  pseudolimnaeus prefers leaves colonized by micro-algae, and has much higher a s s i m i l a t i o n e f f i c i e n c y with t h i s d i e t than with leaves alone (Barlocher and Kendrick, 1975). Untreated Zostera are preferred by Gammarus oceanicus to those treated to remove epibionts (Harrison, 1977). Harrison re f e r s to the work of other authors (Harrison, 1977 c i t i n g 34 Fenchel, 1970, 1973; Hargrave, 1970 and Kristensen, 1972) showing that amphipods have high a s s i m i l a t i o n e f f i c i e n c i e s (50 - 90%) of the micro-organisms attached to Zostera. These workers have suggested that amphipods have a r e l a t i v e l y poor a b i l i t y to digest the plant's s t r u c t u r a l carbohydrates. Epiphytes are also important i n the die t s of Gammarus p a l u s t r i s (Gable and Croker, 1977) and Gammarus pulex (Moore, 1975), among others. Texture of food may be important i n determining i t s a c c e p t a b i l i t y and d i g e s t i b i l i t y . Martin (1966) noted that Marinogammarus accepted a wide v a r i e t y of fresh plant material, e s p e c i a l l y s o f t - t i s s u e d membranaceous or filamentous algae. However, t h i s amphipod preferred decaying food, perhaps due to the softening action of micro-organisms. Pomeroy and Levings (1980) found texture was important i n determining the p a l a t a b i l i t y and ease of manipulation of algae by E. confervicolus. They reported best growth with the filamentous, e a s i l y manipulated Enteromorpha l i n z a and P y l a i e l l a l i t t o r a l i s . Pomeroy (1977) investigated the food preferences of Eogammarus confervicolus, o f f e r i n g starved, f i e l d - c o l l e c t e d amphipods a choice of decayed sedge shoots, d e t r i t u s and several species of macroalgae. Amphipods preferred to feed on filamentous algae ( P y l a i e l l a l i t t o r a l i s and Enteromorpha minima) with some feeding on t h a l l o s e forms.(Monostrema oxyspermum). Carex shoots were lea s t preferred, and were acceptable only when greatly decayed. In the experiments reported here, amphipods grew best on the e a s i l y manipulated diatom Amphiprora and s o f t , decaying Fucus. Enteromorpha p r o l i f e r a -with coarse t h a l l i - gave reduced growth. Pomeroy and Levings (1980) reared E. confervicolus at 4°C and 10°C 35 on a v a r i e t y of plant foods. Experiments were started with two week old j u v e n i l e s , and continued for twelve weeks. E. l i n z a gave best growth and s u r v i v a l - .103 mg dry weight/wk; k' = .002. Good growth and low mort a l i t y were found with Porphyra species (growth rate = .057 mg/wk; k' = .005), P y l a i e l l a l i t t o r a l i s (.058 mg/wk; .005), the diatom Navicula (.045 mg/wk; .010) and Carex lyngbyei (.039 mg/wk; .004). Growth rates reported by Pomeroy and Levings would be expected to be somewhat higher than those reported here, as experiments were run longer (12 vs 10 weeks) and started with older animals (two week old vs newly released j u v e n i l e s ) . E. l i n z a provided considerably higher growth rates than any foods reported here. Other growth rates are quite s i m i l a r to those observed i n these experiments. Pomeroy and Levings 1 r e s u l t s again demonstrate the wide range of foods acceptable to E. confervicolus. Chang and Parsons (1975) reared Anisogammarus pugettensis on a v a r i e t y of plant and animal foods. Good growth was obtained with Enteromorpha species, diatoms (Pseudonitzchia species) and frozen f i s h . Growth at 10°C on Enteromorpha was higher than that reported for E_. conf ervicolus - . 2 mg dry weight/wk. Willoughby and S u t c l i f f e (1976) measured the growth rate of Gammarus pulex, kept at 15°C on a number of foods - elm and oak leaves, M o l i n i a and several fungal and a l g a l species. High growth rates - .16 mg dry weight/wk - were obtained with decaying oak and elm leaves. As these experiments were st a r t e d with juveniles of .3 to 1.7 mg dry weight, growth rates cannot be compared to those reported here. It has been demonstrated that a wide v a r i e t y of plant foods are suita b l e f or the rearing of Eogammarus confervicolus. Best growth i s provided by the diatom Amphiprora and decaying Fucus with associated 36 micro-organisms. These two foods can be suggested as s u i t a b l e f o r mass amphipod c u l t u r e . The former can be grown i n a continuous c u l t u r e using an enriched medium pumped i n t o the amphipod c u l t u r e , as described by Parsons and Bawden (1979). Brown and Parsons (1972) have i n v e s t i g a t e d the production of p h y t o d e t r i t u s - clumps of sedimented phytoplankton w i t h a s s o c i a t e d b a c t e r i a and organic slime - i n an impoundment flushed w i t h seawater. A f l u s h i n g r a t e of 100%/day was found to y i e l d maximum production of phytoplankton, w i t h the formation of l a r g e amounts of sedimented m a t e r i a l . Clumps of a l g a l c e l l s and b a c t e r i a could be e a s i l y manipulated by _E. c o n f e r v i c o l u s , and would be expected to provide good growth. In d e n s i t y experiments, amphipods showed good growth and low mort-a l i t i e s at 1.0 and 2.0 mg/1. Growth slowed and m o r t a l i t i e s increased at higher d e n s i t i e s , although c u l t u r e s were supplied w i t h excess food. Amphipods r e a r e d . i n low d e n s i t y c u l t u r e s took s l i g h t l y longer to reach a d u l t s i z e than amphipods reared i n i s o l a t i o n i n feeding experiments. This r e d u c t i o n i n growth r a t e even at low d e n s i t i e s suggests that the crowding e f f e c t may be associated w i t h amphipod i n t e r a c t i o n s , perhaps through increased a c t i v i t y or disturbance of feeding. Wilder (1940) stud i e d the e f f e c t s of de n s i t y on growth, f e c u n d i t y and m o r t a l i t y i n H y a l e l l a azteca. U n l i k e t h i s study, she found increased growth, f e c u n d i t y and s u r v i v a l at intermediate d e n s i t i e s . However, growth and f e c u n d i t y were i n h i b i t e d and m o r t a l i t y increased at high d e n s i t i e s . Food was hot su p p l i e d i n excess, and may have been l i m i t i n g at high d e n s i t i e s . Wilder suggests accumulation' of carbon d i o x i d e and 37 excretory products, depletion of oxygen and reduced food and space may have played a r o l e i n i n h i b i t i n g growth i n her cultures. In the experiment reported i n Table V, amphipods provided with flowing water (Containers A and B) showed good growth, despite the high densities reached by the end of the experiment. The water flow prevented 0^ depletion and waste accumulation i n the containers. Addition of netting (Container A) to reduce contact had l i t t l e e f f e c t on growth, but reduced mortality somewhat. This suggests that cannibalism - frequently observed i n t h i s species - may be an important source of m o r t a l i t y . More extensive experiments w i l l be necessary before firm conclusions can be drawn about the causes of reduced growth and increased m o r t a l i t y at high d e n s i t i e s . The increased m o r t a l i t i e s , reduced growth and delay i n reaching maturity observed at high d e n s i t i e s suggest that low d e n s i t i e s w i l l be best f o r mass culture. Densities can be increased i n a w e l l flushed system. Further experiments w i l l be necessary to determine maximum densities under such conditions. Three separate generations of Eogammarus confervicolus were observed i n both r e p l i c a t e cylinders i n the long term culture experiment. The growth of these cohorts had d i f f e r e n t c h a r a c t e r i s t i c s than the growth of cohorts supplied with excess food i n the low density cultures. Increased generation time was noted i n the food-limited cohorts, with the f i r s t release of young occurring a f t e r 150 days. Young were f i r s t released i n the low density cohorts 105 days a f t e r cultures were established. Comparison of growth rates a f t e r 120 days shows reduced i n d i v i d u a l growth i n the long term cultures. Growth rates of 0.13 mg dry 38 weight/week i n long-term cultures were s i g n i f i c a n t l y lower than growth rates i n the low density cultures - 0.15 mg/wk for the cohort reared at 2.0 mg/1 and 0.19 mg/wk for the cohort reared at 1.0 mg/1. F i n a l l y , a considerable increase i n i n d i v i d u a l m o r tality was noted i n the food l i m i t e d cultures. Long term cultures had a combined mortality of 0.013 %/day, compared to a combined mortality of 0.002 %/day i n the low density cultures. These comparisons demonstrate the importance of maintaining adequate food supplies i n mass culture. E. confervicolus populations maintained i n long-term culture had a 150 day generation period. Biomass peaked when each generation of amphipods reached maturity (Figure 9). For the f i r s t generation, t h i s biomass maximum coincided with minimum mortality values. In the curve showing f i r s t generation mortality, high i n i t i a l m o r t ality occurred when juveniles competed f or a li m i t e d food supply. M o r t a l i t y values decreased as amphipods grew to maturity, then increased again a f t e r mating, when cannibalism increased. M o r t a l i t y also increased a f t e r young were released, when adults competed f or food with the abundant j u v e n i l e s . Unlike Daphnia, which adjusts to food l i m i t a t i o n by regulation of b i r t h and growth rates without increased m o r t a l i t y (Frank, B o l l and K e l l y , 1957), a l l three population parameters are al t e r e d by food l i m i t a t i o n i n Eogammarus confervicolus. Parsons and Bawden (1979) followed the growth of four populations of Anisogammarus pugettensis for 130 days i n continuous culture. Amphipods were kept i n 2.5 1 cylinders suspended i n an Instant Ocean aquarium with a r e c i r c u l a t i o n system. Amphiprora paludosa was supplied d a i l y i n four d i f f e r e n t volumes, pumped into the amphipod cy l i n d e r s . Population 39 growth i n the cylinder receiving the highest volume of a l g a l culture -14.5 mgC/day - demonstrated many of the same trends noted i n the experiment reported here. A c y c l i c f l u c t u a t i o n i n amphipod numbers was observed, with mortality of the f i r s t generation continuing u n t i l replacement by the second generation. Again, population biomass maximized as the f i r s t generation reached maturity. N i l s s o n (1977) set up cultures of Gammarus pulex, following the growth and m o r t a l i t y of a series of cohorts produced by f i e l d - c o l l e c t e d females. He found that mortality of juveniles was greatest i n the f i r s t four weeks a f t e r hatching. Similar observations were made by Bamstedt and Matthews (1975) on the copepod Euchaeta norvegica. M o r t a l i t y of the young stages (nauplius and copepodite I - IV) balanced growth of i n d i v i d u a l s . No population growth occurred before the moult to stage V. This experiment demonstrates the importance of supplying adequate food to amphipods i n mass culture. Food l i m i t e d populations are characterized by increased generation time, reduced i n d i v i d u a l growth rates and increased i n d i v i d u a l mortality. 40 5. EOGAMMARUS CONFERVICOLUS AS A FOOD FOR FISH 5.1 Introduction Hatchery reared f i s h have been observed to feed more r e a d i l y and grow more e f f i c i e n t l y i f t h e i r d i e t includes l i v e or fresh-frozen invertebrates (Fulton, 1976). Walker ( i n Fulton, 1976) reports that the sandlance Ammodytes hexapterus would not begin to feed on commercial food u n t i l i t s appetite was stimulated by frozen zooplankton. Zoo-plankton supplements added to commercial feeds caused Oncorhynchus species to feed more voraciously (Brett, 1974). To assess the p o t e n t i a l u t i l i t y of E. confervicolus i n f i s h d i e t s , i t was necessary to obtain information about the chemical composition of the amphipod and the growth response of f i s h to amphipod preparations. 5.2 Materials and Methods 5.21 Chemical Analyses The crude protein, l i p i d and ash content of samples of Eogammarus  confervicolus were analysed. Amino acid analysis was also c a r r i e d out. Amphipods used for these determinations were j u v e n i l e s , c o l l e c t e d from the f i e l d i n May, 1978. Amino acid analysis of frozen E. confervicolus was performed under contract by AAA Laboratory, Mercer Island, Washington. Analysis followed hydrolysis for twenty-four hours i n 6 N HC1 at 110°C. This method does not give quantitative recovery of cystine or tryptophan. Proximate analysis of a sample of j u v e n i l e E. confervicolus was kindly supplied by B. Dosanjh of the West Vancouver Laboratory, West Vancouver. Protein determination was performed by autoanalyser; l i p i d was 41 extracted In chloroform:methanol (1:2 by volume). Ash was determined by combustion at 600°C for two hours. For d e t a i l s of l i p i d extraction, r e f e r to the Association of O f f i c i a l A g r i c u l t u r a l Chemists (1960) manual of analysis. 5.22 F i s h Feeding T r i a l s Growth of coho salmon (Oncorhynchus kisutch) on two amphipod preparations was compared to growth on a commercial d i e t . One hundred and sixty-two j u v e n i l e coho were kind l y supplied by Andy Lam, West Vancouver Laboratory, West Vancouver. F i s h were divided into equal groups and placed i n three 200 1 f i b e r g l a s s tanks, kept i n the courtyard of the Biosciences B u i l d i n g at the Un i v e r s i t y of B r i t i s h Columbia. Feeding t r i a l s were ca r r i e d out during July, 1977. I n i t i a l fork length and wet weight values were estimated by sampling 23 f i s h from each tank. The tanks were aerated, and the f l u s h i n g rate was adjusted to maintain a r e l a t i v e l y constant temperature. Mean surface temperature i n the tanks was 11.9°C, with a range of 10° to 13°C. Surface temperatures i n the three tanks were i d e n t i c a l each day. The three f i s h d i e t s included two amphipod preparations - freeze-dried and l i v e amphipods - and a control d i e t of Oregon Moist P e l l e t s . The p a r t i c l e s i z e of the three diets was standardized. Live amphipods, freeze-dried amphipods and p e l l e t s offered to coho a l l passed through a #16 U.S. Standard Sieve (1.19 mm mesh) and were retained on a #30 sieve (.595 mm mesh). Live amphipods selected i n t h i s manner ranged i n s i z e from 0.2 mg to 0.7 mg dry weight. A f i x e d d a i l y r a t i o n f o r each d i e t was set i n excess of expected 42 growth requirements (Parsons and LeBrasseur, 1968). The r a t i o n provided was 25% of i n i t i a l body weight/day, calculated on an equivalent dry weight bas i s . The d a i l y r a t i o n was 14 - 16% of the f i n a l body weight of the f i s h . F i s h were fed once a day for 26 days. A f t e r one day's starvation, to allow f i s h e s ' guts to c l e a r , f i s h were s a c r i f i c e d . Fork length (to nearest .1 cm) and wet weight (to nearest .01 mg) were determined for each f i s h . 5.3 Results 5.31 Chemical Analyses Proximate composition of a sample of j u v e n i l e Eogammarus confervicolus i s shown i n Table VI. TableVII gives the amino acid composition (on % dry weight basis) of a batch of frozen amphipods. E s s e n t i a l amino acid requirements (based on values determined f o r chinook salmon f i n g e r l i n g s ) are also given, converted to % dry weight for a d i e t with 48.6% protein (Mertz, 1972). 5.32 F i s h Feeding T r i a l s Results of the experiment comparing growth of j u v e n i l e coho on two amphipod preparations and Oregon Moist P e l l e t s are summarized i n Table VIII. Growth rate i s calculated as mg wet weight/day and as % body weight/day. The frequency d i s t r i b u t i o n s i n Figure 10 i l l u s t r a t e the i n i t i a l and f i n a l wet weight d i s t r i b u t i o n s of coho i n the feeding t r i a l s . An ANOVA showed no s i g n i f i c a n t differences i n i n i t i a l wet weights of samples taken from the three tanks, and these samples were pooled to give the i n i t i a l 43 Table VI. Proximate composition of Eogammarus c o n f e r v i c o l u s sample. Values expressed as % dry weight. Component % Dry Weight Ash 28.6 Fat 10.3 P r o t e i n 48.6 44 Table V I I . Amino a c i d composition of f r o z e n Eogammarus c o n f e r v i c o l u s sample. Amino A c i d Alanine A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d G l y c i n e H i s t i d i n e I s o l e u c i n e Leucine Lysine Methionine Phenylalanine Tyrosine P r o l i n e Serine Threonine Tryptophan V a l i n e % Dry Weight 2.86 3.36 5.08 Not determined 6.63 2.90 1.18 2.21 3.63 2.80 1.28 2.52 1.92 2.22 2.47 2.24 Not determined 2.56 Requirement (% Dry Weight) 2.92 0.83 1.21 1.90 2.43 0.73 (with adequate c y s t i n e ) 2.48 (phenylalanine & t y r o s i n e ) 1.09 0.24 1.55 c a l c u l a t e d from amino a c i d requirements of chinook salmon f i n g e r l i n g s (values as % d i e t a r y p r o t e i n ) quoted i n Mertz (1972) Values converted f o r d i e t w i t h 48.6% p r o t e i n . Table VIII. Comparisons of mean d a i l y growth rates and i n i t i a l and f i n a l wet weights and forklengths of j u v e n i l e coho of three d i e t groups i n feeding t r i a l . INITIAL Forklength (cm) Wet weight (g) FINAL Forklength (cm) Wet weight (g) GROWTH RATE Mg wet weight/day % body weight/day Live Amphipods Oregon Moist P e l l e t s Freeze-dried Amphipods Mean S.D. Sample Size Mean S.D. Sample Size Mean S.D. Sample Size 6.7 0.9 24 3.25 1.24 24 8.0 1.0 31 6.25 2.45 31 6.6 1.0 24 3.29 1.58 24 7.8 1.2 28 5.89 2.97 28 7.0 1.0 23 3.84 1.59 23 7.7 1.0 31 5.38 2.24 31 0.15 3.2% 0.09 31 0.14 3.1% 0.11 28 0.11 2.4% 0.09 31 46 Figure 10. Frequency d i s t r i b u t i o n of wet weight of j u v e n i l e coho (Oncorhynchus kisutch) before and a f t e r feeding t r i a l s on A. Live amphipods, B. Oregon Moist P e l l e t s and C. Freeze-dried amphipods. INITIAL FINAL A. Live amphipods 18-? 16-814-12-LL 0 10-€L 8-u CD 6-1 4-Z 2-0 1 2 3 4 5 6 7 81 6-4 -2-1 B. Oregon moist pellets C. F reeze dried amphipods 3 4 5 6 7 8 9 1011 12 13 WET WEIGHT (gm) 47 wet weight frequency d i s t r i b u t i o n s . Table IK shows the re s u l t s of ANOVA tests f o r s i g n i f i c a n c e of differences between treatments f o r f i n a l wet weights, f i n a l fork lengths and d a i l y growth rates. Although coho feeding on l i v e amphipods had a mean growth rate of 0.15 mg/day vs 0.14 mg/day for Oregon Moist P e l l e t s and 0.11 mg/day for freeze dried amphipods, no s i g n i f i c a n t treatment e f f e c t s were noted. Within group v a r i a t i o n was high. In freeze-dried amphipod and O.M.P. feeding groups, the standard deviation approached the mean growth rates. 5.4 Discussion Proximate analysis of the composition of E_. confervicolus (Table VI) shows the amphipod to be n u t r i t i o n a l l y adequate to support the growth of coho salmon. Protein (48.6% dry weight) i s within the salmonid dietary requirement at 10°C of 40 to 50% of the r a t i o n dry weight (Mertz, 1969). L i p i d s make up an adequate 10.3% of the sample. Fat c a l o r i e s are r e a d i l y d i g e s t i b l e , and serve to spare p r o t e i n i n the r a t i o n . Ash values are high at 28.6%. The sample analysed was composed of young j u v e n i l e amphipods. Ash content of amphipod samples c o l l e c t e d i n Squamish estuary shows seasonal f l u c t u a t i o n s . Lowest ash content i s found i n summer months, when large numbers of ovigerous females are present (C. D. Levings, pers. comm.). Carbohydrate (12.5% of sample by difference) i s at an allowable dietary l e v e l . D i g e s t i b l e carbohydrate up to l e v e l s of 25% i n the die t are as e f f e c t i v e an energy source as f a t for many f i s h species (Cowey and Sargent, 1979). Eogammarus confervicolus supplies a favorable balance.of e s s e n t i a l 48 Table IX. Summary of ANOVA on f i n a l wet weights and forklengths and growth rates of coho on three test d i e t s . Source of V a r i a t i o n df SS MS F i n a l wet weight treatment 2 12.04 6.02 0.93 ns within treatment 84 542.19 6.45 F i n a l forklength treatment 2 1.64 0.82 0.69 ns within treatment 84 98.78 1.18 Growth rate treatment 2 0.022 0.011 1.22 ns within treatment 84 0.796 0.009 49 amino acids, and i s s u i t a b l e as a source of dietary protein. A decrease i n the t o t a l dietary protein requirement of f i s h i s noted with foods with a good balance of the e s s e n t i a l amino acids (Mertz, 1972). In growth experiments, j u v e n i l e coho fed r e a d i l y on l i v e _E. confervicolus. F i s h were i n i t i a l l y reluctant to feed on the freeze-dried preparation, which f l o a t e d on the surface of the tank. Af t e r one week, they became accustomed to t h i s product and fed r e a d i l y . Due to high v a r i a b i l i t y i n f i s h s i z e , no s i g n i f i c a n t differences i n growth rate were noted between d i e t s . However, i t i s clear that l i v e and freeze-dried E. confervicolus are acceptable to j u v e n i l e coho and produce good short-term growth. Good long-term growth of trout fed exclusive on amphipods has been reported by Surber (1935) and Pentelow (1939) . Surber fed brook trout (Salvelinus f o n t i n a l i s ) and rainbow trout (Salmo gairdneri) l i v e Gammarus  fas c i a t u s . A f t e r f i v e months the f i s h remained healthy, with more b r i l l i a n t c o l o r a t i o n than normal hatchery or wild trout. Brown trout (Salmo trutta) fed e x c l u s i v e l y on Gammarus pulex for up to 595 days showed no evidence of dietary d e f i c i e n c y . Pentelow concludes from h i s experiments that G_. pulex i s a very e f f i c i e n t food for trout. Gammarus l a c u s t r i s serves as the primary food for rainbow trout cultured i n small aquaculture ponds i n Manitoba (Mathias et al., 1977). The growth rates (% body weight/day) obtained i n t h i s experiment can be compared to values reported for other j u v e n i l e salmonids feeding on invertebrate preparations. LeBrasseur (1969) fed chum salmon (Oncorhynchus  keta) an excess d i e t of l i v e Calanus plumchrus at 14° to 15°C, and reported a growth rate of 5.7%/day. His experiment was started with 0.45 50 g wet weight j u v e n i l e chum. Growth of salmon on t h i s copepod i s higher than that reported here for E_. confervicolus, a f i n d i n g which i s p a r t i a l l y a t t r i b u t a b l e to the increased temperatures i n LeBrasseur's experiment. Juvenile chum ( s t a r t i n g weight - 0.405 g) showed a much lower growth rate on excess frozen JZ. plumchrus. This f i n d i n g i s a t t r i b u t e d by Fulton (1976) to rupture of the carapace and subsequent loss of body l i p i d s of the copepod. Heath (1977) obtained growth rates of 2.7%/day and 3.8%/day when feeding j u v e n i l e coho ( i n i t i a l weight - 0.29 g) on excess amounts of frozen and freeze-dried Euphausia p a c i f i c a at 9°C. Despite the lower temperature, these are quite s i m i l a r to the values of 3.2%/day and 2.4%/ day reported here on l i v e and freeze-dried _E. confervicolus. Results from chemical analyses and coho feeding experiments show Eogammarus confervicolus to be a n u t r i t i o n a l l y s a t i s f a c t o r y constituent of f i s h d i e t s , r e a d i l y acceptable to coho and providing good short-term growth. 51 6. EOGAMMARUS CONVERVICOLUS HARVEST MODEL 6.1 Introduction An important aspect of the investigation of the suitability of mass culture of Eogammarus confervicolus as a food for fi s h i s the determination of the maximum harvest that can be regularly obtained from the population. Analysis of the population dynamics of the amphipod can be useful in determining the optimum harvest strategy. Simulation modelling has been used as a management tool by Powers (1973). He investigated the survival and growth of Eogammarus confervicolus under various salinity and temperature regimes. This information was used to model the population dynamics of the amphipod i n a salmon culture pond. Powers used his predictions to suggest management policies designed to maximize availability of amphipods to salmon. By manipulations of pond salinity and amphipod population size, Powers hoped to reduce growth rate of amphipods, i n order to increase the period of time that they are subject to predation by salmon. In this study, information obtained about the growth rates, mortalities and fecundities of individuals under optimum density conditions were used as elements i n a Leslie matrix. This matrix model predicts the stable population structure and weekly yield resulting from various harvest policies. 6.2 Development of Model Leslie (1945, 1948) proposed a deterministic, discrete time matrix model, which describes a growth of a population having discrete age groups with age-specific properties. Age-specific mortalities and 52 fecundities are represented by elements i n a matrix A, which i s m u l t i p l i e d by a column vector a^ of female population age structure at time t, to predict age structure at time t + 1. The model i s : A a t = a t + l or f0 f l f2 Ln-1 n-1 — — nt,o nt+i,o n t , l n t + l , l n t , 2 n t + i , 2 ; n t , 3 n t + l , 3 n _ t+l,n_ where: f ^ (i=0,l,2 n) = fecundity of females i n the i t h age group ( i . e . the number of females i n age group 0 at t+1 born to each female i n the i t h age group at time t) p^ (i=0,l,2 n) = p r o b a b i l i t y that a female i n the i t h age group at time t w i l l survive to the ( i + l ) t h age group at t+1 n^ = number of females i n age group i at time t A l l other matrix elements are zero. The largest p o s i t i v e eigenvalue A of A has a corresponding eigenvector with a l l elements non-negative. When a stable age d i s t r i b u t i o n has been 53 reached, p o p u l a t i o n growth can be described by the equation: A v = Xv where X , the dominant eigenvalue, describes the change i n po p u l a t i o n s i z e per u n i t time and v, the corresponding eigenvector, describes the st a b l e age d i s t r i b u t i o n . A can be r e l a t e d to r , the i n t r i n s i c r a t e of n a t u r a l increase of a p o p u l a t i o n , by the equation: r = In A . I f the dominant eigenvalue X of the L e s l i e matrix i s greater than one, the po p u l a t i o n can withstand a maximum s u s t a i n a b l e harvest r a t e of 100(^ ^ ) percent, i f a p p l i e d e q u a l l y over a l l age c l a s s e s . This e x p l o i t a t i o n may be enhanced when only one age group i s harvested, as the production of the e x p l o i t e d group increases r e l a t i v e to the production of the other age groups (Williamson, 1967). This t h e o r e t i c a l r e s u l t i s supported by the experiments of Watt (1955), who harvested v a r i o u s stages of T r l b o l i u m confusum, and by Slobodkin and Richman (1956), who removed newborn Daphnia p u l i c a r j a . Both s t u d i e s found an increase i n r e l a t i v e abundance of the e x p l o i t e d group. Usher (1972) has reviewed developments of the L e s l i e model: i n c l u s i o n of both sexes (Williamson, 1959), c o n s i d e r a t i o n of s i z e s t r u c t u r e ( L e f k o v i t c h , 1965) and density-dependance of matrix elements (Pennycuick et a l . , 1968). The simple L e s l i e m a t r i x model gives the r a t e of increase of a po p u l a t i o n under unvarying c o n d i t i o n s , w i t h u n l i m i t e d space and no i n t r a s p e c i f i c d e n s i t y e f f e c t s . This model has r e s t r i c t e d a p p l i c a b i l i t y , as Beddington and Taylor (1973) d i s c u s s . I t i s s u i t a b l e f o r a po p u l a t i o n i n which e x p l o i t a t i o n keeps the po p u l a t i o n d e n s i t y constant. In an e x p l o i t e d p o p u l a t i o n , the L e s l i e matrix can be used to determine at what 54 r a t e v a r i o u s age c l a s s e s should be cropped i n order to maximize s u s t a i n a b l e y i e l d . The elements f o r the L e s l i e m a t r i x were obtained from the combined s t a t i s t i c s f o r s u r v i v a l and f e c u n d i t y of the amphipod cohorts reared f o r 26 weeks at 1.0 and 2.0 mg/1 i n d e n s i t y experiments. Data f o r males and females were combined u n t i l week 18. S u r v i v a l data f o r weeks 19 to 25 were obtained f o r females only. Since mean s i z e , s u r v i v a l and number of ovigerous females were determined weekly i n these c u l t u r e s , one week i n t e r v a l s were used i n the L e s l i e m atrix. Weekly percent s u r v i v a l f i g u r e s were used as p. elements i n the matrix: where = p o p u l a t i o n s i z e i n week i . These values provided p^ elements f o r weeks 0 to 25. A new s u r v i v a l element was added to the m a t r i x , representing the p r o p o r t i o n of females remaining i n the l a s t age c l a s s each week. This age c l a s s i n c l u d e s a l l females o l d e r than 25 weeks. In weeks 24 to 26, a l l females i n the c u l t u r e s were i n mating p a i r s or bearing eggs or young. I t was assumed that m o r t a l i t y i n these females was l a r g e l y a s s o c i a t e d w i t h reproduction - cannibalism by the male f o l l o w i n g the female moult, or death of the female f o l l o w i n g r e l e a s e of young. Thus the element representing weekly s u r v i v a l of females o l d e r than 26 weeks was assumed to be i d e n t i c a l to the s u r v i v a l values obtained f o r the previous two weeks. Figure 11 shows the l i n e a r r e g r e s s i o n l i n e (Model I I regression) f i t t e d to data obtained on brood s i z e vs female dry weight i n f i e l d -55 c o l l e c t e d Eogammarus c o n f e r v i c o l u s (Data from Dr. C. D. Levings, West Vancouver Laboratory, West Vancouver). Despite the very wide s c a t t e r of p o i n t s , a t - t e s t (Snedecor, 1956) showed the slope to be s i g n i f i c a n t l y d i f f e r e n t from zero (P < .05). The v a r i a t i o n may be due to d i f f e r e n c e s i n r e a r i n g c o n d i t i o n s experienced by these females. S e n s i t i v i t y of growth r a t e to d i e t and d e n s i t y c o n d i t i o n s has been demonstrated i n previous experiments. A r e g r e s s i o n of brood s i z e on female dry weight obtained from females reared i n c o n d i t i o n s determined f o r mass c u l t u r e would be more s u i t a b l e f o r c a l c u l a t i n g the L e s l i e m a t r i x elements. Incubation time - days from s e p a r a t i o n of mating p a i r u n t i l r e l e a s e of young - was measured at 10°C. Incubation times v a r i e d from 16 to 19 days, with a mean value of 17 days. Weekly values f o r the f r a c t i o n , a^, of females bearing eggs or young were obtained from the low density c u l t u r e s . Fecundity of females of each age c l a s s (F^) was c a l c u l a t e d w i t h the brood size:female dry weight r e g r e s s i o n equation (Figure 11), using the mean dry weight of amphipods i n each age c l a s s . F_^  values were d i v i d e d by two, assuming a 1:1 sex r a t i o i n broods. f. values were thus c a l c u l a t e d as: 1 2.43 where 2.43 i s the i n c u b a t i o n time i n weeks at 10°C. Table X gives a l l f^ and p^ elements f o r the L e s l i e m atrix. Values f o r mean dry weight of each age c l a s s are a l s o given. Weekly harvests were simulated by s u b t r a c t i n g a harvest term - h -from the s u r v i v a l elements - p_^  - f o r each age c l a s s . Thus, harvest terms can be a p p l i e d to a l l amphipods i n the p o p u l a t i o n or to s e l e c t e d age c l a s s e s . Eigenvalues and corresponding eigenvectors can be c a l c u l a t e d f o r each 56 Figure 11. R e l a t i o n s h i p between brood s i z e and dry weight f o r Eogammarus c o n f e r v i c o l u s females, w i t h 95% confidence l i m i t s . (Dr. C. D. Levings) 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. DRY WEIGHT (mg) 57 Table X. Leslie matrix elements for Eogammarus conferviculus. Fecundity elements (fjO and survival elements (pp are given for each age class. An accumulation element is given for age class 26 A l l other matrix elements are zero. Mean dry weight i s given for each age class. Week i i Mean dry weight(mg) 0 0. .98 .016 1 0. .98 .03 2 0. .99 .04 3 0. .96 .05 4 0. .98 .07 5 0. .99 ' .11 6 0. 1.0 .19 7 0. .99 .36 8 0. .97 .50 9 0. 1.0 .62 10 0. .96 .71 11 0. .99 .87 12 0. .99 1.04 13 9. 1.0 1.15 14 1.8 .97 1.47 15 3.0 .98 1.70 16 3.3 .97 1.95 17 4.7 .97 2.16 18 5.1 .95 2.46 19 5.6 .98 2.81 20 6.6 .95 2.98 21 10.5 .95 3.53 22 12.7 .94 3.98 23 14.2 .90 4.15 24 15.3 .88 4.47 25 15.9 .88 5.10 26 16.4 .88 5.40 58 modified L e s l i e matrix. In each case, the dominant eigenvalue A describes the weekly change i n population s i z e , while i t s corresponding eigenvector describes the stable age d i s t r i b u t i o n . Harvest s t r a t e g i e s can be selected to give A values of 1 . 0 0 , providing a sustainable y i e l d with constant population s i z e . I t i s u n r e a l i s t i c to harvest Eogammarus confervicolus populations by age classes, as these cannot be distinguished i n a mixed population. I t i s , however, a r e l a t i v e l y simple matter to separate amphipods by s i z e classes. These can be equated with age classes with some inherent error due to the v a r i a b i l i t y i n s i z e of each age c l a s s . A further s i m p l i f i c a t i o n was introduced to the model by assuming equal harvest of males and females i n the population. For c a l c u l a t i o n of weekly y i e l d (mg/wk), a stable age d i s t r i b u t i o n and t o t a l population dry weight of 2 0 0 0 mg was assumed. Y i e l d (Y) i s calculated as: Y = Ih..2n..dw. . 1 X 1 l where h = harvest applied to i t h age group, n^ = number of females i n age group i , dw_^  = mean dry weight of females of age class i 2Zn..dw. = 2 0 0 0 mg . 1 1 i 6.3 Results and Discussion Table XI summarizes the r e s u l t s of various harvesting strategies applied to the Eogammarus confervicolus population. The unexploited population has a dominant eigenvalue of 1 . 2 2 , i n d i c a t i n g an exponential 59 Table XI. Predicted weekly y i e l d from various harvest p o l i c i e s . Weekly harvest Dominant Eigenvalue(A) Y i e l d (mg/wk) No harvest 1.22 18% of a l l age classes 1.00 362 40% of age classes 0 - 8 •001 - .5 mg) 1.00 158 41% of age classes 9 - 1 7 (.5 - 2.2 mg) 1.00 436 90% of age classes 18 - 26 (. >2.2 mg) and 15% of a l l other age classes 1.00 382 60 increase i n p o p u l a t i o n s i z e i n the absence.of h a r v e s t i n g . The p o p u l a t i o n could s u s t a i n a maximum weekly harvest of 100 - ( ^ — ^ 5 P . ) or 18%, i f a p p l i e d e q u a l l y over a l l age c l a s s e s . Next, the L e s l i e m a t r i x was modified by t h i s harvest r a t e and the eigenvalues and eigenvectors were r e c a l c u l a t e d . The new dominant eigen-value i s 1.00, i n d i c a t i n g a s t a b l e p o p u l a t i o n s i z e , as expected. With a s t a b l e p o p u l a t i o n s i z e of 2000 mg dry weight, t h i s harvest s t r a t e g y gives a weekly y i e l d of 362 mg, i n c l u d i n g amphipods of a l l s i z e s . The p r e d i c t e d s t a b l e age d i s t r i b u t i o n f o r t h i s p o p u l a t i o n shows a decrease i n amphipod numbers w i t h each subsequent age c l a s s . However, maximum biomass i s found at ages 7 to 18 weeks, as amphipods reach m a t u r i t y and s t a r t to reproduce. Figure 12 shows the p r e d i c t e d age d i s t r i b u t i o n f o r the harvested f r a c t i o n of the pop u l a t i o n . T o t a l numbers and dry weights of amphipods are given f o r each harvested age c l a s s . Equal harvest of a l l age c l a s s e s produces a good weekly y i e l d , w i t h maximum biomass obtained from the middle age c l a s s e s (weeks 8 to 14). A wide range of s i z e s i s represented i n the harvested f r a c t i o n of the amphipod p o p u l a t i o n . With the harvest a p p l i e d only to young amphipods - age c l a s s e s 0 to 8 - an e x p l o i t a t i o n r a t e of 40% per week can be sustained by the popu l a t i o n . Although the number harvested i s h i g h , the y i e l d i n dry weight i s considerably reduced, to 158 mg/week. The p r e d i c t e d s t a b l e age d i s t r i b u t i o n under these c o n d i t i o n s shows a great decrease i n amphipod numbers w i t h i n c r e a s i n g age i n age cl a s s e s 0 through 8. Uniform numbers with, i n c r e a s i n g biomass are seen i n the higher age c l a s s e s , w i t h increased numbers and maximum biomass i n the l a s t age c l a s s . With no harvest of reproductive a d u l t s , a high r a t e of production of young i s 61 Figure 12. P r e d i c t e d age d i s t r i b u t i o n of harvested f r a c t i o n w i t h 18% harvest a p p l i e d to a l l age c l a s s e s . fcleu 0 5 10 15 20 W E E K S 62 found. However, the dry weight y i e l d i s very low. Figure 13 shows, the p r e d i c t e d age d i s t r i b u t i o n of harvested amphipods. Only a narrow range of s m a l l s i z e s (.02 to .5 mg) i s represented i n the harvested f r a c t i o n , with, highest numbers obtained from the youngest age c l a s s e s . With e x p l o i t a t i o n r e s t r i c t e d to age cl a s s e s 9 to 17, the po p u l a t i o n can s u s t a i n a harvest r a t e of 41% per week. This s t r a t e g y produces an increased y i e l d of 436 mg/week. The s t a b l e age d i s t r i b u t i o n p r e d i c t e d f o r t h i s p o p u l a t i o n shows maximum numbers i n the lower, u n e x p l o i t e d age c l a s s e s , w i t h maximum biomass between 6 and 13 weeks. Biomass shows a second increase i n the f i n a l age c l a s s . In Figure 14, the p r e d i c t e d age d i s t r i b u t i o n of the harvested amphipods, a l a r g e range of s i z e s (0.6 to 2.2 mg) i s seen to be included i n the harvest. Maximum numbers and biomass values are obtained from the younger age cl a s s e s i n the harvest. High harvest r a t e s l i m i t e d to amphipods older than 17 weeks d i d not reduce the dominant eigenvalue of the modified m a t r i x to 1.00. With a 90% harvest of amphipods l a r g e r than 2.2 mg, a r e l a t i v e l y l a r g e s u s t a i n a b l e harvest r a t e of 15% could s t i l l be imposed on a l l other age c l a s s e s . This s t r a t e g y gives a s l i g h t increase i n y i e l d (to 382 mg/week) over the p o l i c y of equal 18% harvest of a l l age c l a s s e s . The p r e d i c t e d s t a b l e age d i s t r i b u t i o n shows a steady decrease i n numbers w i t h i n c r e a s i n g age, w i t h maximum biomass at age c l a s s e s 6 to 17. Numbers and biomasses of age cl a s s e s 18 and up are very low. Figure 15 shows the p r e d i c t e d y i e l d f o r t h i s s t r a t e g y , demonstrating the la r g e s i z e range i n c l u d e d i n t h i s harvest. Maximum biomass i n the harvested f r a c t i o n i s obtained from age c l a s s 18. The best harvest s t r a t e g y i s the 41% weekly harvest of amphipods i n the age cl a s s e s 9 to 17. This p o l i c y produces the best y i e l d - 436 mg/ 63 Figure 13. P r e d i c t e d age d i s t r i b u t i o n of harvested f r a c t i o n w i t h 40% harvest a p p l i e d to age c l a s s e s 0 to 8. N U M B E R c (7» O o o o O c. M G DRY W E I G H T 64 Figure 14. P r e d i c t e d age d i s t r i b u t i o n of harvested f r a c t i o n w i t h 41% harvest a p p l i e d to age c l a s s e s 9 to 17. 1800 1600 1400 1200 1000 M l CO ZD z 800 600 400 200 6«y ow {4Kof a ge c lasses 9 - 17 ) ^ , 8 0 160. 65 Figure 15. P r e d i c t e d age d i s t r i b u t i o n of harvested f r a c t i o n w i t h 90% harvest a p p l i e d to age c l a s s e s >17; 15% harvest a p p l i e d to age c l a s s e s 0 to 17. 6 & 1 8 0 0 v 15°/ o f a g e c l a s se s 0 - 1 7 1600 1400 1200 1000 LU CO 800 600 400 200 ^50% of a g e classes > 17 v 180. 160. 140. 120. 100. 80. 60. X O LU >-Q O 3 40. 20. 0. 25 66 week - and i n c l u d e s a wide s i z e range - 0.6 to 2.2 mg - of amphipods i n the harvest. The conclusions of t h i s i n v e s t i g a t i o n of the e f f e c t of e x p l o i t a t i o n on p o p u l a t i o n s i z e and s t r u c t u r e are t e n t a t i v e . The assumptions and s i m p l i f i c a t i o n s made i n the c o n s t r u c t i o n of the L e s l i e matrix should he evaluated by comparing p r e d i c t e d y i e l d and p o p u l a t i o n s t r u c t u r e to those obtained i n l a r g e s c a l e c u l t u r e s , subjected to the harvest p o l i c i e s suggested by the model. 67 7. SUMMARY AND CONCLUSIONS This study i n v e s t i g a t e d s u i t a b l e c o n d i t i o n s f o r mass c u l t u r e of Eogammarus c o n f e r v i c o l u s , assessed the p o t e n t i a l of the amphipod as a d i e t c o n s t i t u e n t of young f i s h and, w i t h a L e s l i e m a t r i x model, explored the e f f e c t of v a r i o u s harvest s t r a t e g i e s on y i e l d and p o p u l a t i o n s t r u c t u r e . _E. c o n f e r v i c o l u s demonstrated wide s a l i n i t y and temperature t o l e r a n c e s , w i t h best s u r v i v a l at low s a l i n i t i e s (5 to 10^/00) and temperatures (5° to 10°C). Growth and s u r v i v a l was reduced at high p o p u l a t i o n d e n s i t i e s (>2 mg/1), de s p i t e p r o v i s i o n of excess food. The amphipod may be capable of good growth at increased d e n s i t y w i t h a flow-through system. Further experiments are necessary to determine s u i t a b l e d e n s i t i e s f o r mass c u l t u r e . E. c o n f e r v i c o l u s showed good growth and s u r v i v a l on a v a r i e t y of algae and a s s o c i a t e d epiphytes, demonstrating the broad d i e t of the s p e c i e s . Clumping diatoms or p h y t o d e t r i t u s are recommended as s u i t a b l e foods f o r l a r g e s c a l e c u l t u r e . Populations were maintained over three generations, demonstrating the f e a s i b i l i t y of long term c u l t u r e of t h i s amphipod. Chemical analyses performed on Eogammarus c o n f e r v i c o l u s samples show the amphipod to be a n u t r i t i o n a l l y s a t i s f a c t o r y c o n s t i t u e n t of f i s h d i e t s . I n feeding t r i a l s , amphipod preparations were r e a d i l y acceptable to j u v e n i l e coho, and provided good short-term growth, comparable to that provided by standard hatchery (Oregon Moist P e l l e t ) d i e t . F i n a l l y , i n f o r m a t i o n about growth, m o r t a l i t y and f e c u n d i t y of E_. c b r i f e r v i c o l u s under optimal d e n s i t y c o n d i t i o n s was used to develop a 68 L e s l i e matrix model. Harvest terms were a p p l i e d to v a r i o u s age c l a s s e s , and p r e d i c t i o n s made about r e s u l t i n g p o p u l a t i o n s t r u c t u r e and weekly y i e l d . A 41% harvest a p p l i e d to amphipods between 0.6 and 2.2 mg dry weight gave the best s u s t a i n a b l e y i e l d of the s t r a t e g i e s examined. Further experiments t e s t i n g the p r e d i c t i o n s of the L e s l i e m a t r i x model are recommended. 6 9 8. LITERATURE CITED A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r e Chemists. 1960. O f f i c i a l methods of a n a l y s i s . 9th ed. Washington, D.C. 12 p. Bamstedt, U. and J . B. L. Matthews. 1975. Studies of the deep-water p e l a g i c community of K o r s f j o r d e n , Western Norway. 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