POTENTIAL FOR MASS CULTURE OF THE ESTUARINE AMPHIPOD
EOGAMMARUS CONFERVICOLUS
by
JOAN CATHERINE SHARP
B.A., McGill University, Montreal, Quebec, 1972
B.Sc, McGill University, Montreal, Quebec, 1976
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES
(Department of Zoology)
We accept this thesis as conforming
to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
March, 1980
(c) Joan Catherine Sharp, 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study.
I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of "2-OOLOQ
The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS
Date P\rw\\ 3Q , R^Q ii
ABSTRACT
The gammarid amphipod Eogammarus confervicolus (Stimpson) was
investigated as a potential mass culture organism, with utility as a
diet supplement for artificially reared fish. Suitable conditions for
large-scale culture were determined in a series of experiments. E_.
confervicolus demonstrated wide salinity and temperature tolerances, with best survival at low salinities (5 - 10^/00) and temperatures
(5 - 10 C). Populati on densities greater than 2 mg/1 reduced amphipod
growth and survival, although densities may be increased with a flow-
through system. .E. confervicolus showed good growth and survival on a variety of algae and associated epiphytes, demonstrating the broad diet
of the species. Clumping diatoms or phytodetritus were suggested as
suitable foods for mass culture. Maintenance of populations over three generations showed the feasibility of long term culture of this amphipod.
Short term growth rates of juvenile coho at 12°C were similar on
live amphipods (3.2%/day), freeze-dried amphipods (2.4%/day) and Oregon
Moist Pellets (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 nutritionally satisfactory component of fish diets.
A Leslie matrix model was developed from information about growth, mortality and fecundity of Eogammarus confervicolus under optimal
conditions, and was used to test various harvest strategies. Highest yield 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 Leslie matrix model were recommended. iii
TABLE OF CONTENTS
Page
ABSTRACT ii
TABLE OF CONTENTS iii
LIST OF FIGURES v
LIST OF TABLES vi
ACKNOWLEDGEMENTS vii
1. INTRODUCTION 1
2. DESCRIPTION OF EOGAMMARUS CONFERVICOLUS 5
3. FIELD COLLECTION AND MAINTENANCE 8
3.1 Description of collection site 8
3.2 Collection 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 27
4.4 Discussion 31 iv
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 Fish Feeding Trials 41
5.3 Results 42
5.31 Chemical Analyses 42
5.32 Fish Feeding Trials 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. Mortality at various salinities 15
4. Mortality at various temperatures 17
5. Relationship between head length and dry weight 18
6. Growth in weight and survival on various diets 19
7. Growth in weight at five experimental densities 24
8. Changes in numbers of E_. confervicolus in replicate
cultures 29
9. First generation mortality and changes in biomass in
replicate cultures 30
10. Frequency distributions of wet weight of coho before and
after feeding trials 46
11. Relationship between brood size and dry weight 56
12. Predicted age distribution of harvested fraction with
18% harvest applied to all age classes 61
13. Predicted age distribution of harvested fraction with
40% harvest applied to age classes 0 to 8 63
14. Predicted age distribution of harvested fraction with
41% harvest applied to age classes 9 to 17 64
15. Predicted age distribution of harvested fraction with
90% harvest applied to age classes >17; 15% harvest
applied to age classes 0 to 17 65 vi
LIST OF TABLES
Page
I. Growth rates and mortality coefficients for E_.
confervicolus reared on various diets 20
II. Significance of differences in pairwise comparisons of
mean growth rates on six diets 23
III. Growth rates and mortality coefficients for E_.
confervicolus reared at five experimental densities 25
IV. Significance of differences in pairwise comparisons of
mean growth rates at five densities 26
V. Growth and mortality coefficients and final densities for
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 initial and final wet
weights and forklengths of coho of three diet groups 45
IX. Summary of ANOVA on final wet weights and forklengths and
growth rates of coho on three test diets 48
X. Leslie matrix elements 57
XI. Predicted weekly yield from harvest strategies 59 vii
ACKNOWLEDGEMENTS
Many people have contributed to the development of this thesis. I
am grateful to my thesis supervisor, Dr. T. R. Parsons, for his
encouragement, advice and patience throughout the study. I thank my
fellow graduate students, Alan Carruthers and Brenda Harrison, for their very helpful comments on the thesis. Eric Woodsworth and Pam Mace provided cheerful assistance in field collections, for which I am
grateful. Thanks are also due to Julie Oliviera, for her identification
of algal species.
I appreciate the assistance provided by researchers at the West
Vancouver Laboratory, West Vancouver. Dr. C. D. Levings was very helpful throughout the project, providing information, equipment and
advice. B. Dosanjh gave generous assistance in laboratory analyses.
I would like to thank my thesis committee members, Drs. C. D.
Levings and D. McPhail for their advice and suggestions in improving the manuscript. Finally, I acknowledge the financial assistance of the
National Research Council of Canada. 1
1. INTRODUCTION
Cultivation of marine or brackish water invertebrates in large numbers under controlled conditions is of interest both for biological research and for artificial rearing of fish. Many researchers have established breeding populations of crustaceans, either as a preliminary
to commercial mariculture or to provide a continuous supply of animals
of known physiological status for further experimentation.
Fewer than 2% of marine organisms can be reared through their
entire life cycle under controlled laboratory conditions (Kinne, 1970).
Ryther and Bardach (1968) have summarized the biological characteristics
of organisms lending themselves to intensive culture. They should
reproduce readily in captivity, and the eggs and juveniles should be
hardy and capable of hatching and developing under controlled conditions.
They should demonstrate good growth on a wide variety of inexpensive and
abundantly available foods, and should be tolerant of high density
conditions. Further desirable attributes include wide salinity and
temperature tolerances and year-round reproduction with high fecundity
(Chang and Parsons, 1975).
Several steps must be taken prior to establishment of a large
scale culture. Foods of suitable quality to allow reproduction and
development of the culture organism must be identified (Nassogne, 1970).
The temperatures and salinities allowing best survival must be
determined (Sastry, 1970). Sensitivity of the species to high density,
and its effects on feeding, growth and behaviour should be assessed
(Ryther and Bardach, 1968).
There is a need for a suitable organism to be cultivated to serve
as a diet supplement for artificially reared fish. The brine shrimp, 2
Artemia salina, is available for this purpose, but at a very high price.
Young hatchery-reared fish frequently need live or fresh-frozen invertebrate preparations to stimulate their appetite before they will accept commercial foods (Walker, in Fulton, 1976). With diet supplements of invertebrate preparations added to the standard hatchery diet,
Oncorhynchus species have been observed to feed more voraciously and grow more quickly (Brett, 1974). In western Norway, an experimental fishery has been established for the copepod Calanus finmarchicus.
Calanus is used as a diet supplement in salmonid rearing, to increase coloration of fish flesh (Heath, 1977).
The potential of' the gammarid amphipod Eogammarus confervicolus
(Stimpson) for mass culture and its utility as a diet supplement for artificially reared fish was explored in this study. The utility of amphipods as stocking organisms in impoundments and reservoirs has been investigated by several researchers. Ioffe (1972) introduced 49 invertebrate species, including 18 amphipod species, into man-made reservoirs and storage lakes in an attempt to enrich the food supply for commercial fish. The amphipods established themselves successfully and were widely utilized by demersal fish. Fish production estimates for
the Tsimlianskoe storage lake before and after introduction of invertebrates indicated that increased growth rate and improved production of commercial fish species resulted from these introductions.
Ivanova and Abrosimova (1975) were able to increase carp production at
decreased cost per kilogram of product by stocking fish fattening ponds with mysids and gammarid amphipods. Eogammarus confervicolus served
as the primary food source for coho salmon (Oncorhynchus kisutch) 3
reared in natural enclosures (Powers, 1973). Powers also modelled the
growth and survival of the amphipod to assess suitable conditions for maximum productivity.
The possibility of mariculture of amphipods ha* been discussed by
Zebchenko (1975), who recommended the development of methods for the mass culture of Gammarus balcanicus, described as a high quality food
for domestic animals and fish. Tenore, Browne and Chesney (1974) set
cu up a polyspecies aqua lture system in which a Corophium species formed part of a successful detritus feeding component, utilizing pseudo-feces
from oyster culture. They proposed this amphipodas a food source in the production of fish species such as winter flounder.
The present study proposes Eogammarus confervicolus as a suitable
organism for mass culture, possessing the desirable biological
characteristics outlined earlier and having potential as a diet supplement
for young fish. To assess the suitability of the amphipod as a mariculture
organism, its salinity and temperature tolerances were explored. Amphipods were reared on various foods and food combinations, and the resulting growth and survival were compared. Effects of density on growth rate, mortality and fecundity were investigated in cohorts reared at five experimental densities. The causes of reduced growth at high density were briefly explored. Replicate cultures were maintained for three generations
(close to one year) and demonstrate-the feasibility of long term
culture.
To assess the potential utility of El. confervicolus as a diet
supplement for fish, chemical analyses were performed to determine the
composition of amphipod tissue. The short term growth response of 4
juvenile coho to amphipod preparations was assessed in feeding trials.
Finally, information about growth rate, mortality and fecundity of amphipods reared with excess food at low density was used to construct a Leslie matrix model of the amphipod's population dynamics. Various harvest rates and policies were modelled to predict the resulting population age structure and weekly yield. 5
2. DESCRIPTION OF EOGAMMARUS CONFERVICOLUS
Eogammarus confervicolus (Stimpson), the most abundant and widely distributed amphipod on the North American Pacific coast, is 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 its recent reclassification by Bousfield (1979), is described by Barnard
(1954) and Bousfield (1958, 1979). An adult male is diagrammed in
Figure 1. The head bears two pairs of antennae, the first with an accessory flagellum. The mandibular molars are strong and well-developed, with a cutting edge with two to three teeth. Gnathopods on the first two pereon segments function in food handling and as precopulatory grasping appendages. Pereopods 3 to 7 are used for walking and clinging. Coxal gills are found on pereon segments 2 to 7. The female marsupium is formed by brood plates (oostegites) attached to coxal margins 2 to 5.
The three pleon segments bear pleopods, used for swimming and gill ventilation. Uropods on the three urosome segments are also used for swimming.
Males of E_. conf ervicolus can be recognized by their larger gnathopods, mature females by the presence of oostegites. Mating in this species is typical of gammarid amphipods, as described by Hynes (1955).
Mating pairs are formed following a random encounter between a male and a receptive female (Holmes, 1903), the male using his gnathopods to grasp the female's thoracic segments. Amplexus may last for up to a week.
Copulation follows the female moult, then the male and female separate. 6
Figure 1. Diagram of adult Eogammarus confervicolus (male) (from
Bousfield, 1979).
A, antennae (2 pairs); AF, accessory flagellum (on first
antennae); G, gnathopods (2 pairs); HL, head length; P,
pereopods (5 pairs); PL, pleopods (3 pairs); U, uropods (3
pairs); T, telson. u 7
The female ovulates, releasing her eggs into the marsupium, where they are fertilized and develop until hatching. The marsupium is ventilated 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 is not expressed until maturity, reached at a dry weight of between 1.1 and 1.2 mg.
Common in British Columbia estuaries and coastal areas with fresh• water influence, E. confervicolus is adapted to low salinity habitats, but can survive over a wide range of salinities (Levings at al., 1976).
Waldichuk (1969) notes that _E. confervicolus can tolerate low dissolved oxygen levels (1.92 mg/1) and can survive for several days at temperatures as high as 20°C. This amphipod is abundant within the zone of influence of the Port Mellon pulp mill, suggesting a high tolerance for toxicants from bleached kraft mill effluent (Harger and Nassichuk, 1974).
_E. confervicolus is found in association with vascular plants, benthic algae and detritus, with field abundance directly correlated with the presence of a sedge rhizome habitat (Hoos and Void, 1975). Benthic algae and detritus are utilized as food (Pomeroy, 1977). E-. conf ervicolus is capable of rapid colonization of new habitats, and is widely dispersed by the spring freshet.
In the central delta of Squamish estuary,where Eogammarus confervicolus is present in high abundance, amphipod biomass is greatest in April and
May (Levy and Levings, 1976). Juveniles and ovigerous females are present in the population year round, with juveniles predominating in the early winter months (September to January). 8
3. FIELD COLLECTION AND MAINTENANCE
3.1 Description of collection site
Amphipods were collected from the tidal flats of the central basin of Squamish estuary (49°41'N, 123°10'W) at the head of Howe Sound, a large fjord in southern British Columbia (Figure 2). The central basin has received no direct freshwater input since dyke construction in 1972 restricted the flow of the Squamish river to the west side of the central delta. Water movements in the central basin are due to tidal flows.
Surface temperatures in the central basin ranged from 4.4°C (winter) to 14.7°C in late summer. Surface salinities ranged from 30^/00
(winter) to 3.4^/00 (early summer) (Levings, 1976B). The middle and upper intertidal zones in the central delta (1.6 to 4.0 m O.D.) are covered by sedge meadows (Carex lyngbyei). Benthic algae, including diatoms and attached seaweeds (primarily Enteromorpha and Fucus), grow on logs, pilings, exposed sediment and sedges. The most abundant benthic invertebrates in the intertidal are the amphipod Eogammarus confervicolus and the isopod Exosphaeroma oregonensis. E. confervicolus is found primarily in the middle intertidal zone of the central delta. Its abundance is greatest in sedge rhizomes, which form a refuge at low tide
(Levings, 1976B). The biomass of E_. confervicolus fluctuates over the year, with maximum values occurring in mid-April (Levings and Levy, 1976).
Juveniles and mating pairs are found throughout the year.
Other amphipod species recorded in the Squamish estuary include
Ramellogammarus ramellus (formerly Anisogammarus ramellus),
Locustogammarus locustoides (formerly A. locustoides), Anisogammarus pugettensis, Lagunogammarus setosus (formerly Gammarus setosus) and 9
Figure 2. Major features of the Squamish estuary (courtesy of Dr.
C. D. Levings, 1973).
10
Corophium spinicorne, a tube-dwelling amphipod (Levings, 1973).
Eogammarus confervicolus is the major food organism for juvenile salmonids in the estuary, and also serves as prey for herring, sculpin and flounder (Goodman and Vroom, 1972).
3.2 Collection methods
Amphipods were collected by horizontal plankton tows at high tide. 2
A 0.25 m , 350 ym SCOR/UNESCO net was towed just over the sedge mat. Two other methods of collecting amphipods were tried. Nylon net bags were filled with Fucus and suspended from pilings at the 2.5 m tide level, as described by Levings (1976A). Amphipods swimming in the water column accumulated in the bags at high tide. After two to three weeks, bags were collected and amphipods rinsed from the Fucus. Individual amphipods were collected by dip net from tide pools and by hand from sedge rhizomes and from under debris at low tide. These methods proved less successful than net tows at providing large numbers of _E. confervicolus.
3.3 Maintenance
Amphipods were maintained in fully aerated five and ten gallon aquaria prior to use. Nylon mesh was provided for cover, and animals were fed excess Enteromorpha prolifera. Rhizoclonium served as the maintenance food source during density experiments. Before tolerance experiments, animals were maintained at 10^/00 and 5°C. For all other experiments, salinity was kept at 7^/00 and temperature at 10°C. The maintenance light regime was 12:12. 11
4. REARING CONDITIONS
4.1 Introduction
A major aim of this study was to determine suitable conditions for mass culture of E_. confervicolus. Salinity and temperature tolerances and growth at different densities were investigated. Growth and survival of amphipods reared on various foods and food combinations were
compared. Populations were maintained over three generations to demonstrate the feasibility of long-term culture.
4.2 Materials and Methods
4.21 Tolerance Experiments
Salinity and temperature tolerances of E. confervicolus were
evaluated in laboratory experiments. Animals were collected from the
field in June and July, 1977 and maintained at 10^/00 and 5°C in the
seven days before experiments were initiated.
Healthy juvenile amphipods of roughly equal size (0.6 to 0.8 mg dry
weight) were placed in individual compartments (5x5x5 cm) of a
covered plexiglass box. Animals were provided with 100 mis of water
and excess food (Enteromorpha prolifera). Photoperiod was 12:12.
Trial salinities and temperatures were selected to include the range
of values experienced by animals in the field. For the salinity series,
fifteen animals were kept in a 10°C coldroom at each of six experimental
salinities: 0, 5, 10, 15, 20 and 25^/00. For the temperature series,
salinity was maintained at 10^/00 for each group of fifteen animals.
Animals were placed in cold rooms kept at 5°-and 10°C, and in incubators
at.18° and.22°C. , Effects of interactions between temperature and 12
salinity were not investigated.
Each week, the amphipods were transferred to clean water and supplied with fresh excess food. Weekly mortalities were noted. An amphipod was considered dead when no movement was observed despite prodding with a glass rod. Experiments were terminated after four weeks.
4.22 Feeding Experiments
To determine diets suitable for rearing of Eogammarus confervicolus, newly released juveniles (.016 mg dry weight) were raised on six foods or food combinations. One food offered was Amphiprora paludosa, a benthic diatom forming clumps that can be easily manipulated by the amphipod.
Three algal species abundant in the Squamish estuary were selected: two green algae, Enteromorpha prolifera and Rhizoclonium, and the brown alga
Fucus, with associated epiphytes. Herring meal and a combination diet of
Fucus, _E. prolifera and herring meal were also tested.
Juveniles were placed in individual plexiglass containers containing
100 mis of 7^/00 water, maintained at 10°C with a photoperiod of 12:12.
Ten to twelve amphipods were started on each food type. Head length was measured at weekly intervals and converted to dry weight by the empirical relationship:
log (dry weight) = 0.2125 + 3.4783 log (head length)
Animals were returned to clean water with fresh excess food following measurement. Growth and survival were monitored for ten to eleven weeks, when mean size of amphipods reared on three of the diets exceeded minimum adult size (1.15 mg dry weight). 13
4.23 Density Experiments
The effects of density on growth, mortality and fecundity in
Eogammarus confervicolus was explored to determine suitable densities for mass culture. The amphipods used were newly hatched juveniles of known size, released from field collected ovigerous females. Cultures were established at five experimental densities - 1., 2., 5., 7.5 and 10. mg dry weight/1. The lowest density culture was established with twenty juveniles, while sixty juveniles were started at each of the other four densities.
Amphipods were maintained at 10°C, with 7^/00 seawater and excess
Rhizoclonium. Weekly head length measurements were made of a subsample of ten amphipods from each culture. Amphipods were transferred to a fresh container in order of size, with individuals being selected at regular intervals for measurement of head length. Head length measure• ments were converted to dry weight. The number of amphipods surviving and presence of mating pairs and ovigerous females were also noted weekly. Females bearing young were removed from the culture and the number of young released was determined. Animals were replaced in clean water. Water volume was adjusted for changes in total dry weight of each culture, to maintain constant densities (mg/1).
Initially, cultures were grown in glass stacking dishes holding a maximum volume of 350 mis. As the animals grew, cultures were transferred to large glass stacking dishes (maximum volume 1800 mis), small plastic pails (4 liters) and large plastic pails (10 liters).
4.24 Long Term Culture
Duplicate E. confervicolus cultures were established, and growth, 14
mortality and reproduction of the amphipod populations were followed for one year. Each culture was kept in a 2.5 1 cylinder made of plexiglass, (R) with open ends covered with 35 ym Nitex^ mesh. The cylinders were suspended in an InstantOcean ® aquarium filled with 7^/00 seawater, maintained at 10°C. Recirculation of water within the aquarium kept water flowing through the cylinders.
Fifty newly released E. confervicolus were placed in each cylinder and supplied with twisted nylon net for cover. Each cohort was fed daily with equal amounts of the benthic diatom Amphiprora paludosa. This diatom forms clumps that can be easily manipulated by the amphipod. Approximately
18.5 mg C were supplied to each cylinder daily.
The Amphiprora was grown as a unialgal culture in one liter flasks in a cold room maintained at 15°C. Each day, 325 mis were removed from each flask, and the culture rediluted to 650 mis with Enriched Seawater
(McLachlan, 1973).
Weekly observations were made to note the presence of mating pairs or the release of young in the cylinders. Each month, amphipods in each cylinder were measured and sexed. Cultures were followed for one year, allowing time for three separate generations.
4.3 RESULTS
4.31 Tolerance Experiments
i. Salinity Tolerance
Mortality of Eogammarus confervicolus at various salinities is illustrated in Figure 3. _E. confervicolus tolerates a wide range of salinities (from 5 to 25^/00) with maximum survival at 5^/00 and 10^/00. 15
Figure 3. Mortality in E. confervicolus at various salinities. 0°/oa
20%
25°/oO " • 15°/oo •
2 WEEKS 16
The species shows poor survival in fresh water,
ii. Temperature Tolerance
Low temperatures (5° - 10°C) proved optimal for survival of E.
confervicolus (Figure 4). Although short term temperature elevations
could be tolerated without high mortality, low temperatures would be most
suitable for long term maintenance.
4.32 Feeding Experiments
A linear relationship is demonstrated in the logarithmic plot of head
length vs dry weight for Eogammarus confervicolus, shown in Figure 5.
(Data from Dr. C. D. Levings, West Vancouver Laboratory, West Vancouver).
Growth and survival of E. confervicolus varied with food type. Weekly values for mean size and number surviving for each diet are given in
Figure 6. Since 100% mortality ended the herring meal experiment after
ten weeks, only the first ten weeks were used to calculate the growth
rates and mortality coefficients listed in Table I.
Growth coefficients (%/day) were calculated as the coefficients of
daily exponential growth k according to the equation:
fc wt = w/
(Mullin and Brooks, 1967), where
W^ = dry weight at time t
WQ = initial dry weight (.016 mg)
t = time in days
Linear regression of loge dry weight on time was calculated for each food
type, to obtain the values for the growth coefficient k.
Growth rates (mg/wk) were calculated by dividing the increase in dry 17
Figure 4. Mortality in E. confervicolus at various temperatures. 17a 18
Figure 5. Relationship between head length and dry weight for
Eogammarus conf ervicolus (Dr. CD. Levings).
(Base 10 logarithms)
19
Figure 6. Growth in weight and survival of E. confervicolus raised
on various diets. Mean dry weight, 95% confidence limits
and number surviving given for each week. WEEKS
B. Herring Meal
1-40-
1-00-
E •60-
1
UJ
>- • 20- 3 T3 ,3 1 Q 12 10 9 —i 1— i— i 10 11 0 4 5 6 8 9 WEEK C. Enteromorphg proliferg
1-4Ch
1-OCH
0 1 2 3 4 5 6 7 8 9 10 11
WEEKS F. Rhizoclonium
1-40-
1-20-
" T r 1 1 1 1 1 1 1 i i 1 0 123456789 10 11 12
WEEKS Table I. Growth rates and mortality coefficients for Eogammarus confervicolus reared on various diets.
f Days Growth coefficient (k) Growth rate Mortality coefficient (k ) (to adult size) %/day mg/wk %/day
A. Amphiprora paludosa 75 .063 .082 .001
B. Herring meal .025 .005 .035
C. Enteromorpha prolifera 93 .051 .040 .001
D. Fucus 75 .063 .079 0.
E. Choice 80 .059 .076 .008
F. Rhizoclonium 88 .054 .056 21
weight of individual amphipods over ten weeks by the number of weeks.
Mean values for growth on each food type are presented in Table I.
Mortality coefficients (%/day) were calculated as k' from the expression: -k 't
NFC = N^e , where
= number surviving at week ten
NQ = initial number
t = 70 days
Exponential growth coefficients k were used to predict days to adult size - 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 still showed low mortality. 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 mortalities were noted with diets B - herring meal alone, (k' =
0.035 %/day) - and E - choice of Fucus, Enteromorpha prolifera and herring meal, (k' = .008 %/day). This suggests that inclusion of herring meal in diet E increased the mortality of animals otherwise provided with a satisfactory diet allowing good growth (k = .059%/day).
The assumption of homoscedasticity of growth rates (mg/wk) of the experimental diet groups (calculated to week 9) was evaluated with the F test (Sokal and Rohlf, 1969). As variances of the six groups were max 22
not significantly different, an analysis of variance was performed using individual growth rates. The Student-Neuman-Keuls procedure (Sokal and
Rohlf, 1969) was used to evaluate significance of differences between means. Results of pairwise comparisons of mean growth rates are summarized in Table H> showing level of significance of differences between each pair of means.
4.33 Density Experiments
Figure 7 shows weekly values for mean size of E_. conf ervicolus reared at five densities - 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 first twenty weeks were used to calculate the growth rates and mortality coefficients listed in TableIII,due to the removal of large numbers of ovigerous females from low density cultures in subsequent weeks. Growth (k) and mortality (k') coefficients and mean growth rates
(mg/wk) were calculated as previously described (Section 4.32). The time taken by each cohort to reach a mean size of 1.15 mg is given as
'days to adult size'. Growth of E_. conf ervicolus decreases with increasing density, while mortalities 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 significance of differences between means. Results of pairwise
comparisons of mean growth rates in Table IV show the level of significance
of differences between each pair of means. Growth at 1.0 and 2.0 mg/1 were not significantly different. However, amphipods showed significantly 23
Table II. Significance of differences in pairwise comparisons of mean
growth rates of Eogammarus confervicolus on six diets.
Herring meal Enteromorpha Rhizoclbnium Choice Fucus
Enteromorpha *
Rhizoclonium * ns
Choice * ns ns
Fucus ** * ns ns
Amphiprora ** ** * ns ns
* P < .05
** P < .01 24
Figure 7. Growth in weight of Eogammarus confervicolus reared at five
experimental densities. Mean dry weight and 95% confidence
limits given for each week. 24a
24c
E - 10.0 mg I
IS WEEKS Table III. Growth rates and mortality coefficients of Eogammarus confervicolus reared at five experimental
densities.
Density pays Growth coefficient(k) Growth rate Mortality coefficient^') (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 in pairwise comparisons of mean
growth rates of Eogammarus confervicolus at five experimental
densities.
1.0 mg/1 2.0 mg/1 5.0 mg/1 7.5 mg/1
2.0 mg/1 ns
5.0 mg/1
7.5 mg/1 •k* ns
10.0 mg/1 ns ns
* P < .05
** P < .01 27
reduced growth at the three higher densities - 5.0, 7.5 and 10.0 mg/1.
The factors causing reduced growth and increased mortality of E_. confervicolus at high densities were briefly investigated in three cultures, each started with 48 newly released juveniles. Two cultures -
A and B - were set up in 2.5 1 plexiglass cylinders, suspended in an
aquarium, with recirculating water flow preventing accumulation of and excretory products, and depletion of 0^. Culture
A was provided with excess food (Rhizoclonium). Twisted nylon net was supplied for cover, reducing interactions between amphipods. Culture B was supplied with excess Rhizoclonium only. Culture C was set up in a plastic pail, 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 for 95 days. Growth and mortality coefficients (%/day) and final densities are given in Table V.
T tests (Sokal and Rohlf, 1969) were used to assess significance of differences between mean growth coefficients (k) in the three cultures.
Amphipods in container C showed slowest growth and highest mortality; reduction of growth was highly significant (P < .01) in comparisons with growth in cultures A and B. Growth of amphipods in cultures A and B was not significantly different, but mortality was increased in container B.
Mean dry weights of amphipods after 95 days - 1.26 mg (A) and 1.17 mg
(B) - were very close to those of amphipods reared at low densities - 1.27 mg at 1.0 mg/1; 1.32 mg at 2.0 mg/1.
4.34 Long Term Cultures
Data are presented in Figures 8 and 9 for the two replicate amphipod 28
Table V. Growth and mortality coefficients and final densities for
Eogammarus confervicolus reared in three experimental cultures.
Culture Growth coefficient Mortality coefficient Final density (fc) ) %/day %/day mg/1
.044 .006 .13.7
.043 .008 10.5
.032 .012 2.6 29
Figure 8. Changes in numbers of Eogammarus confervicolus in replicate
cultures.
30
Figure 9. First generation mortality and changes in biomass in
replicate Eogammarus confervicolus cultures.
31
populations in which growth, mortality and reproduction were monitored
for 340 days. Three separate generations of Eogammarus confervicolus were followed over this period.
Figure 8 shows the changes in amphipod numbers in the two cylinders over the year. The first appearance of mating pairs and the first release of young is noted for each generation. All adults of the previous generation died before their offspring reached maturity, so no interbreeding between generations occurred. Generation time in the populations was approximately 150 days.
Figure 9 shows the changes in total biomass in the two cylinders over the course of the experiment. Mortality of the first generation amphipods is also illustrated. Biomass of the amphipod population reaches a maximum as the first and second generation amphipods reach maturity.
The maximum biomass for the first generation coincides with the minimum mortality values for this generation. Highest mortalities are seen as juvenile amphipods compete for a limited food resource, with, mortality values decreasing as amphipods grow to maturity. A second increase in mortality is seen after mating and release of young, as large males cannibalize moulting females and compete with abundant juveniles for food.
4.4 DISCUSSION
Eogammarus confervicolus has wide salinity and temperature tolerances, with best survival at low salinities (5 - 10^/00) and temperatures (5 - 10°C). This species would be well able to tolerate fluctuations in salinity (especially increased salinity) and temperature 32
in culture.
Levings et al. (1976), investigating survival of E_. conf ervicolus in seawater and bleached kraft mill effluent at various temperatures and salinities, found maximum juvenile survival at 8^/00, with adults surviving best at 16^/00. He also noted that field observations show
E. confervicolus is adapted to low salinities, and is 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 salinities, 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
little 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 al. (1976), who found that low
temperatures (3° - 10°C) provided optimum conditions for survival of E.
confervicolus. Again, this corresponds with the field 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 its high nutritive value for fish. Amphipods ate it, but demonstrated poor growth and high mortality. Martin (1966) reports that Marinogammarus species readily ingested undigestible materials, and concludes that nutritive value of foods seems of little importance in food selection.
Results of the feeding experiments demonstrate the wide diet of
Eogammarus confervicolus. A number of algal species appear suitable for rearing this amphipod.
Characteristic bacteria, micro-algae and protozoans associated with each algal species are also used as a food source by the amphipod, and may be important in determining growth. Pomeroy and Levings (1980) reared
_E. confervicolus on a number of diets, and mentioned epiphytes as a major food source. The high growth rate demonstrated with Fucus may be largely due to epiphytic growth. Watanabe (pers. comm.) reported that Fucus provides shelter for Lagunogammarus setosus but is not used for food.
Epiphytes serve as the main food source for many amphipod species.
Hargrave (1970A) reported that growth of Hyalella azteca was proportional to the amount of microflora in the diet. He also reported high (> 50%) assimilation efficiency of naturally 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 assimilation efficiency with this diet than with leaves alone
(Barlocher and Kendrick, 1975). Untreated Zostera are preferred by
Gammarus oceanicus to those treated to remove epibionts (Harrison, 1977).
Harrison refers to the work of other authors (Harrison, 1977 citing 34
Fenchel, 1970, 1973; Hargrave, 1970 and Kristensen, 1972) showing that amphipods have high assimilation efficiencies (50 - 90%) of the micro•
organisms attached to Zostera. These workers have suggested that
amphipods have a relatively poor ability to digest the plant's
structural carbohydrates. Epiphytes are also important in the diets of
Gammarus palustris (Gable and Croker, 1977) and Gammarus pulex (Moore,
1975), among others.
Texture of food may be important in determining its acceptability
and digestibility. Martin (1966) noted that Marinogammarus accepted a
wide variety of fresh plant material, especially soft-tissued
membranaceous or filamentous algae. However, this amphipod preferred
decaying food, perhaps due to the softening action of micro-organisms.
Pomeroy and Levings (1980) found texture was important in determining
the palatability and ease of manipulation of algae by E. confervicolus.
They reported best growth with the filamentous, easily manipulated
Enteromorpha linza and Pylaiella littoralis. Pomeroy (1977) investigated
the food preferences of Eogammarus confervicolus, offering starved,
field-collected amphipods a choice of decayed sedge shoots, detritus
and several species of macroalgae. Amphipods preferred to feed on
filamentous algae (Pylaiella littoralis and Enteromorpha minima) with
some feeding on thallose forms.(Monostrema oxyspermum). Carex shoots were
least preferred, and were acceptable only when greatly decayed. In the
experiments reported here, amphipods grew best on the easily manipulated
diatom Amphiprora and soft, decaying Fucus. Enteromorpha prolifera -
with coarse thalli - gave reduced growth.
Pomeroy and Levings (1980) reared E. confervicolus at 4°C and 10°C 35
on a variety of plant foods. Experiments were started with two week old juveniles, and continued for twelve weeks. E. linza gave best growth and survival - .103 mg dry weight/wk; k' = .002. Good growth and low mortality were found with Porphyra species (growth rate = .057 mg/wk; k'
= .005), Pylaiella littoralis (.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 juveniles).
E. linza provided considerably higher growth rates than any foods reported here. Other growth rates are quite similar to those observed in these experiments. Pomeroy and Levings1 results again demonstrate the wide range of foods acceptable to E. confervicolus. Chang and Parsons (1975) reared Anisogammarus pugettensis on a variety of plant and animal foods.
Good growth was obtained with Enteromorpha species, diatoms (Pseudonitzchia species) and frozen fish. Growth at 10°C on Enteromorpha was higher than that reported for E_. conf ervicolus - . 2 mg dry weight/wk. Willoughby and
Sutcliffe (1976) measured the growth rate of Gammarus pulex, kept at 15°C on a number of foods - elm and oak leaves, Molinia and several fungal and algal species. High growth rates - .16 mg dry weight/wk - were obtained with decaying oak and elm leaves. As these experiments were started 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 variety of plant foods are suitable for the rearing of Eogammarus confervicolus. Best growth is provided by the diatom Amphiprora and decaying Fucus with associated 36
micro-organisms. These two foods can be suggested as suitable for mass amphipod culture. The former can be grown in a continuous culture using an enriched medium pumped into the amphipod culture, as described by
Parsons and Bawden (1979). Brown and Parsons (1972) have investigated the production of phytodetritus - clumps of sedimented phytoplankton with associated bacteria and organic slime - in an impoundment flushed with seawater. A flushing rate of 100%/day was found to yield maximum production of phytoplankton, with the formation of large amounts of sedimented material. Clumps of algal cells and bacteria could be easily manipulated by _E. confervicolus, and would be expected to provide good growth.
In density experiments, amphipods showed good growth and low mort• alities at 1.0 and 2.0 mg/1. Growth slowed and mortalities increased at higher densities, although cultures were supplied with excess food.
Amphipods reared.in low density cultures took slightly longer to reach adult size than amphipods reared in isolation in feeding experiments.
This reduction in growth rate even at low densities suggests that the crowding effect may be associated with amphipod interactions, perhaps through increased activity or disturbance of feeding.
Wilder (1940) studied the effects of density on growth, fecundity and mortality in Hyalella azteca. Unlike this study, she found increased growth, fecundity and survival at intermediate densities. However, growth and fecundity were inhibited and mortality increased at high densities. Food was hot supplied in excess, and may have been limiting at high densities. Wilder suggests accumulation' of carbon dioxide and 37
excretory products, depletion of oxygen and reduced food and space may have played a role in inhibiting growth in her cultures. In the experiment reported in 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 in the containers. Addition of netting
(Container A) to reduce contact had little effect on growth, but reduced mortality somewhat. This suggests that cannibalism - frequently observed in this species - may be an important source of mortality. More extensive experiments will be necessary before firm conclusions can be drawn about the causes of reduced growth and increased mortality at high densities.
The increased mortalities, reduced growth and delay in reaching maturity observed at high densities suggest that low densities will be best for mass culture. Densities can be increased in a well flushed system. Further experiments will be necessary to determine maximum densities under such conditions.
Three separate generations of Eogammarus confervicolus were observed in both replicate cylinders in the long term culture experiment. The growth of these cohorts had different characteristics than the growth of cohorts supplied with excess food in the low density cultures.
Increased generation time was noted in the food-limited cohorts, with the first release of young occurring after 150 days. Young were first released in the low density cohorts 105 days after cultures were established. Comparison of growth rates after 120 days shows reduced individual growth in the long term cultures. Growth rates of 0.13 mg dry 38
weight/week in long-term cultures were significantly lower than growth rates in 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. Finally, a considerable increase in individual mortality was noted in the food limited cultures. Long term cultures had a combined mortality of 0.013
%/day, compared to a combined mortality of 0.002 %/day in the low density cultures. These comparisons demonstrate the importance of maintaining adequate food supplies in mass culture.
E. confervicolus populations maintained in long-term culture had a
150 day generation period. Biomass peaked when each generation of amphipods reached maturity (Figure 9). For the first generation, this biomass maximum coincided with minimum mortality values. In the curve showing first generation mortality, high initial mortality occurred when juveniles competed for a limited food supply. Mortality values decreased as amphipods grew to maturity, then increased again after mating, when
cannibalism increased. Mortality also increased after young were released, when adults competed for food with the abundant juveniles. Unlike
Daphnia, which adjusts to food limitation by regulation of birth and growth rates without increased mortality (Frank, Boll and Kelly, 1957),
all three population parameters are altered by food limitation in
Eogammarus confervicolus.
Parsons and Bawden (1979) followed the growth of four populations of
Anisogammarus pugettensis for 130 days in continuous culture. Amphipods were kept in 2.5 1 cylinders suspended in an Instant Ocean aquarium with a recirculation system. Amphiprora paludosa was supplied daily in
four different volumes, pumped into the amphipod cylinders. Population 39
growth in the cylinder receiving the highest volume of algal culture -
14.5 mgC/day - demonstrated many of the same trends noted in the experiment reported here. A cyclic fluctuation in amphipod numbers was observed, with mortality of the first generation continuing until replacement by the second generation. Again, population biomass maximized as the first generation reached maturity. Nilsson (1977) set up cultures of Gammarus pulex, following the growth and mortality of a
series of cohorts produced by field-collected females. He found that mortality of juveniles was greatest in the first four weeks after hatching. Similar observations were made by Bamstedt and Matthews
(1975) on the copepod Euchaeta norvegica. Mortality of the young stages
(nauplius and copepodite I - IV) balanced growth of individuals. No
population growth occurred before the moult to stage V.
This experiment demonstrates the importance of supplying adequate
food to amphipods in mass culture. Food limited populations are
characterized by increased generation time, reduced individual growth
rates and increased individual mortality. 40
5. EOGAMMARUS CONFERVICOLUS AS A FOOD FOR FISH
5.1 Introduction
Hatchery reared fish have been observed to feed more readily and grow more efficiently if their diet includes live or fresh-frozen invertebrates (Fulton, 1976). Walker (in Fulton, 1976) reports that the sandlance Ammodytes hexapterus would not begin to feed on commercial food until its 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 potential utility of E. confervicolus in fish diets, it was necessary to obtain information about the chemical composition of the amphipod and the growth response of fish to amphipod preparations.
5.2 Materials and Methods
5.21 Chemical Analyses
The crude protein, lipid and ash content of samples of Eogammarus confervicolus were analysed. Amino acid analysis was also carried out.
Amphipods used for these determinations were juveniles, collected from the field in 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 in 6 N HC1 at 110°C. This method does not give quantitative recovery of cystine or tryptophan.
Proximate analysis of a sample of juvenile E. confervicolus was kindly supplied by B. Dosanjh of the West Vancouver Laboratory, West
Vancouver. Protein determination was performed by autoanalyser; lipid was 41
extracted In chloroform:methanol (1:2 by volume). Ash was determined
by combustion at 600°C for two hours. For details of lipid extraction,
refer to the Association of Official Agricultural Chemists (1960) manual
of analysis.
5.22 Fish Feeding Trials
Growth of coho salmon (Oncorhynchus kisutch) on two amphipod
preparations was compared to growth on a commercial diet. One hundred
and sixty-two juvenile coho were kindly supplied by Andy Lam, West
Vancouver Laboratory, West Vancouver. Fish were divided into equal groups
and placed in three 200 1 fiberglass tanks, kept in the courtyard of the
Biosciences Building at the University of British Columbia. Feeding trials were carried out during July, 1977. Initial fork length and wet weight values were estimated by sampling 23 fish from each tank.
The tanks were aerated, and the flushing rate was adjusted to maintain a relatively constant temperature. Mean surface temperature in
the tanks was 11.9°C, with a range of 10° to 13°C. Surface temperatures in the three tanks were identical each day.
The three fish diets included two amphipod preparations - freeze-dried and live amphipods - and a control diet of Oregon Moist Pellets. The particle size of the three diets was standardized. Live amphipods, freeze-dried amphipods and pellets offered to coho all 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 in this manner ranged in size from 0.2 mg to 0.7 mg dry weight.
A fixed daily ration for each diet was set in excess of expected 42
growth requirements (Parsons and LeBrasseur, 1968). The ration provided was 25% of initial body weight/day, calculated on an equivalent dry weight basis. The daily ration was 14 - 16% of the final body weight of the fish.
Fish were fed once a day for 26 days. After one day's starvation, to allow fishes' guts to clear, fish were sacrificed. Fork length (to nearest .1 cm) and wet weight (to nearest .01 mg) were determined for each fish.
5.3 Results
5.31 Chemical Analyses
Proximate composition of a sample of juvenile Eogammarus confervicolus
is shown in Table VI. TableVII gives the amino acid composition (on % dry weight basis) of a batch of frozen amphipods. Essential amino acid
requirements (based on values determined for chinook salmon fingerlings)
are also given, converted to % dry weight for a diet with 48.6% protein
(Mertz, 1972).
5.32 Fish Feeding Trials
Results of the experiment comparing growth of juvenile coho on two
amphipod preparations and Oregon Moist Pellets are summarized in Table VIII.
Growth rate is calculated as mg wet weight/day and as % body weight/day.
The frequency distributions in Figure 10 illustrate the initial and
final wet weight distributions of coho in the feeding trials. An ANOVA
showed no significant differences in initial wet weights of samples taken
from the three tanks, and these samples were pooled to give the initial 43
Table VI. Proximate composition of Eogammarus confervicolus sample.
Values expressed as % dry weight.
Component % Dry Weight
Ash 28.6
Fat 10.3
Protein 48.6 44
Table VII. Amino acid composition of frozen Eogammarus confervicolus
sample.
Amino Acid % Dry Weight Requirement (% Dry Weight)
Alanine 2.86
Arginine 3.36 2.92
Aspartic acid 5.08
Cystine Not determined
Glutamic acid 6.63
Glycine 2.90
Histidine 1.18 0.83
Isoleucine 2.21 1.21
Leucine 3.63 1.90
Lysine 2.80 2.43
Methionine 1.28 0.73
(with adequate cystine) Phenylalanine 2.52 2.48 (phenylalanine & tyrosine) Tyrosine 1.92
Proline 2.22
Serine 2.47
Threonine 2.24 1.09
Tryptophan Not determined 0.24
Valine 2.56 1.55
calculated from amino acid requirements of chinook salmon fingerlings (values as % dietary protein) quoted in Mertz (1972) Values converted for diet with 48.6% protein. Table VIII. Comparisons of mean daily growth rates and initial and final wet weights and forklengths
of juvenile coho of three diet groups in feeding trial.
Live Amphipods Oregon Moist Pellets Freeze-dried Amphipods
Mean S.D. Sample Size Mean S.D. Sample Size Mean S.D. Sample Size
INITIAL
Forklength (cm) 6.7 0.9 24 6.6 1.0 24 7.0 1.0 23
Wet weight (g) 3.25 1.24 24 3.29 1.58 24 3.84 1.59 23
FINAL
Forklength (cm) 8.0 1.0 31 7.8 1.2 28 7.7 1.0 31
Wet weight (g) 6.25 2.45 31 5.89 2.97 28 5.38 2.24 31
GROWTH RATE
Mg wet weight/day 0.15 0.09 31 0.14 0.11 28 0.11 0.09 31
% body weight/day 3.2% 3.1% 2.4% 46
Figure 10. Frequency distribution of wet weight of juvenile coho
(Oncorhynchus kisutch) before and after feeding trials on
A. Live amphipods, B. Oregon Moist Pellets and C. Freeze-
dried amphipods. INITIAL FINAL A. Live amphipods
81 B. Oregon moist pellets 18- 6- ? 16- 4- 814- 2- 12- 1 LL 0 10- €L 8- u C. Freeze dried amphipods CD 6- 1 4- Z 2-
0 12 3 4 5 6 7 3 4 5 6 7 8 9 1011 12 13 WET WEIGHT (gm) 47
wet weight frequency distributions.
Table IK shows the results of ANOVA tests for significance of differences between treatments for final wet weights, final fork lengths and daily growth rates. Although coho feeding on live amphipods had a mean growth rate of 0.15 mg/day vs 0.14 mg/day for Oregon Moist Pellets and 0.11 mg/day for freeze dried amphipods, no significant treatment effects were noted. Within group variation 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 nutritionally adequate to support the growth of coho salmon. Protein (48.6% dry weight) is within the salmonid dietary requirement at 10°C of 40 to 50% of the ration dry weight (Mertz, 1969).
Lipids make up an adequate 10.3% of the sample. Fat calories are readily digestible, and serve to spare protein in the ration. Ash values are high at 28.6%. The sample analysed was composed of young juvenile amphipods.
Ash content of amphipod samples collected in Squamish estuary shows seasonal fluctuations. Lowest ash content is found in summer months, when large numbers of ovigerous females are present (C. D. Levings, pers. comm.). Carbohydrate (12.5% of sample by difference) is at an allowable dietary level. Digestible carbohydrate up to levels of 25% in the diet are as effective an energy source as fat for many fish species (Cowey and
Sargent, 1979).
Eogammarus confervicolus supplies a favorable balance.of essential 48
Table IX. Summary of ANOVA on final wet weights and forklengths and
growth rates of coho on three test diets.
Source of Variation df SS MS
Final wet weight
treatment 2 12.04 6.02 0.93 ns
within treatment 84 542.19 6.45
Final 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 is suitable as a source of dietary protein. A decrease in the total dietary protein requirement of fish is noted with foods with a good balance of the essential amino acids (Mertz, 1972).
In growth experiments, juvenile coho fed readily on live _E. confervicolus. Fish were initially reluctant to feed on the freeze-dried preparation, which floated on the surface of the tank. After one week, they became accustomed to this product and fed readily. Due to high variability in fish size, no significant differences in growth rate were noted between diets. However, it is clear that live and freeze-dried
E. confervicolus are acceptable to juvenile 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 fontinalis) and rainbow trout (Salmo gairdneri) live Gammarus fasciatus. After five months the fish remained healthy, with more brilliant coloration than normal hatchery or wild trout. Brown trout
(Salmo trutta) fed exclusively on Gammarus pulex for up to 595 days showed no evidence of dietary deficiency. Pentelow concludes from his experiments
that G_. pulex is a very efficient food for trout. Gammarus lacustris serves as the primary food for rainbow trout cultured in small aquaculture ponds in Manitoba (Mathias et al., 1977).
The growth rates (% body weight/day) obtained in this experiment can be compared to values reported for other juvenile salmonids feeding on
invertebrate preparations. LeBrasseur (1969) fed chum salmon (Oncorhynchus keta) an excess diet of live 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 juvenile chum. Growth of salmon on this copepod is higher than that reported here for E_. confervicolus, a finding which is partially attributable to the increased temperatures in LeBrasseur's experiment.
Juvenile chum (starting weight - 0.405 g) showed a much lower growth rate on excess frozen JZ. plumchrus. This finding is attributed by Fulton
(1976) to rupture of the carapace and subsequent loss of body lipids of the copepod. Heath (1977) obtained growth rates of 2.7%/day and 3.8%/day when feeding juvenile coho (initial weight - 0.29 g) on excess amounts of frozen and freeze-dried Euphausia pacifica at 9°C. Despite the lower temperature, these are quite similar to the values of 3.2%/day and 2.4%/ day reported here on live and freeze-dried _E. confervicolus.
Results from chemical analyses and coho feeding experiments show
Eogammarus confervicolus to be a nutritionally satisfactory constituent of fish diets, readily 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 fish is 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 in 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, in 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 in 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 in a matrix A, which is multiplied by a column vector a^ of female population age structure at time t, to predict age structure at time t + 1. The model is:
Aat = at+l or — —
n f0 fl f2 Ln-1 t,o nt+i,o
nt,l nt+l,l
n n t,2 t+i,2;
nt,3 nt+l,3
n n-1 _ t+l,n_ where:
f^ (i=0,l,2 n) = fecundity of females in the ith age group (i.e.
the number of females in age group 0 at t+1
born to each female in the ith age group at
time t)
p^ (i=0,l,2 n) = probability that a female in the ith age group
at time t will survive to the (i+l)th age group
at t+1
n^ = number of females in age group i at time t
All other matrix elements are zero.
The largest positive eigenvalue A of A has a corresponding eigenvector with all elements non-negative. When a stable age distribution has been 53
reached, population growth can be described by the equation:
A v = Xv where X , the dominant eigenvalue, describes the change in population size per unit time and v, the corresponding eigenvector, describes the stable age distribution. A can be related to r, the intrinsic rate of natural increase of a population, by the equation:
r = In A .
If the dominant eigenvalue X of the Leslie matrix is greater than one, the population can withstand a maximum sustainable harvest rate of
100(^ ^) percent, if applied equally over all age classes. This exploitation may be enhanced when only one age group is harvested, as the production of the exploited group increases relative to the production of the other age groups (Williamson, 1967). This theoretical result is supported by the experiments of Watt (1955), who harvested various stages of Trlbolium confusum, and by Slobodkin and Richman (1956), who removed newborn Daphnia pulicarja. Both studies found an increase in relative abundance of the exploited group.
Usher (1972) has reviewed developments of the Leslie model: inclusion of both sexes (Williamson, 1959), consideration of size structure
(Lefkovitch, 1965) and density-dependance of matrix elements (Pennycuick et al., 1968). The simple Leslie matrix model gives the rate of increase of a population under unvarying conditions, with unlimited space and no intraspecific density effects. This model has restricted applicability, as Beddington and Taylor (1973) discuss. It is suitable for a population in which exploitation keeps the population density constant. In an exploited population, the Leslie matrix can be used to determine at what 54
rate various age classes should be cropped in order to maximize
sustainable yield.
The elements for the Leslie matrix were obtained from the combined
statistics for survival and fecundity of the amphipod cohorts reared for
26 weeks at 1.0 and 2.0 mg/1 in density experiments. Data for males and
females were combined until week 18. Survival data for weeks 19 to 25 were obtained for females only. Since mean size, survival and number of
ovigerous females were determined weekly in these cultures, one week
intervals were used in the Leslie matrix. Weekly percent survival
figures were used as p. elements in the matrix:
where = population size in week i. These values provided p^ elements
for weeks 0 to 25.
A new survival element was added to the matrix, representing the
proportion of females remaining in the last age class each week. This
age class includes all females older than 25 weeks. In weeks 24 to 26,
all females in the cultures were in mating pairs or bearing eggs or young.
It was assumed that mortality in these females was largely associated with
reproduction - cannibalism by the male following the female moult, or
death of the female following release of young. Thus the element
representing weekly survival of females older than 26 weeks was assumed
to be identical to the survival values obtained for the previous two
weeks.
Figure 11 shows the linear regression line (Model II regression)
fitted to data obtained on brood size vs female dry weight in field- 55
collected Eogammarus confervicolus (Data from Dr. C. D. Levings, West
Vancouver Laboratory, West Vancouver). Despite the very wide scatter of
points, a t-test (Snedecor, 1956) showed the slope to be significantly
different from zero (P < .05). The variation may be due to differences
in rearing conditions experienced by these females. Sensitivity of
growth rate to diet and density conditions has been demonstrated in
previous experiments. A regression of brood size on female dry weight
obtained from females reared in conditions determined for mass culture
would be more suitable for calculating the Leslie matrix elements.
Incubation time - days from separation of mating pair until release
of young - was measured at 10°C. Incubation times varied from 16 to 19
days, with a mean value of 17 days.
Weekly values for the fraction, a^, of females bearing eggs or young
were obtained from the low density cultures. Fecundity of females of each
age class (F^) was calculated with the brood size:female dry weight
regression equation (Figure 11), using the mean dry weight of amphipods
in each age class. F_^ values were divided by two, assuming a 1:1 sex
ratio in broods. f. values were thus calculated as:
1 2.43 where 2.43 is the incubation time in weeks at 10°C.
Table X gives all f^ and p^ elements for the Leslie matrix. Values
for mean dry weight of each age class are also given.
Weekly harvests were simulated by subtracting a harvest term - h -
from the survival elements - p_^ - for each age class. Thus, harvest terms
can be applied to all amphipods in the population or to selected age classes.
Eigenvalues and corresponding eigenvectors can be calculated for each 56
Figure 11. Relationship between brood size and dry weight for
Eogammarus confervicolus females, with 95% confidence
limits. (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
All other matrix elements are zero. Mean dry weight is given
for each age class.
Mean dry weight(mg) Week ii 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 Leslie matrix. In each case, the dominant eigenvalue A describes the weekly change in population size, while its corresponding eigenvector describes the stable age distribution. Harvest strategies can be selected to give A values of 1.00, providing a sustainable yield with constant population size.
It is unrealistic to harvest Eogammarus confervicolus populations by age classes, as these cannot be distinguished in a mixed population.
It is, however, a relatively simple matter to separate amphipods by size
classes. These can be equated with age classes with some inherent error due to the variability in size of each age class. A further simplification was introduced to the model by assuming equal harvest of males and
females in the population.
For calculation of weekly yield (mg/wk), a stable age distribution
and total population dry weight of 2000 mg was assumed. Yield (Y) is
calculated as:
Y = Ih..2n..dw. .1X1
l where h = harvest applied to ith age group,
n^ = number of females in age group i,
dw_^ = mean dry weight of females of age class i 2Zn..dw. = 2000 mg .11 i
6.3 Results and Discussion
Table XI summarizes the results of various harvesting strategies
applied to the Eogammarus confervicolus population. The unexploited
population has a dominant eigenvalue of 1.22, indicating an exponential 59
Table XI. Predicted weekly yield from various harvest policies.
Weekly harvest Dominant Eigenvalue(A) Yield (mg/wk)
No harvest 1.22
18% of all age classes 1.00 362
40% of age classes 0-8 1.00 158 •001 - .5 mg)
41% of age classes 9-17 1.00 436 (.5 - 2.2 mg)
90% of age classes 18 - 26 1.00 382 (. >2.2 mg) and 15% of all other age classes 60
increase in population size in the absence.of harvesting. The population could sustain a maximum weekly harvest of 100 -(^—^5P.) or 18%, if applied equally over all age classes.
Next, the Leslie matrix was modified by this harvest rate and the eigenvalues and eigenvectors were recalculated. The new dominant eigen• value is 1.00, indicating a stable population size, as expected. With a stable population size of 2000 mg dry weight, this harvest strategy gives a weekly yield of 362 mg, including amphipods of all sizes. The predicted stable age distribution for this population shows a decrease in amphipod numbers with each subsequent age class. However, maximum biomass is found at ages 7 to 18 weeks, as amphipods reach maturity and start to reproduce. Figure 12 shows the predicted age distribution for the harvested fraction of the population. Total numbers and dry weights of amphipods are given for each harvested age class. Equal harvest of all
age classes produces a good weekly yield, with maximum biomass obtained
from the middle age classes (weeks 8 to 14). A wide range of sizes is
represented in the harvested fraction of the amphipod population.
With the harvest applied only to young amphipods - age classes 0 to
8 - an exploitation rate of 40% per week can be sustained by the
population. Although the number harvested is high, the yield in dry weight is considerably reduced, to 158 mg/week. The predicted stable
age distribution under these conditions shows a great decrease in
amphipod numbers with increasing age in age classes 0 through 8. Uniform
numbers with, increasing biomass are seen in the higher age classes, with
increased numbers and maximum biomass in the last age class. With no
harvest of reproductive adults, a high rate of production of young is 61
Figure 12. Predicted age distribution of harvested fraction with 18%
harvest applied to all age classes. fcleu
0 5 10 15 20 WEEKS 62
found. However, the dry weight yield is very low. Figure 13 shows, the predicted age distribution of harvested amphipods. Only a narrow range of small sizes (.02 to .5 mg) is represented in the harvested fraction, with, highest numbers obtained from the youngest age classes.
With exploitation restricted to age classes 9 to 17, the population can sustain a harvest rate of 41% per week. This strategy produces an increased yield of 436 mg/week. The stable age distribution predicted for this population shows maximum numbers in the lower, unexploited age classes, with maximum biomass between 6 and 13 weeks. Biomass shows a second increase in the final age class. In Figure 14, the predicted age distribution of the harvested amphipods, a large range of sizes (0.6 to 2.2 mg) is seen to be included in the harvest. Maximum numbers and biomass values are obtained from the younger age classes in the harvest.
High harvest rates limited to amphipods older than 17 weeks did not reduce the dominant eigenvalue of the modified matrix to 1.00. With a
90% harvest of amphipods larger than 2.2 mg, a relatively large sustainable harvest rate of 15% could still be imposed on all other age classes. This strategy gives a slight increase in yield (to 382 mg/week) over the policy of equal 18% harvest of all age classes. The predicted stable age distribution shows a steady decrease in numbers with increasing age, with maximum biomass at age classes 6 to 17. Numbers and biomasses of age classes 18 and up are very low. Figure 15 shows the predicted yield for this strategy, demonstrating the large size range included in this harvest. Maximum biomass in the harvested fraction is obtained from age class 18.
The best harvest strategy is the 41% weekly harvest of amphipods in the age classes 9 to 17. This policy produces the best yield - 436 mg/ 63
Figure 13. Predicted age distribution of harvested fraction with
40% harvest applied to age classes 0 to 8. NUMBER
c
(7»
o O o o O c. MG DRY WEIGHT 64
Figure 14. Predicted age distribution of harvested fraction with
41% harvest applied to age classes 9 to 17. 6«y ow
age classes 9 - 17) ^ ,80 1800 {4Kof
1600 160.
1400
1200
1000
Ml CO
ZD z
800
600
400
200 65
Figure 15. Predicted age distribution of harvested fraction with
90% harvest applied to age classes >17; 15% harvest
applied to age classes 0 to 17. 6&
1800 v 15°/ of age classes 0-17 ^50% of age classes > 17 v 180.
1600 160.
1400 140.
1200 120.
1000 100.
LU X CO O LU
800 80. >-
Q O 600 60. 3
400 40.
200 20.
0. 25 66
week - and includes a wide size range - 0.6 to 2.2 mg - of amphipods in
the harvest.
The conclusions of this investigation of the effect of exploitation
on population size and structure are tentative. The assumptions and
simplifications made in the construction of the Leslie matrix should he
evaluated by comparing predicted yield and population structure to those
obtained in large scale cultures, subjected to the harvest policies
suggested by the model. 67
7. SUMMARY AND CONCLUSIONS
This study investigated suitable conditions for mass culture of
Eogammarus confervicolus, assessed the potential of the amphipod as a
diet constituent of young fish and, with a Leslie matrix model, explored
the effect of various harvest strategies on yield and population
structure.
_E. confervicolus demonstrated wide salinity and temperature
tolerances, with best survival at low salinities (5 to 10^/00) and
temperatures (5° to 10°C). Growth and survival was reduced at high
population densities (>2 mg/1), despite provision of excess food. The
amphipod may be capable of good growth at increased density with a
flow-through system. Further experiments are necessary to determine
suitable densities for mass culture.
E. confervicolus showed good growth and survival on a variety of
algae and associated epiphytes, demonstrating the broad diet of the
species. Clumping diatoms or phytodetritus are recommended as suitable
foods for large scale culture. Populations were maintained over three
generations, demonstrating the feasibility of long term culture of this
amphipod.
Chemical analyses performed on Eogammarus confervicolus samples show
the amphipod to be a nutritionally satisfactory constituent of fish diets.
In feeding trials, amphipod preparations were readily acceptable to juvenile coho, and provided good short-term growth, comparable to that provided by standard hatchery (Oregon Moist Pellet) diet.
Finally, information about growth, mortality and fecundity of E_. cbrifervicolus under optimal density conditions was used to develop a 68
Leslie matrix model. Harvest terms were applied to various age classes, and predictions made about resulting population structure and weekly yield. A 41% harvest applied to amphipods between 0.6 and 2.2 mg dry weight gave the best sustainable yield of the strategies examined.
Further experiments testing the predictions of the Leslie matrix model are recommended. 69
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