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The influence of environmental factors on the nitrogenous excretion of the spot prawn, Pandalus platyceros Quarmby, Lynne Marie 1983

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C . ( THE INFLUENCE OF ENVIRONMENTAL FACTORS ON THE NITROGENOUS EXCRETION OF THE SPOT PRAWN, PANDALUS PLATYCEROS by LYNNE MARIE QUARMBY B.Sc.,University Of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Deparment Of Zoology We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1983 © Lynne Marie Quarmby, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D F - f i (7/7Q) i i Abstract There exist many c o n f l i c t i n g reports on the influence of environmental factors on the nitrogenous excretion of marine invertebrates. In an attempt to resolve these paradoxes, the composition and rates of nitrogenous excretion of the spot prawn, Pandalus platyceros, were examined under c a r e f u l l y controlled conditions. The animals were incubated for r e l a t i v e l y short periods (1 to 2 hours), keeping b a c t e r i a l density to a minimum: excretion was measured by the accumulation of nitrogenous compounds in the incubation water. Although the incubation water inhibited the a c t i v i t y of urease, t h i s i n h i b i t i o n was overcome by the use of increased concentrations of urease in urea analysis. Ammonium excretion rates of 0 to 10.5 Mg -at NHfl+-N g _ 1 h " 1 were observed, while the rate of excretion of urea ranged from 0 to 5.7 Mg-at Urea-N g~ 1h" 1. These rates were highly dependent on temperature, s a l i n i t y and diet with the effects of each factor determined by l i f e stage. Response to temperature was more complex than changes in metabolic rates and, s i m i l a r l y , reduction in the rate of metabolism due to sa l t stress was not enough to explain the effects of s a l i n i t y on nitrogenous excretion. Young prawns did not excrete as much ammonium as expected on the basis of body si z e . Excretion of dissolved organic nitrogen (DON), other than urea, occassionally constituted a s i g n i f i c a n t proportion of the nitrogen excreted but was highly irregular and on average did not contribute s i g n i f i c a n t l y to the nitrogneous excretion of any study group. Diet influenced the composition and rates of nitrogenous excretion of the euphausiid, Euphausia p a c i f i c a , the amphipod Ampithoe simulans, and the shrimp Eualus pusiolus as well as P.  platyceros. In each case the effect was correlated with the ra t i o of protein to carbohydrate in the d i e t . Urea was an important excretory product of each of the four species of marine Crustacea examined. iv Table of Contents Abstract i i L i s t of Tables v L i s t of Figures v i Acknowledgements v i i I. INTRODUCTION 1 II . MATERIALS AND METHODS 8 A. ANIMALS 8 B. TEMPERATURE 10 C. SALINITY 10 D. DIET 11 E. DRY WEIGHTS 13 F. BACTERIAL COUNTS 13 G. EXCRETION MEASUREMENTS 14 I I I . RESULTS 17 A. TEMPERATURE AND BODY SIZE 17 B. SALINITY AND BODY SIZE 33 C. NUTRITION 51 D. DISSOLVED ORGANIC NITROGEN 59 IV. DISCUSSION 60 A. TEMPERATURE AND BODY SIZE 61 B. SALINITY AND BODY SIZE 63 C. NUTRITION AND SPECIES 66 D. DISSOLVED ORGANIC NITROGEN 7 3 V. CONCLUSIONS ; 7 5 BIBLIOGRAPHY 78 L i s t of Tables Food sources used to vary the n u t r i t i o n a l value, as indicated by the r a t i o of protein to carbohydrate, of diets fed to the study animals 56 v i L i s t of Figures 1. Size dependent e f f e c t of temperature on ammonium excretion at 2 7 ° / o o . A: small males A ; B; large males C: females D. Mean rates (± standard deviation) of A-C . . . . . 2 0 2. Size dependent e f f e c t of temperature on urea excretion at 27°/oo • A: small males A ; B: large males C; large females •; D.Mean rates (± standard deviation) of A-C . . .22 3. Size dependent e f f e c t of temperature on ammonium:urea ( ' 2 7 ° / 0 0 ) . A: small males A ; B; large males •; Cs large females D.Mean rates (± standard deviation) .24 4. Size dependent ef f e c t of temperature on t o t a l N excretion at 2 7 ° / o o . A: small males A ; B: large males C: large females •; D-Mean rates (± standard deviation) 26 5. Dependence of excretion rate on body size at 2.5°C and 2 7 ° / o o • A - Ammonium excretion B. Urea excretion C. Ammonium:urea D. Total nitrogen excretion. . .28 6. Dependence of excretion rate on body size at 5.5°C and 2 7 ° / 0 0 . A. Ammonium excretion B. Urea excretion C. Ammonium:urea D.Total nitrogen excretion. ...........30 7. Dependence of excretion rate on body size at 9.5°C and 2 7 ° / o o • A« Ammonium excretion B. Urea excretion C. Ammonium:urea D.Total nitrogen excretion. 32 8. Size dependent ef f e c t of s a l i n i t y on ammonium excretion at 9.5°C. A: small males A ; B: large males «; C: large females •; and D. Mean rates (± standard deviation) ." .36 9. Size dependent effect-of s a l i n i t y on rate of urea excretion at 9.5°C. A: small males A ; B: large males •; C: large females and D. Mean rates (± standard d e v i a t i o n ) . 38 10. Size dependent ef f e c t of s a l i n i t y on t o t a l N excretion at 9.5°C. A: small males A ; B: large males •; C: large females •»; and D. Mean rates (± standard deviation) 40 11. Size dependent ef f e c t of s a l i n i t y on ammonium:urea (9 .5°C). A: small males A ; B: large males •; C: large females and D. Mean rates (± standard deviation) .42 12. Dependence of ammonium excretion on body size at 9.5°C. A. 2 2 / 0 0 ; B. 2 5 ° / 0 0 ; C. 2 7 ° / 0 0 ; and D. 2 9 ° / o o 44 13. Dependence of excretion of urea on body size at 9.5°C. A. 22°/oo; B. 2 5 0 / 0 0 ; C. 2 7 ° / 0 0 ; D. 29°/oo 46 14. Dependence of rate of t o t a l nitrogen excretion on body size at 9.5°C. A. 22°/oo? B. 2 5 % o ; C. 2 7 % 0 ; and D. 2 9 ° / 0 0 48 15. Dependence of r a t i o of ammonium to urea excretion on body size at 9.5°C. A. 2 2 ° / 0 0 ; B. 2 5 ° / 0 0 ; C. 2 7 ° / 0 0 ; and D. 2 9 / 0 0 • • 50 v i i 16. The influence of n u t r i t i o n on nitrogenous excretion. - ammonium; = - urea (error bars are standard dev i a t i o n s ) . . .TT .....54 17. D i e l migration of euphausiids in Saanich I n l e t , 22-25 June 1981, as observed on an echo sounder at 200 KHz. J represents the depth range of the zooplankton 58 v i i i Ac knowledgement I am very grateful for the freedom of exploration given me by Dr. T.R. Parsons, and for his invaluable assistance when problems arose. To Baron Carswell I am deeply indebted for his supply of prawns and l i v e -holding f a c i l i t i e s . A debt of gratitude i s also owed to Dr. P.J. Harrison for his continued interest in the progress of t h i s work. Dr. A.G. Lewis was especially helpful in the organization of the June, 1981 cruise and I wish to thank him here. As well, a special thanks i s owed to those who worked many long hours on my behalf during that cruise: Maura McKeag, Elaine Nutbrown, Fiona Parkinson, Neil Price, Andy Thomas, Peter Thompson, and Niko Zorkin. To these people I also offer an apology that so l i t t l e of the data they helped to c o l l e c t i s presented in this thesis. I thank Maggie Hampong for her assistance in the preparation of figures. For f i n a n c i a l support during the progress of t h i s thesis I thank the Natural Sciences and Engineering Research Council of Canada for post-graduate scholarships; the department of zoology, University 'of B r i t i s h Columbia, for teaching assistantships; and the faculty of graduate studies, University of B r i t i s h Columbia, for a University Graduate Fellowship. 1 I . INTRODUCTION Regenerated nitrogen is important to primary productivity in the sea (for review see Harrison, 1980). B i o l o g i c a l regeneration may be the most important source of nitrogen for algae in temperate regions in the summer ( e.g. Butler et. a l . , 1970; Vargo, 1979; Butler, 1979) in t r o p i c a l and subtropical oceanic regions ( e.g. Eppley and Peterson, 1979; McCarthy and Goldman, 1979), as well as in estuaries ( e.g. Welsh, 1975; Smith, 1978; Caperon et a l . , 1979). The r e l a t i v e annual contributions of new and regenerated nitrogen varies greatly between areas (for example compare Eppley and Peterson, 1979, with Ikeda and Motoda, 1978, and with Smith, 1978) but regeneration i s often e c o l o g i c a l l y s i g n i f i c a n t . The contribution of marine invertebrate excretion to the pool of regenerated nitrogen i s also highly variable (Caperon et a l . , 1979; Szyper et a l . , 1976; Jawed,1973; Ikeda et a l . , 1982b) and d i f f i c u l t to assess (Mullin et a l . , 1975; Ikeda and Motoda, 1978). Part of this d i f f i c u l t y i s due to the v a r i a b i l i t y of the species composition of marine invertebrate communities and part i s due to the plethora of factors complexly af f e c t i n g the nitrogenous excretion of these organisms. Temperature has long been recognized as a factor a f f e c t i n g ammonium excretion by marine invertebrates. Although investigators have reported increases in the rate of excretion of ammonium with increased temperature (Corner et a l . , 1965, for Calanus spp.; Kremer, 1977, for a ctenophore; Mann and Glomb, 1978, for a bivalve mollusc), the patterns and rates of increase 2 reported are variable. Further, Mace and Ansell (1982) report the rate of ammonium excretion of two gastropod molluscs to be independent of temperature in the range 10 to 20°C. The rate of excretion of free amino acids has also been reported to increase with temperature (Johannes and Webb, 1965; Webb and Johannes, 1967), but S t i c k l e and Bayne (1982) report no change in the rate of excretion of amino acids while ammonium excretion of Thais  l a p i l l u s increased with temperature. L i t t l e is known about the e f f e c t s of s a l i n i t y on the nitrogenous excretion of marine invertebrates. Emerson (1969) found that Thai s lamellosa excreted more ammonia in 100% seawater (35°/ 0 0 ) than in 50% or 150% seawater. Two echinoids were studied by Sabourin and S t i c k l e (1981); they reported no ef f e c t of s a l i n i t y on the nitrogenous.excretion of a sea urchin, while the t o t a l nitrogen excreted by a sea cucumber decreased as the s a l i n i t y decreased from 3 0 ° / o o to 1 5 ° / 0 0 • The drop in t o t a l nitrogen excreted consisted of a large decrease in ammonium released, no change in urea excretion and an increase in amino acid released. S t i c k l e and Bayne (1982) report that the rate of ammonium excretion of Thais l a p i l l u s did not "vary systematically with s a l i n i t y " . However, they did report a peak in ammonium excretion occurring between 20 and 2 5 ° / 0 0 . Amino acid release in their study was not related to s a l i n i t y . It has been c l e a r l y demonstrated that nitrogen excretion rates depend on feeding status (Takahashi and Ikeda, 1975; Ikeda, 1977; Mayzaud and Poulet, 1978). It i s therefore desirable to measure excretion while animals are feeding on 3 concentrations of food comparable to those found in the natural environment. However, phytoplankton uptake kinetics are also variable, and depend on nitrogen le v e l s , nutrient past history, the form of nitrogen available (Eppley and Renger, 1974; Conway and Harrison, 1977; McCarthy and Goldman, 1979), as well as the patchiness of the supply (Turpin and Harrison, 1979; Quarmby e_t a l . , 1982). R e a l i s t i c estimates of zooplankton excretion rates are therefore confounded by the interactions of a l g a l growth, nutrient uptake, grazing and nutrient release. Lehman (1980) has suggested a technique for measuring zooplankton excretion which could overcome these d i f f i c u l t i e s . Our current understanding of algal physiology suggests that nutrient uptake and transport systems of al g a l c e l l s always become saturated when concentrations are high enough. Using t h i s , Lehman (1980) conducted his experiments at saturating le v e l s of nutrients (ammonium and phosphate), where nutrient excretion i s the net rate of change of the nutrient concentrations ( i . e . zooplankton excretion, or net change, was the measured or gross change in nutrients plus the amount removed by phytoplankton uptake which could be calculated from uptake k i n e t i c s ) . Although zooplankton abundance i s generally constant in an experiment of a few hours, phytoplankton abundance may change due to growth and grazing. By integrating over the time period of an experiment, i t is possible to estimate the excretion rates of the zooplankton (Lehman, 1980). Unfortunately, the method described by Lehman (1980) is only v a l i d i f ammonium is the sole component of the nitrogenous 4 excretion. The excretion of an unknown mixture of ammonium, urea, amino acids, and other organic forms of nitrogen prohibits the calculation of phytoplankton uptake. Further, excreted organic nitrogen would not be distinguishable from that exuded by the phytoplankton or released as a result of c e l l damage during grazing e.g. Lampert (1978) reports that 17% of the al g a l carbon ingested by Daphnia pulex was lost as dissolved organic carbon from algae damaged during feeding. For a comprehensive study of nitrogen excretion i t is therefore necessary to incubate the animals without food. E f f e c t s of starvation can be minimized by shortening the incubation time. In larger animals (such as the prawn) th i s i s not a problem but to study the nitrogenous excretion of smaller organisms (such as amphipods), shorter incubation times must be accompanied by smaller incubation volumes for detectable guantities of excretory products to accumulate. These smaller volumes and smaller quantities of product are more e f f e c t i v e l y handled by automated analysis ( e.g. Hawkins and Keizer, 1982). Since nitrogenous wastes result from protein and nucleic acid metabolism one would expect nitrogen excretion to be a function of diet quality as well as quantity. To my knowledge only three studies have examined the e f f e c t of diet on the nitrogenous excretion of marine invertebrates: Corner e_t a l . , 1965, found the ammonium excretion of Calanus spp. to depend on al g a l species, fed, but they did not examine the n u t r i t i o n a l composition of the various a l g a l species; Nelson e_t a_l. , 1979, found the ammonium excretion of an estuarine shrimp to vary with 5 die t , but the excretion was not correlated with the protein content of the diet ; Diehl and Lawrence, 1979, report that ammonium and urea excretion of an asteroid were both unaffected by levels of dietary protein. Differences in d i g e s t a b i l i t y of the d i f f e r e n t diets may have confounded the results of each of these studies. Most investigations of the nitrogenous excretion of marine invertebrates have involved only the measurement of ammonium. In studies where concentrations of dissolved organic nitrogen (DON) have been measured, the DON fraction i s often highly variable ( e.g. Smith, 1978) or not examined in conjunction with measurements of ammonium ( e.g. Webb and Johannes, 1967; Butler et a l . , 1969). Szyer et a l . (1976) and Corner and Newell (1967) found that the amount of DON excreted was increased when the animals were crowded either prior to or during excretion experiments. It i s unclear whether th i s effect was due to food a v a i l a b i l i t y or increased membrane permeability due to stress. Johannes and Webb (1965) and Webb and Johannes (1967) have shown release rates of amino acids by zooplankton to be p o s i t i v e l y correlated with temperature. However, Corner and Newell (1967) f e l t that these results were seriously confounded by the eff e c t s of crowding and found excretion of amino acids to be unimportant. Webb and Johannes (1967) claimed that no effect of the d i f f e r e n t experimental densities could be detected s t a t i s t i c a l l y and suggested that the results of Corner and Newell (1967) were due to decomposition during their longer incubation time (8 to 24 hours). Mayzaud (1973) and Corner and 6 Davies (1971) present data suggesting that the true value of DON excretion l i e s somewhere between the high levels of Johannes and Webb (1965) and the low levels of Corner and Newell (1967) due to the effects of crowding and b a c t e r i a l contamination respectively. However, Corner et a l . (1976) found the same low result with 4 hour incubations, and LeBorgne (1973) found high organic excretion (50%) in 24 hour experiments in the Mauritanian upwelling. LeBorgne (1979) did, however, fi n d excretion of DON decreased while ammonium excretion remained constant over a 24 hour period. Our understanding of the nitrogenous excretion of marine invertebrates i s obviously far from complete. Many of the paradoxical results b r i e f l y reviewed here remain unresolved because important factors have not been controlled. Most commonly, diets were uncharacterized, b a c t e r i a l l e v e l s were not determined, and l i f e stage was not distinguished. Bacteria are important because they take up both amino acids (Hollibaugh, 1978) and urea (Remson et a l . , 1971). Conover and Corner (1968) report nitrogen excretion to be l i f e stage dependent. Further, the importance of urea, amino acids and other forms of dissolved organic nitrogen was rarely recognized. By measuring only ammonium, the ef f e c t of the perturbation on t o t a l nitrogen excretion w i l l remain unknown and t o t a l nitrogen regeneration estimates for nitrogen budgets and dynamic models w i l l be low. In addition, because urea is used by both phytoplankton (Antia et a l . , 1977; Horrigan and McCarthy, 1981 and 1982; Kristiansen, 1983) and bacteria (Remsen et ajL., 1971), the potential exists 7 for the composition of regenerated nitrogen to influence bacteria/phytoplankton as well as phytoplankton/phytoplankton competition in much the same way as the l i m i t i n g nutrient ratios of Tilman (1977). 1 Antia et a l . (1980) go so far as to suggest that the excretion of s p e c i f i c organic nitrogen compounds (e.g. hypoxanthine) may provide opportunities for the co-evolution of zooplankton (e.g. c i l i a t e s ) and favored phytoplankton species (e.g. f l a g e l l a t e s ) . The purpose of the present study was to examine the effects of temperature, s a l i n i t y , n u t r i t i o n and l i f e stage on rates and composition of the nitrogenous excretion of the spot prawn, Pandalus platyceros under controlled conditions. The effects of nu t r i t i o n on the excretion of the amphipod Ampithoe simulans, the euphausiid Euphausia pac i f i c a , and the shrimp Eualus  pusiolus are included for comparison. 1 When species are limited by the same nutrient, the outcome of competition may depend on uptake c a p a b i l i t i e s (uptake rates and a f f i n i t i e s ) . The better competitor for one form of nitrogen may be less successful i f a di f f e r e n t form i s ava i l a b l e . 8 II. MATERIALS AND METHODS A. ANIMALS Pandalus platyceros Brandt, were collected from long-line traps set for 24 hour periods near Horseshoe Bay, West Vancouver, in February and A p r i l , 1983. The prawns were transported in natural seawater back to the West Vancouver Laboratory where they were maintained in an aerated flow-through system of f i l t e r e d (Millipore 5 Mm) natural seawater. Each day an excess of the mussel Mytilus edulis was fed to the prawns. After a minimum holding period of f i v e days, individual animals from three size classes were selected on the basis of carapace length: small males 15-18 mm; large males 25-34 mm; and unegged females 36-43 mm. Animals were henceforth maintained in individual tagged cages in a c i r c u l a t i n g system of aerated, f i l t e r e d (Millipore 0.22 urn) natural seawater under controlled conditions of diet, temperature and s a l i n i t y . The average dry weights of the individuals studied were: small males 0.57 g; large males 4.54 g; and large females 10.5 g. Specimens of the amphipod Ampithoe simulans were co l l e c t e d from mud f l a t s in the booming grounds of the north arm of the Fraser River estuary, Vancouver, B.C., during spring 1982. A 'trapping' method described by Levings (1976) and modified by D. Levy (personal communication) was used: Mesh bags were stuffed with freshly c o l l e c t e d Fucus spp. from English Bay, Vancouver, B. C. and then suspended from a rope between two 2"X 4"X 4' boards at about the middle of the l i t t o r a l zone. Traps were 9 l e f t in the f i e l d from two weeks to one month. In the laboratory the amphipods were gently shaken from Fucus into 20 1 aquaria supplied with a small amount of Fucus serving as cover for the amphipods and substrate for their diet of epiphytic diatoms. Tanks were continuously aerated in a cold room at 10°C under a 12L:12D l i g h t regime. Approximately one-third of the water was replaced twice a week. After a two week holding period (Ikeda and Skjoldal, 1980), the animals were randomly di s t r i b u t e d among experimental treatments. The experimental tanks were supplied with mobius s t r i p s (made of Nitex netting) to provide cover from cannabalization, and to provide substrate for the growth of a pennate diatom described below. The average dry weight of amphipods studied was 0.095 g/animal. In November, 1982, specimens of the shrimp Eualus pusiolus Kroyer were coll e c t e d from the same mesh bags which had been used to c o l l e c t the amphipods. In t h i s case, the bags were suspended from the K i t s i l a n o Yacht Club dock in Vancouver, B.C. at a minimum depth of 2 meters. The shrimp were maintained under the cold room conditions described above for the amphipods including a two week holding period. The average dry weight of the shrimp studied was 0.038 g/animal. Euphausia pac i f ica was studied in s i t u in Saanich Inlet, Vancouver Island, B.C., June, 1981. Animals were coll e c t e d by v e r t i c a l hauls, sorted, placed in containers of f i l t e r e d seawater and returned to depth. The average dry weight of euphausiids studied was 0.013 g/animal. 10 B. TEMPERATURE Animals were maintained at the study temperature for a minimum of 3 days before excretion rates were measured. During this time the measured temperature fluctuated less than ± 0.5°C. In the prawn studies, temperatures of 8.0 and 9.5°C were the result of the natural seawater flow-through system at the West Vancouver Laboratory. With the aid of a r e f r i g e r a t i o n unit, temperatures of 5.5 and 2.5°C were obtained with the prawns experiencing changes of less than 0.2°C per hour. Although the animals are not l i k e l y to experience 2.5°C in nature, the potential for l i v e holding these animals for human consumption made t h i s an interesting temperature to examine. The amphipod and e u l i d experiments were a l l done in the cold room at 10°C. C. • SALINITY S a l i n i t y was measured with a YSI Model 33 conductivity meter. A l l animals were maintained and studied in f i l t e r e d natural seawater and the s a l i n i t i e s are reported with the results of each experiment. In addition, in two of the prawn experiments, s a l i n i t y was lowered by d i l u t i o n with well water from the West Vancouver Laboratory. The prawns experienced changes of less than 1 ° / 0 0 per hour, and were held at the study s a l i n i t y for a minimum of 3 days before excretion rates were measured. To study the changes in nitrogenous excretion of Pandalus  platyceros I chose the lower s a l i n i t y of 22°/ 0o as t h i s i s close to the lower tolerance l i m i t reported by Whyte and Carswell 11 (1982) and the upper s a l i n i t y of 2 9 ° / 0 0 as this is close to the upper s a l i n i t y in which platyceros is found in nature (30.8°/ o o i Butler, 1980). D. DIET Food quality was characterized by the protein to carbohydrate r a t i o of the diet of each group of animals. A l l animals were fed excess food for a minimum of 7 days (for adaptation of digestive enzymes, Mayzaud and Poulet, 1978) before the effects of diet were examined. Scott's (1980) modification of the Folin-phenol method of Lowry and colleagues (1951) was used for protein determination. Diatoms from 100 ml of culture were co l l e c t e d on a 35 mm diameter surface of glass f i b r e f i l t e r . From t h i s , two 6 mm diameter discs (representing 3 mis of culture each) were analyzed. Two blanks (unused f i l t e r paper) and two standards (50 Mg albumin) were analyzed with, each set. The mussels, p e l l e t s and f i s h (described below) were homogenized in buffered seawater and the homogenate treated in the same way as the phytoplankton culture. Replicate 6 mm discs from the sample described for protein were analyzed for carbohydrate by a modification of the anthrone method described by Strickland and Parsons (1972). The discs were placed in disposable test tubes to which 1 ml of deionized d i s t i l l e d water was added, the tubes were mixed with a tube buzzer, allowed to s i t for 0.5 hours, mixed again, then analyzed. The mussel Mytilus edulis, c o l l e c t e d fresh from the dock at 12 the West Vancouver Laboratory, served as the high protein:carbohydrate diet (P:C = 2.34) in the prawn experiments; Oregon Moist Pellets (P:C = 0.40) served as the low protein to carbohydrate d i e t . The temperate, estuarine diatom, Amphiprora hyalina Eulenstein, Northeast P a c i f i c Culture Co l l e c t i o n s t r a i n #B266, University of B r i t i s h Columbia, was used as a food source for the amphipods and the shrimp. This pennate diatom is only approximately 10 um in length but forms aggregates which both species of animals grazed successfully. The diatoms were grown in natural seawater enriched with modified ES medium (Harrison et a_l. , 1980) further modified by replacing Na 2glycerophosphate with NaH2POa to reduce b a c t e r i a l growth and by adding a l l iron in the form FeCl 3-6H 20 to eliminate the addition of ammonium ( a l l nitrogen was supplied in the form of NaN0 3). The diatoms were cultured in the same temperature and l i g h t regime as the amphipods and shrimp. The protein:carbohydrate composition of the c e l l s was manipulated by nitrogen supply, l i g h t quantity and quality (P.J. Harrison, personal communication; Wallen and Geen, 1971) High protein, low carbohydrate c e l l s were obtained by culturing in f u l l enrichment medium, under low intensity blue l i g h t . (The blue l i g h t was obtained by f i l t e r i n g white l i g h t through blue plexiglass.) Low protein, high carbohydrate c e l l s resulted from growth on background levels of nitrogen ( i . e . no nitrogen enrichment) and high intensity white l i g h t . These measurements are reported with the r e s u l t s . The proteintcarbohydrate r a t i o was increased in the Eualus 1 3 pusiolus experiments by feeding one group on fresh frozen 3-spined sticklebacks Gasterosteus aculeatus with a protein:carbohydrate r a t i o of 6.6. Euphausia p a c i f i c a was studied after feeding on naturally occurring phytoplankton. The chlorophyll content of the water column was measured by the ir\ v i t r o fluorometric method as outlined by Strickland and Parsons (1972). E. DRY WEIGHTS Amphipods, eualids and euphausiids were oven-dried at 50°C u n t i l a constant weight was obtained. Prawns were freeze-dried. A l l excretion rates are expressed per gram dry weight. F. BACTERIAL COUNTS Four ml samples were stained for 2 min with 0.2 ml acridine orange solution (1 mg acridine orange in 1 ml 0.22 Nucleopore f i l t e r e d d i s t i l l e d water) f i l t e r e d onto a charcoal-stained Nucleopore (0.22 jum) f i l t e r and counted within 24 h using a Zeiss epifluorescent microscope. Blanks consisted of 2 ml of f i l t e r e d acridine orange solution. In a l l laboratory experiments, the incubation water was checked for b a c t e r i a l density. In the amphipod and eualid studies, incubation water was autoclaved prior to incubation and in the prawn studies i t was f i l t e r e d through 0.22 urn M i l l i p o r e f i l t e r s . Only t r i a l s where the b a c t e r i a l density was not s i g n i f i c a n t l y above background are reported here. 1 4 G. EXCRETION MEASUREMENTS Prawns were incubated i n d i v i d u a l l y in f i l t e r e d seawater in Erlynmeyer f l a s k s . Small males were incubated in 0.5 1 of water for three hours, large males in 1.0 1 for two hours, and large females in 1.0 1 for one hour. Each prawn incubation was i n i t i a t e d at approximately 0900h. Amphipods were incubated in 1 1 screw-top jars with 16-20 animals per 1.0 1 of water for 5-7 hours. Eualids were also incubated in 1 1 jars, with two animals per 1.0 1 of water for 5-7 hours. Two to six euphausiids were incubated in 1.0 1 f i l t e r e d seawater in p l a s t i c bottles at capture depths (see figure 17) for 1-2 hours. The incubation water was always maintained at the study temperature and s a l i n i t y for the incubation period. No food was available to the animals during incubation. Each experiment was terminated by f i l t e r i n g through glass fi b r e f i l t e r s to remove fecal p e l l e t s . F i l t e r s were prewashed with 1 l i t e r of unused incubation water to remove any. ammonium and urea present. Animals were co l l e c t e d for dry weight determination, and the f i l t r a t e immediately analyzed for ammonium, urea and t o t a l dissolved organic nitrogen (DON). Ammonium was analyzed by the phenol-hypochlorite method of Solorzano (1969), as modified by Strickland and Parsons (1972), for a l l experiments. Urea concentrations were determined by the urease method of McCarthy (1970) as outlined by Strickland and Parsons (1972). During the prawn study, urease e f f i c i e n c y was monitored by spiking a subsample of each urea sample with 10 ixq-at urea-N 1 _ 1 15 and analyzing for urea. The excretory products of the prawn apparently inhibited urease a c t i v i t y but t r i p l i n g the concentration of the dil u t e urease solution was usually s u f f i c i e n t to overcome this i n h i b i t i o n . In instances where i n h i b i t i o n was observed the urea concentration was corrected accordingly. Because replicate samples of ammonium and urea were consistently found to be within less than 5% of one another, the precision of these methods r e l a t i v e to the b i o l o g i c a l variance was judged to be such as to make replicate analyses of each incubation unnecessary. The large amount of b i o l o g i c a l variance, however, necessitated replicate incubations. The accuracy of the ammonium analysis was evaluated on three separate occassions by analyzing subsamples of.incubation water from ongoing experiments of another researcher, Mr. Baron Carswell. He measured ammonium with a probe and, in each case, our measurements were within 7% of one another. Due to the problems of i n h i b i t i o n , the accuracy of each urea sample was evaluated i n d i v i d u a l l y r e l a t i v e to a standard. In the case of both urea and ammonium, most of the variance reported here was unexplained b i o l o g i c a l variance rather than experimental error. Dissolved organic nitrogen (DON) was analyzed by the persulphate method as described by Solorzano and Sharp (1980). I found oxidation e f f i c i e n c i e s of 95 to 100% for each of ammonium, urea, and glutamic acid: t h i s i s in contrast to the highly variable and i n e f f i c i e n t (15-27%) oxidation of urea I found by the u l t r a v i o l e t method. Strickland and Parsons (1972) 1 6 also report incomplete oxidation of urea by this method. In addition, Smart and colleagues (1981) report that persulphate digestion i s more precise and more accurate than the Kjeldahl method. 1 7 II I . RESULTS A. TEMPERATURE AND BODY SIZE Temperature had a s t a t i s t i c a l l y s i g n i f i c a n t (two-way ANOVA) effect on the rates of excretion of both ammonium (p=0.0G"l) and urea (p=0.031) by prawns fed on mussels at 26.5°/ 0o • The effect was dependent however, on the size of the prawns (p=0.005 for ammonium and p=0.009 for urea). Large males and large females 1 both showed increases in rates of ammonium excretion (Figures 1B and C), comparable to those observed by Ganf and Blazka (1974) for freshwater zooplankton, while the small males decreased their output of ammonium with increased temperature (Figure 1A). A corresponding increase in urea excretion was observed in the males (Figures 2A and B), but the females excreted less urea at higher temperatures (Figure 2C). Figure 3 i l l u s t r a t e s t h i s change in the composition of the nitrogneous excretion: the ra t i o of ammonium to urea excreted decreased with increasing temperature in the small males (Figure 3A) and increased in the large males and females (Figures 3A and B). The small males' dry weight s p e c i f i c rate of urea excretion was greater than the large animals' at a l l temperatures studied (Figure 2D), but at 9.0°C their dry weight s p e c i f i c rate of ammonium excretion was lower than that of the larger prawns (Figure 1D). Total nitrogenous excretion increased with 1 Ei. platyceros i s male when young and undergoes sex change between i t s second and t h i r d years (Butler,1980,p.141). The females in this study are new females, but I refer to them as large because, r e l a t i v e to the males, they are. 18 temperature in the large animals, and was not influenced by temperature in the small males (Figure 4). 1 Urea excretion decreased with dry weight at a l l three temperatures (Figures 5B, 6B and 7B) as did ammonium at 2.5 and 5.5°C (Figures 5A and 6A). At 9.0°C there i s a peak in ammonium excretion of the mid-size animals (Figure 7A). However, when to t a l nitrogenous excretion was examined, there was a consistent decrease with increasing dry weight at a l l temperatures studied. This t o t a l was composed primarily of ammonium in the larger animals and mainly of urea in the smaller animals as the temperature was increased from 2.5 to 9.0°C. The change in slope of the ammonium:urea versus dry weight plots (Figures 5C,6C, and 7C) from negative to positive with increasing temperature i l l u s t r a t e t h i s change. 1 Although s i g n i f i c a n t quantities of DON were excreted by some prawns, the average contribution to t o t a l excretion by any group was i n s i g n i f i c a n t . Hence " t o t a l " refers to the sum of urea and ammonium excretion. 19 Figure 1 - Size dependent e f f e c t of temperature on ammonium excretion at 2 7 ° / 0 0 • A: small males A ; B: large males •; C: females D. Mean rates (± standard deviation) of A-C. 3 T3 a c "I o A m m o n i u m E x c r e t i o n ( / j g - a t Nl-I^-N g ' V 1 ) p o M pi-uT Ul CO b p • • • r o Q) to ui I to o I A m m o n i u m E x c r e t i o n ( / ug -a t N l j j - N p _» w , n 0) IA) J I • • • • 10 Ul Ul b 0) V 1 ) CD 21 Figure 2 - Size dependent ef f e c t of temperature on urea excretion at 2 7 ° / 0 0 • A: small males A ; B: large males large females •; D.Mean rates (± standard deviation) of A--22-5.204 4 .624 o ii 1_ ? s c g L u •X LU o £ 4.04 4 3.46 4 2 . 8 5 A* 2 . 3 0 -2 . 5 (A) i 5 5 i 9 . 0 Tempera tu re C C ) 4.804 JC a a 1^2.884 o ai ~ 1.924 c o L. U X LU a i) i. 23 0 .96 0.00 4 (B) 2 5 5 .5 8 . 0 9 .2 T e m p e r a t u r e (°C) a 0 . 8 0 H« 0 .6 4 ^ (C) - 0 . 4 8 4 9 3 c o «> i_ u X LU a 0 . 3 2 4 0.164 0 . 0 0 H 2 .5 5 . 5 T e m p e r a t u r e C C ) 9 . 0 z a ai 3 c o u X UJ a 4.54 3.04 5 5 T e m p e r a t u r e C O 8 . 0 9.0 23 Figure 3 - Size dependent e f f e c t of temperature on ammonium:urea (27°/ 0 0 ). A: small males A ; B: large males large females D.Mean rates (+ standard deviation). Ammonium Excretion.Urea Excretion p o p p • _. O Kj cn O to to CD p Ol J _ _I_ -L 3 T J a Ui Ui" n P' > Ammonium Excretion: Urea Excretion P M A-CD IV) 8 Ul p o CD 25 Figure 4 - Size dependent e f f e c t of temperature on t o t a l N excretion at 27°/ 0o • A: small males A ; B: large males •; C large females D-Mean rates (± standard deviation). -26-Toi a i 3 c o t> U X UJ c <u Ol o o +-> ,o 6.8 H G.2A 50 H (A) • fc H 3.8 H 2-5 5.5 Temperature ( ° C ) 9.0 - 13.00' oi z ? 10.54' O) *—* c 2 8.08 • CD 1_ u & 5.62 H c cu O) o •fc 3.16-•2 0.70-« (B) 1 1 2.5 . 5.5 Temperature C C ) —I r— 8.0 9.2 Temperature C C ) Temperature C C ) 27 Figure 5 - Dependence of excretion rate on body size at 2.5°C and 2 7 ° / 0 0 . A. Ammonium excretion B. Urea excretion C. Ammonium:urea D. Total nitrogen excretion. -28--^10.0 + oi z 2 8.0 + O) - 6.0 + c o CO l _ o X u E 3 'c o E E < 4.0 + 2.0 + 0.0 + (A) 00 2.6 5.2 78 10.4 13.0 Dry Weight (grams) ~ 6.0 + Z i o to l_ 3 4.8 + Z 3.6 + oi 3. V -c o t> (. u X LU o i> i_ 3 2.4 + 12 + 0.0 + (B) * n n I I I I I I I I I 0 0 2.6 5.2 7.8 10.4 13.0 Dry Weight (grams ) | 1 3 0 u u x w 10.4 a o i_ D c o % L. U X LU = 2 6 + 7.8 + 5.2 + 0.0+, (C) 0.0 I 1 1 I I I I 2.6 5.2 7.8 10.4 Dry Weight (grams) 13.0 * i 16.0 + i oi 5 12.8 + o Ol c o n L. U X LU 9.6 + 6.4 b 3.2 + 0.0 0.0 (D) 2.6 5.2 7.8 10.4 Dry Weight (grams ) 13.0 29 Figure 6 - Dependence of excretion rate on body size at 5.5°C and 2 7 ° / 0 0 . A. Ammonium excretion B. Urea excretion C. Ammonium:urea D.Total nitrogen excretion. i -30-' o> 10.0 + z I 5 8 . 0 + 6 . 0 + (A) 4 . 0 + •? 2 0 -0 . 0 + , 0 . 0 2.6 5.2 7.8 1 0 . 4 13.0 Dry Weight (grams) T _ 6.0 + Z 4 8 + o ~ 3.6 + oi 3. c g a> L. U X LU 2.4 1 2 + <D 3 0 .0- fe (B) H—I—I—I—h 0.0 2.6 5.2 7.8 10.4 13.0 Dry Weight (grams) § 1 3 . 0 -U LU 10.4-a u L. (C) c g o L. U X u E C o E E < 7.8 + 5.2 + 2.6 + 0.0+, 0 .0 2.6 5.2 7.8 10 .4 13.0 Dry Weight (grams) 16.0 + (D) oi Z 12.8 + cn 3 c g a; L. U X LU c ai o L. 9.6 + 6.4 + b 3.2 + a +^  ,o 0 . 0 + . 0 . 0 2.6 5.2 7.8 1 0 . 4 13.0 Dry Weight (grams) 31 Figure 7 - Dependence of excretion rate on body size at 9.5°C and 2 7 ° / 0 0 . A. Ammonium excretion B. Urea excretion C. Ammonium:urea D.Total nitrogen excretion. -32-10.0-z z o 8.0-Ol c o 6.0-Excret Excret 4.0-ionium 2.0-Amnr 0.0-~ ** * ( A ) _ 6.0 + M—I—hH—I I I I I | I 0.0 2.6 5.2 7.8 10.4 13.0 Dry Weight (grams ) _ 3.6 oi z a 0) i_ o I Ol 3 C g <u t-u X LU 4 8 + ** 2 4 + 12 + o i . o.o-h (B) « 0.0 2.6 5.2 7.8 10.4 13.0 Dry Weight (grams) c c ~ 13.0 + u X LU O c g L. U X £ 3 10.4 + 7 8 + 5.2 E 2.6 + (C) ^ 16.0 o . o + i . M l I I I I I I I 0.0 2.6 5.2 7.8 10.4 Dry Weight (grams) 13.0 ci. Z 12.8 + oi | 9.6 + o u x 6.4-u c il Ol 2 3.2 I 0.0' (D) M i l l — I I I I—I—I—h 0.0 2.6 5.2 7.8 10.4 13.0 Dry Weight (grams) 33 B. SALINITY AND BODY SIZE Ammonium and urea excretion of prawns fed mussels at 9.0°C were both s i g n i f i c a n t l y affected by s a l i n i t y (two-way ANOVA, P<0.001 for both). The effect was highly dependent on the size of the animal for both ammonium and urea (p<0.00l). Both size classes of males showed a maximum in ammonium excretion at 2 5 ° / O 0 (Figures 8A, B and D). Females on the other hand showed a minimum rate of ammonium excretion at 2 7 ° / 0 0 increasing as s a l i n i t y increased or decreased. This was accompanied by a minimum in urea excretion at 2 5 ° / 0 0 for the small males (Figure 9A), a maximum at 2 5 ° / 0 0 for the large males (Figure 9B), and a decrease in urea excretion with s a l i n t i y for the large females (Figure 9C). The large males excreted as much or more ammonium than the other size classes at a l l s a l i n i t i e s studied (Figure 8D) whereas they excreted more urea than the others only at the lower s a l i n i t i e s -- at 2 9 ° / 0 0 the small males excreted the most urea (Figure 9D). Total nitrogen excretion in the. small males did not seem to be influenced by s a l i n i t y (Figure 10A and D) while the large males showed a peak at 2 5 ° / 0 0 (Figure 10B) and the large females a minimum at 2 7 ° / 0 0 (Figure 10C). The large females excreted an increasing proportion of ammonium at increasing s a l i n i t i e s (Figure 11C and D). The small males excreted generally more urea than ammonium at a l l s a l i n i t i e s studied (Figure 1IA) and, conversely, the large males excreted generally more ammonium than urea (Figure 11B). At a l l s a l i n i t i e s the highest ammonium excretion rates were 34 observed in the mid-size range (Figure 12). Urea excretion showed this same pattern at 25°/oo but increased with dry weight at 27 and 29°/ 0o and was apparently not effected by body size at 2 2 ° / 0 0 (Figure 13). Total nitrogenous excretion decreased with increasing dry weight at 27 and 29°/ 0o (Figures 14C and D), but at the lower s a l i n i t i e s animals in the mid-size range excreted the most nitrogen (Figures 14A and B). The r a t i o of ammonium excretion to urea excretion does not seem to be affected by s a l i n i t y except at 29°/ 0o where there i s a dramatic increase in the r e l a t i v e amount of ammonium excreted by larger animals(Figure 15). 35 Figure 8 - Size dependent ef f e c t of s a l i n i t y on ammonium excretion at 9.5°C. A: small males A ; B; large males C large females and D. Mean rates (± standard deviation). A m m o n i u m E x c r e t i o n ( ug-cit NH^- N g 1 h 1 ) D O Iv) 6 rv> IA) V) ro Q 3 CO »-*• K l\J ^ CD o IV) 0 —l\) N 0) IV) ."0 b o oo IV) J l_ to -fc> _J_ O) 0) o CO IV) o n A m m o n i u m E x c r e t i o n ( / ug -a t NI -^-N g 1 h 1 ) -* I V ) t d J > m O ) - g C O ( O o o o b o o o o o o b A m m o n i u m E x c r e t i o n ( j j g - a t NH - N g h ) 4 P ^ N w A o» O iv> J>. <D rv> o i> CO IV) cn o IS o IV) CO J> (/) ro o 5 CO *< IV) _o 0) 0 ro ro >J O) ro (0 b J _ _ l _ J L J_ _ l _ -L. > A m m o n i u m E x c r e t i o n ( / u g - a t N H ^ - N g " 1 h"1) - » (V) CO CO fO '-• co b) to '-» io O © IV) CO .J> O I CO I 37 Figure 9 - Size dependent ef f e c t of s a l i n i t y on rate of urea excretion at 9.5°C. A: small males A ; B: large males • large females «»; and D. Mean rates (± standard deviation) -38-5 .20 + (A) O) 4 1 6 -F o o O) 3 A 312 + c 2 . 0 8 + o % 1. 0 4 + LU O £ 0 . 0 0 + , ' I I I 1 I I I I 22.0 2 3 4 2 4 8 2 6 . 2 27.6 S a l i n i t y (•/<,.) 2 9 . 0 .c • 6 0 0 + 4 8 0 + . Z 3 .60 + O) 3 C o L u X LU 3 2 . 4 0 + 1. 2 0 -0 . 0 0 -(B) ) I I I i l I 2 2 . 0 2 3 . 4 24 .9 2 6 . 3 27 .8 2 9 . 2 S a l i n i t y ( °/oo ) 39 Figure 10 - Size dependent e f f e c t of s a l i n i t y on t o t a l N excretion at 9.5°C. A; small males A ; B: large males large females »; and D. Mean rates (± standard deviation) -40-'01 9.10 + 01 3 C o U X LU c <D Oi o 0 —' , o 7 . 4 8 + 5 . 8 6 + 4.2 4 + •t 2 .62 + 1 oo+_ I I I I I I 22.0 2 3 . 4 ( A ) 7 15 .40 + 2 4 . 8 26 .2 27.6 S a l i n i t y (•/„.) 2 9 . 0 12.64 + 3 J 9 8 8 + cu c o ^ 7 .12+ -c cu oi o - 4 . 3 6 + o 1.60 + (B) It 'I I I I I | I | | 22.0 2 3 . 4 2 4 . 9 26 .3 2 7 8 S a l i n i t y (•/,.) 29.2 o i z o Ol 3 C o e u X LU c <u Ol o a , o 5 . 4 0 + 4. 4 2 + 3 . 4 4 + 2 . 4 6 1.48 + 0.50 + (C) 22 0 23 .4 2 4 8 26.2 2 7 . 6 2 9 . 0 S a l i n i t y (•/,.) oi z Ol 3 C o c cu Ol o O o S a l i n i t y (%<>) 41 Figure 11 - Size dependent ef f e c t of s a l i n i t y on ammonium:urea (9.5°C). A: small males A ; B: large males C: large females and D. Mean rates (± standard deviation). -42-S a l i n i t y > S a l i n i t y {•/.. ) 43 Figure 12 - Dependence of ammonium excretion on body size 9.5°C. A. 22/ 0 0 ; B. 25°/00; C. 27°/00; and D. 2 9 % o -44-10.0 + 8 . 0 + 6 . 0 + 4 . 0 + 2 . 0 + * ( A ) 0 . 0 + i 1 I I I +—I h 0 0 2 .6 5.2 7 . 8 10 .4 13.0 D r y Weight ( g r a m s ) cn Z . z cn ' c o to L. o X LU E 3 C o E E < 10 .0 + 8 . 0 + 6 .0 + 4 . 0 + 2 0 + (B) 0 . 0 + . —1—I—I—I—I—I—I—P-+ 0 0 2 . 6 5.2 7 .8 10.4- 13.0 Dry Weight ( g r a m s ) 10.0 + 8 . 0 + 6 .0 + 4.0+^ » 2.0+i 0.0 + I I I I (C) * , *». 0 0 2 .6 5 .2 7.8 1 0 . 4 13.0 D r y Weight ( g r a m s ) cn Z 9 cn 3 c o o X LU E 3 C o E E < 1 0 . 0 + • 8 . 0 + -6 . 0 + 4-.0+-2.04-0 . 0 + • . (D) 0 . 0 2 .6 5 .2 7 . 8 10.4- 13 .0 D r y Weight ( g r a m s ) 45 Figure 13 - Dependence of excretion of urea on body size 9 . 5 ° C . A . 2 2 % o ; B. 2 5 ° / 0 0 ; C 2 7 ° / o o ; D. 2 9 ° / o o -46-oi 6 . 0 + (A) 6.0 + (B) o co 3 O) 3 C g co L. U X LU O CO 1_ 3 4 . 8 + 3 . 6 -2 - 4 + 1 2 + 0 . 0 + 1 1 I I I 1 I 1 1 | | 0 . 0 2.6 5.2 7 .8 10.4 D r y We igh t ( g r a m s ) 13.0 2 4 8 ' o CO ~ 3.6 + O) 3 C o u X LU • CO (. 2 4 + 1 2 + 0 . 0 + \ 1 1 1 1 1 1 1 h-0 .0 2 .6 5 .2 7 . 8 10 .4 D r y We ight ( g r a m s ) 13.0 ~ 6 . 0 + O) a 10 1_ 3 3 C o 1_ o X LU O <0 t_ 3 4 . 8 3 6 + 2 . 4 + * 1.2 0 . 0 + , * * » (C) I i i i r 0 . 0 I I I I 2.6 5.2 7 .8 1 0 . 4 D r y W e i g h t ( g r a m s ) 13.0 6 . 0 + ai Z 4 8 + a co 1 3 . 6 + * o 3 ) 3 C o •4-1 CO [_ o X LU D CO !_ Z) 2 . 4 + 1 2 + 0 . 0 + (D) H — I — I 1 — I — I — I h — 0.0 2 . 6 5 . 2 7 . 8 1 0 . 4 D r y W e i g h t ( g r a m s ) 1 3 . 0 47 Figure 14 - Dependence of rate of t o t a l nitrogen excretion on body size at 9.5°C. A. 22°/00; B. 2 5 ° / 0 0 ; C. 2 7 ° / 0 0 ; and D. 29°/oo • -48-7c 16.0-i cn Z S 12.8 + 9 . 6 + S.4 + c o i) 1_ u UJ c (1) cn O h 3 2 + Z G £ 0 . 0 + ^ 0 .0 (A) T 16.0 + cn r 12.8 + cn 3 c o % 9 6+ l_ U X UJ c O) O 1_ H— I 1—I—I 1—I—I—p 2 .6 5.2 7.8 10.4 13.0 D r y W e i g h t (grams) 6.4 + | 3.2 + a £ op+ (B) I—I—I—I—I—I—I—I—I-0.0 2.6 5.2 7.8 1 0 4 D r y Weight ( g r a m s ) 13.0 16.0 + 3 12.8 + o 9 6 -UJ 6.4-3 2 + £ 0 . 0 + ^ 0 .0 * (C) it* -I—I—I—I—I—I—r-2.6 5 .2 7 .8 10 .4 D r y W e i g h t ( g r a m s ) 13.0 ' r 16.0 + r — I cn Z o 12.8 + i cn 3 c o -4—1 <u L. u X UJ c il cn o .2 9 6 + 6.4 + i 3.2 + 0.0 + (D) 0.0 4 1 1 1 1 1 1 1 1 h 2 .6 5 .2 7.8 10.4 D r y W e i g h t ( g r a m s ) 13.0 49 Figure 15 - Dependence of r a t i o of ammonium to urea excretion on body size at 9.5°C. A. 22°/00; B. 2 5 ° / 0 0 2 7 ° / 0 0 ; and D. 29/ 0 0 . -50-§ 13.0 + (A) CO L . u x L U O CO L. 10.4 + 7.8 + c o ? 5.2 + u x L U E 'c o 2 6 + 0.0- * * * 0.0 I I I I I 2 .6 5.2 7.8 10.4 D r y W e i g h t ( g r a m s ) 13.0 J 13.0 + I 10.4 + o co L . c g CO (_ ( J X L U E 3 'c o E < 7.8 + 5 2 + 2 . 6 + * 0.0+, (B) 0.0 H r— I 1—+ 2.6 5 . 2 7 . 8 10.4 D r y W e i g h t ( g r a m s ) 13.0 c o o L. O X L U o CO L. c o c o E E < 13.0+ 10.4+ 7 . 8 + (C) h 5 . 2 + 2 6 + 0.0-0.0 H — I — I — I — F H — | — \ 2.6 5.2 7.8 10.4 D r y Weight ( g r a m s ) 13.0 c o 13.0 + co o * 10.4-L U CO e O CO (_ u X L U 7.8. I + 5 2 + 5 2 . 6 + (D) * .-4. 0.04. 0.0 2 6 5.2 H I 1 b 7.8 10.4 13.0 D r y Weight ( g r a m s ) 51 C. NUTRITION A l l four species of crustaceans studied showed changes in their nitrogenous excretion with a change of diet (Figure 16). Large male prawns at 9°C and 2 8 ° / 0 0 consistently excreted more ammonium than urea and both components decreased as the protein to carbohydrate r a t i o of the diet was decreased (Figure 16A and Table 1). After one week of fasting, the animals were excreting s l i g h t l y less than those fed on the diet of p e l l e t s . The shrimp (studied at 10°C and 30°/ Oo ) excreted more ammonium than urea when fed a high protein diet of dead sticklebacks (see table 1 for descriptions of d i e t s ) . But when the protein content of the diet was lowered by feeding phytoplankton to the animals, the rate of excretion of ammonium decreased proportionately more than that of urea so that the two products were excreted at approximately the same rate (Figure 16C). The amphipods (studied at.10°C and 2 1 ° / 0 0 ) were fed four n u t r i t i o n a l l y d i f f e r e n t cultures of Amphiprora hyalina (Table 1). The t o t a l nitrogenous excretion of the amphipods decreased consistently as the protein content of the diet decreased. With the higher protein:carbohydrate.this decrease was due mainly to a decrease in the amount of urea excreted; with lower protein content in the diet, the decrease in excretion was due primarily to a drop in ammonium excretion (Figure 16D). The euphausiids were studied _in_ s i t u (9°C and 3 0 ° / o o ) and a n u t r i t i o n a l analysis of their diet is not available. The differences in nitrogenous excretion reported in figure 16B are 52 associated with d i e l migration of the euphuasiids (Figure 17). During the night the euphausiids were in the phytoplankton layer (0-30 m) and during the day they were below i t ; thus a large part of the d i e l differences in nitrogenous excretion may be due to feeding status. At dawn the animals would presumably have been feeding during the night and most recently on phytoplankton c e l l s with the highest protein to carbohydrate r a t i o . It is unlikely that the animals were feeding a c t i v e l y during the day, while at dusk they would have fed only minimally, and on phytoplankton with the lowest protein to carbohydrate r a t i o . The euphausiids excreted only low levels of ammonium and thi s did not d i f f e r between dawn and dusk. However they did excrete large amounts of urea and the rate of t h i s excretion was considerably lower at dusk than at dawn (Figure 16B). 53 Figure 16 - The influence of n u t r i t i o n on nitrogenous excretion. - ammonium; ==- urea (error bars are standard deviations) . -54--—s '.C cn Z 6 _ +-> o cn 5 3 —" ion 4 -+•> 0) i_ 3 — u X LU 2 — c a CO o 1 _ z (A) Pcindalus p l a t v c e r o s P e l l e t s M u s s e l s F a s t e d cn z a i cn 3 1) L. O X UJ c <D cn o 30 4 25 H o 20 H 15 " J (B) Euphausia p a c i f i c a D a w n Dusk i cn S 90 cn 3 C o <D 1_ u X LU c 0; co o SOH 30H 118.2 | Eua lus pus io lus (C) F i s h P h y t o p l a n k t o n «1~ 'cn z 9-c cn 3 C 6-o ID (_ u X LU 3-C CO o Z 15.9 T (D) A m p i t h o e s i m u l a n s i -4-4 2.5 1.6 0.4 P r o t e i n : C a r b o h y d r a t e of D i a t o m 55 Table I - Food sources used to vary the n u t r i t i o n a l value, as indicated by the r a t i o of protein to carbohydrate, of diets fed to the study animals. -56-Food Sources An imal D i e t P:C Prawns M u s s e l s P e l l e t s 2.3 0.4 E u a l i d s A . h y l i n a 0.6 S t i c k l e b a c k 6.6 Amphipods A . h y l i n a 4.4 A . h y l i n a 2.5 A . h y l i n a 1 .6 A . h y l i n a 0.4 E u p h a u s i i d s P h y t o p l a n k t o n ? h i g h P h y t o p l a n k t o n ?low Comments f r e s h d a i l y Oregon M o i s t P e l l e t s no N added 100 uEin nr 2 s~ 1 f r e s h l y thawed N - e n r i c h e d 20 vEin m - 2 s" 1 , N - e n r i c h e d 15 u E i n n r 2 s" 1 , no N added 130 MEin nr 2 s" 1 no N added 100 MEin nr 2 s" 1 , white b lue b lue , white , white end of n i g h t c a . 46Mg c h l a 1"1 end of day c a . 46Mg c h l a 1"1 1 P :C i s P r o t e i n r C a r b o h y d r a t e r a t i o . 57 Figure 17 - D i e l migration 22-25 June 1981, as observed represents the depth of euphausiids in Saanich Inlet, on an echo sounder at 200 KHz. range of the zooplankton. -58-O i 20 H 40 H f 60 Q 80 H 100 H t t • 120 1 1 1 | 1 I I I | I I | I | | | | | | | | | ! | | 0100 0500 1000 1500 2000 2500 Time (hours) 59 D. DISSOLVED ORGANIC NITROGEN Excretion of dissolved organic nitrogen (DON), excluding urea, was not correlated with any of the factors studied here. Although not many animals excreted measurable quantities of DON, a few animals from each study group, and each set of conditions, did. DON excretion (other than urea) measured in the present study ranged from below detection to 12.9 yg-at N g~ 1h~ 1. In a few experiments measurements were not possible due to high background levels of DON. 60 IV. DISCUSSION Nitrogenous excretion is a dire c t result of protein and nucleic acid metabolism and i t can therefore be expected that excretory rates w i l l be influenced by the same factors which influence metabolism. As well as being influenced by environmental conditions, species, body size and n u t r i t i o n (factors studied here), metabolic rate is also affected by various rhythms and by voluntary a c t i v i t y . Because the molt cycle could p o t e n t i a l l y influence excretion (Regnault, 1979), the molts of each individual prawn were recorded and later related to excretion. Eleven prawns molted during the study, but I was unable to a l l o t any variance in excretory rates to stage of molt cycle. Seasonality also influences the metabolism of many animals (Lockwood, 1968) and has been reported to affe c t the excretion of zooplankton (Conover and Corner, 1968; Bayne and Scullard, 1977; Hawkins et a l . , 1983). The diet studies with the prawn were done in the winter while the temperature and s a l i n i t y experiments were conducted in the spring. This may account for the s l i g h t differences in measurements made under the same conditions in the two studies but does not affe c t the discussion of results as each experiment in autonomous. This fact should however be kept in mind when making comparisons of absolute rates with other studies. Each excretion measurement was made at mid-morning to decrease differences due to dail y rhythms. Unfortunately, the prawns seemed to be on an irregular 2-3 day cycle. Excess fresh mussel was made available to each prawn everyday but individuals would often go one or two days 61 without eating. Whether or not an individual had been eating over a given period was noted and these notes can explain some of the residual variance. To minimize differences due to voluntary a c t i v i t y , a l l animals were handled minimally and incubated in the dark. A. TEMPERATURE AND BODY SIZE The metabolic processes of poikliothermic animals could be expected to increase with temperature in the same way as other chemical reactions. However, some crustaceans have been shown to have homeostatic regulation of their metabolic rate, the degree of regulatory a b i l i t y depending on species and time given for adjustment (Lockwood, 1968, pp. 136-138). G i l l e s (1975a) reviewed more recent work which demonstrated that metabolic acclimation to temperature was more complex than changes in rates. The results presented here support t h i s conclusion. The large males increased their output of both ammonium and urea exponentially with increasing temperature indicating an absence of s i g n i f i c a n t regulatory a b i l i t y for r e l a t i v e l y short time -scale changes in temperature (their metabolism may adjust seasonally). The small males on the other hand showed no change in t o t a l nitrogen excretion associated with changes in temperature. There was however a change in the composition of the nitrogenous excretory products: with increased temperature these animals excreted more urea and less ammonium. The increase in urea excretion could be simply a result of increased rates of metabolic a c t i v i t y including formation of urea via the ornithine cycle, the action of arginase on dietary arginine, or 62 from a l l a n t o i c acid acted on by allantoicase (Prosser, 1961). Since this increase in urea excretion is associated with a decrease in ammonium excretion, these young animals may lack urease. In the metabolism of purines, a l l a n t o i c acid breaks down to urea which, in the presence of urease, is broken down to ammonium. In the temperature range studied, the a c t i v i t y of urease would increase with temperature. Since ammonium excretion was decreasing with temperature i t i s unlikely that urease was present. It i s possible that ammonium, from the deamination of amino acids, i s being converted to urea by the ornithine cycle hence decreasing the amount of ammonium excreted and increasing the excretion of urea. However, the large males showed increases in both urea and ammonium excretion. Total nitrogenous excretion by the small males did not increase with temperature indicating that either anabolism was increasing to match the increase in metabolism or metabolism was limited by something other than temperature. Since the composition of the nitrogenous waste changed, the f i r s t explanation is most l i k e l y ( i . e . 'waste' ammonium i s picked up before being excreted and used to bui l d amino acids and proteins). The metabolism of the large females also seemed to compensate for a short term temperature change. Although t o t a l nitrogen excreted by these animals increased with temperature, the increase was not as great as i t was for the large males (Q 1 0 of ca. 3 compared to ca. 8 for the large males). This was in part due to a decrease in the output of urea with increasing temperature. An increase in urease a c t i v i t y ( i f thi s enzyme is present in these older 63 animals) could explain this r e s u l t . However, the increase in ammonium was not s u f f i c i e n t to account for a direct e f f e c t of temperature on protein and purine metabolism (Q 1 0 for ammonium of ca. 4 compared to ca. 6 for the large males). This difference could be accounted for by a switch to reproductive metabolism at the higher temperatures. Mann and Glomb (1978) reported a switch in metabolism of a bivalve mollusc from one concerned with accumulation of reserve material to one of gamete formation at elevated temperatures. Although a l l females used in this study were unegged, i t is possible that at the higher temperatures more nitrogen was directed to reproductive development. The expected relationship between metabolism and body size ( e.g. Parsons et aj^. , 1 977 , p. 135) was observed for urea and to t a l nitrogen excretion at a l l temperatures. Ammonium excretion was also observed to decrease with increasing body size at the lower temperatures, but at 9°C the smaller animals excreted less ammonium than expected. This i s explained above as an increased allotment of nitrogen to growth. B. SALINITY AND BODY SIZE Prawns are reported to be poor osmoregulators: Panikkar (1968) found Pandalus montagui isosmotic from 20 to 3 5 ° / 0 0 an<3 Whyte and Carswell (1982) observed 50% mortality of adult Pandalus platyceros after 72 hours at 2l.6°/ 0 0 and at >35°/ 0o • Although these animals may be isosmotic with their environment, the ionic composition of their blood d i f f e r s from that of seawater (Lockwood, 1968, pp.10-13). Changes in s a l i n i t y could 64 be expected to influence the nitrogenous excretion of prawns for several reasons: (1) As tolerance le v e l s of s a l i n i t y are approached, nitrogenous waste from protein metabolism would be expected to decrease as a result of lowered rates of metabolism. (2) These animals may have limited osmoregulatory a b i l i t y and respond to lower s a l i n i t y by deamination, or excretion, of osmotically active amino acids (reported in molluscs by Bartberger and Pierce, 1976, and in Neomysis by Armitage and Morris, 1982) and a corresponding increase in ammonium excretion. This increase in ammonium production could be accompanied by an increase in urea excretion i f the ornithine cycle i s active. (3) The ammonium ion may be excreted in an active exchange for other cations (such as the sodium ion), and the need for thi s type of exchange may increase as s a l i n i t y decreases. At 9.5°C, I found a peak in both ammonium and urea excretion by large males at 2 5 ° / 0 0 • S t i c k l e and Bayne (1982) observed the same pattern in the ammonium excretion of Thais  lamellosa. This could be explained as an optimal s a l i n i t y for metabolism, except that Whyte and Carswell (1982) found a steady increase in metabolism (measured as oxygen uptake) from 20 to 30°/oo at 10°C for large male E\ platyceros. The lower rates of excretion at 2 2 ° / 0 0 are l i k e l y d i r e c t l y related to the drop in metabolism. The decrease in nitrogenous excretion which occurred at the higher s a l i n i t i e s may have been due to a decreased need to exchange ammonium for other cations, and/or a decreased rate of deamination of amino acid pools in response to 65 decreased osmotic pressure. The concurrent decrease in urea excretion was l i k e l y due to increased urease a c t i v i t y associated with increased metabolism. The constant two-to-one r a t i o of ammonium excretion to urea excretion suggests that the ornithine cycle was active in these animals. The small males also showed a peak in ammonium excretion at 25°/oo i but urea excretion was at a minimum at t h i s same s a l i n i t y . The increasing rate of urea excretion at higher s a l i n i t i e s is probably d i r e c t l y related to purine metabolism. This uncoupling of urea and ammonium excretion i s further evidence for the lack of urease a c t i v i t y in the young animals as discussed above. The increase in urea at lower s a l i n i t i e s may be a mechanism of ion regulation ( i . e . excretion of a neutral molecule rather than a cation). The a c t i v i t y of urease in the older animals may preclude t h i s option for ion regulation. It should be noted that the s a l i n i t y tolerances reported by Whyte and Carswell (1982) were for large male prawns, and that juveniles are often found in shallower water of lower s a l i n i t i e s (Butler, 1980). The urea excretion of large females decreases steadily over the range of s a l i n i t i e s studied, probably in di r e c t r e l a t i o n to increased urease a c t i v i t y . Ammonium excretion on the other hand shows a minimum at 2 7 ° / 0 0 . This suggests that the large females may have a greater low s a l i n i t y tolerance than the large males studied by Whyte and Carswell (1982). While the increase in ammonium excretion at higher s a l i n i t i e s was l i k e l y a direct function of increasing metabolism, the increase at lower 66 s a l i n i t i e s may be a mechanism of ionic balance. The dramatic increase in the ra t i o of ammonium excretion to urea excretion with increases in metabolism (with increased s a l i n i t y and with increased temperature) i s good evidence for the presence of active urease in these animals. At a l l s a l i n i t i e s studied, ammonium excretion of smaller animals was less than would be expected based on the relationship between metabolism and body size (Parsons et a l . , 1977, p.135). However, th i s i s not surprising as the smaller animals are also the younger and nitrogen excretion i s only d i r e c t l y related to basal metabolism. Where active growth i s occurring, anabolism of 'waste' ammonium accounts for the difference between 'expected' and actual excretion. What i s interesting i s the. lack of thi s difference in urea excretion at the higher s a l i n i t i e s . Since ammonium may be synthesized d i r e c t l y into amino acids, whereas urea must f i r s t be broken down to ammonium, I suggest that the lack of relationship between urea excretion and anabolism i s further evidence for the lack of active urease in the young prawn. C. NUTRITION AND SPECIES It is well established that an increase in the quantity of food ingested results in an increase in nitrogenous excretion. Takahashi and Ikeda (1975) measured the excretion of Euphausia  pac i f ica and Me t r i d i a pac i f ica fed on di f f e r e n t concentrations of phytoplankton; ammonium excretion increased with the amount of chlorophyll ingested. S i m i l a r l y , Corner and colleagues (1965) observed increases in the ammonium excretion of Calanus 67 spp. with greater concentrations of food; LaRow and colleagues (1975) found the same result when studying herbivorous 1imnoplankton as did Nelson and colleagues (1979) in studies of Crangon franciscorum. These observations were based on the increase in quantity of a single food type. Because the composition of the diet i s not changing and because the animals would be expected to assimilate required nutrients in constant proportions, i t is not surprising that ammonium excretion increases with quantity consumed. The more obscure influence of food quality on excretion may be as ec o l o g i c a l l y important as the ef f e c t s of food quantity. Nitrogenous waste results primarily from tissue catabolism and metabolism of dietary proteins and nucleic acids. Because nucleic acids compose only 1-5% of ingested nitrogen, depending on diet (Prosser, 1961), a reasonable estimate of dietary nitrogen can be obtained from measurements of protein content. I assumed that tissue catabolism in well fed animals would be minimal and constant, and that by feeding my laboratory animals to saturation, I would be able to attribute changes in nitrogenous excretion to changes in the protein:carbohydrate r a t i o of the d i e t . Higher rates of nitrogen excretion were observed in a l l four crustaceans when on diets of greater protein content. This result does not agree with the results of similar studies reported in the l i t e r a t u r e . Nelson and colleagues (1977) found that the protein content of the diet of a freshwater prawn did not influence i t s rate of excretion of ammonium; neither 68 ammonium nor urea excretion by an asteroid was affected by dietary protein content (Diehl and Lawrence, 1979); DeJorge and colleagues (1969) found an increase in the urea excreted by a t e r r e s t r i a l gastropod related to an increase in dietary nitrogen, but they reported no change in the rate of excretion of ammonium. The rate of excretion of nitrogen is expected to increase in proportion to protein metabolized. Available protein may not be ingested, or upon ingestion, may not be digested. Further, of the protein digested, the amino acid composition may not match the requirements of the animal -- those present in excess would be broken down and excreted. One way to control these differences i s to feed the animals a single type of diet in which the protein content has been manipulated. The present study of amphipods took t h i s approach. Although the t o t a l nitrogen excreted decreased consistently with decreasing protein content, the composition of the excretory products changed. The i n i t i a l drop consisted primarily of a decrease in the rate of excretion of urea. This suggests that arginine was one of the most important constituents lost in the decrease of protein content. At the lower lev e l s of protein to carbohydrate the decrease in production of waste nitrogen resulted from a decrease in ammonium excretion. Most of thi s ammonium i s probably coming from deamination of amino acids. The uncoupling of ammonium and urea excretion indicates that the enzymes of the ornithine cycle and urease are not highly active, and supports the suggestion that the urea excreted by thi s animal results 69 p r i m a r i l y from d i e t a r y a r g i n i n e . The r a t e s of ammonium e x c r e t i o n r e p o r t e d here are comparable to those r e p o r t e d by Hawkins and Reizer (1982) f o r a benthic marine amphipod when a p p r o p r i a t e c o r r e c t i o n s f o r s i z e and temperature are made (1.7 to 9.6 jug-at NH 4 + g~ 1h~ 1 ) . The euphausiids were s t u d i e d j j n s i t u and the change i n t h e i r d i e t can only be i n f e r r e d . I t may c o n s i s t of changes i n food type and q u a n t i t y as w e l l as changes i n q u a l i t y . There may a l s o be f a c t o r s other than n u t r i t i o n a l s t a t u s which a f f e c t d i f f e r e n c e s i n e x c r e t i o n at dawn and dusk. However, the lower r a t e of e x c r e t i o n of urea (the main nitrogenous waste product of these animals) at dusk c o u l d be e x p l a i n e d by a lower r a t e of p r o t e i n i n g e s t i o n . The amount of p r o t e i n i ngested being a f u n c t i o n of food a v a i l a b i l i t y and p r o t e i n content. The importance of urea i n the nitrogenous e x c r e t i o n of t h i s animal suggests an a c t i v e o r n i t h i n e c y c l e and i n a c t i v e or absent urease. Takahashi and Ikeda (1975) s t u d i e d the nitrogenous e x c r e t i o n of Euphausia pac i f i c a , but they measured only ammonium. The rate obtained at a food c o n c e n t r a t i o n of 45 ixq c h l a l " 1 ( s i m i l a r to the c h l o r o p h y l l measurements made i_n s i t u i n t h i s study) was 1.2 yg-at a n i m a l " 1 day" 1 (6.2 uq-at NH„ + -N g~ 1h~ 1) approximately twice the value obtained i n the present study. T h i s d i f f e r e n c e i s e a s i l y accounted f o r by d i f f e r e n c e s in methodology. In the f i r s t p l a c e , Takahashi and Ikeda (1975) incubated t h e i r animals f o r 24 hours i n the presence of b a c t e r i a which may have converted some urea to ammonium (Remson e_t a_l. , 1971). Secondly, c a l c u l a t i o n of the r a t e s r e p o r t e d by Takahashi 70 and Ikeda (1975) necessarily included inferred uptake by phytoplankton. If urea was also present, the phytoplankton may have taken up less ammonium than expected. Further, the average dry weight of the euphausiids studied by Takahashi and Ikeda (1975) was approximately half that of those in the present study. (This size difference may have been because my study was conducted in June, the i r s in October.) Having reconciled these two estimates, I am puzzled to report that both Jawed (1969) and Ikeda (1977) found ammonium excretion rates for p a c i f i c a approximately f i f t e e n times higher than those reported here. Ikeda (1977) does not discuss the f i f t e e n - f o l d difference of his e a r l i e r results (Takahashi and Ikeda, 1975). Although the average dry weight of E.  p a c i f i c a studied by Ikeda (1977) was only about one f i f t h that of the animals studied by Takahashi and Ikeda (1975), the E. p a c i f i c a used by Jawed (1969) weighed on average s l i g h t l y more than in the present study. Takahashi and Ikeda (1975) worked at 8°C, Ikeda (1977) and Jawed (1969) at 10°C and the present study was conducted at 9°C. Only the animals studied by Jawed were starved prior to measurement. A l l animals except those of Takahashi and Ikeda (1975) were co l l e c t e d in the summer. The animals used by Takahashi and Ikeda (1975) as well as those used by Ikeda (1977) were given a period of adjustment to laboratory conditions prior to the measurement of excretion rates. At the present time I can only offer that i f the experiments of Jawed (1969) and Ikeda (1977) were conducted in the presence of urea-decomposing bacteria (Remson et a_l., 1971), and each molecule of 71 urea excreted was decomposed to two molecules of ammonium, the remaining difference could be accounted for by differences in body size and temperature. When fed a diet of dead f i s h , with a protein to carbohydrate r a t i o eleven times higher than the alg a l diet, t o t a l nitrogen excretion by the shrimp increased by less than a factor of two. The shrimp may have fed to sa t i a t i o n on a smaller quantity of f i s h , been less able to digest the f i s h protein, or a l t e r n a t i v e l y , the levels of excretion observed on the phytoplankton diet may have been basal levels and included some tissue catabolism (see discussion of starvation below). Nevertheless, a greater rate of nitrogen excretion was observed in the group fed on a higher protein d i e t . The rates of ammonium excretion reported here are within the range reported by Nelson and colleagues (1979) for an estuarine shrimp (43 to 239 jug-at NH« + -N g" 1 h " 1 ) . In spite of being fed two di f f e r e n t forms of food, the nitrogenous excretion of the prawns was also related to the protein content of their food. The ra t i o of ammonium excretion to urea excretion was the same for each of the di e t s , indicating a balance dictated by some other factor such as temperature (enzyme a c t i v i t y ) or s a l i n i t y (osmoregulation). As noted above for the shrimp, the change in rates of excretion due to the change in diet was not as large as the change in the r a t i o of protein to carbohydrate. In the case of the prawns, the similar rates of excretion in fasted animals provides further evidence to suggest that tissue catabolism i s at least part of the 72 explanation. I was able to study the contribution of tissue catabolism during starvation only for the prawns. The shrimp did not survive starvation, the amphipods became cannabalistic soon after food was removed, and the euphausiids were studied in  s i t u . Diehl and Lawrence (1979) report that " i t is generally concluded that starvation results in an increase in excretion rates due to tissue metabolism". Although Emerson (1969) reported t h i s result for a sea urchin as did Ikeda (1977) for three species of zooplankton, Quentin and colleagues (1980) found a decrease in ammonium excretion with the starvation of mysids. Si m i l a r l y Szyper (1981) reports that the ammonium excretion of a chaetognath decreased with starvation; Hernandorena and Kaushik (1981) observed the same effect with the starvation of Artemia. The relationship between nitrogen excretion and starvation i s more complex than i t was thought to be in 1979. In 1981 Regnault reported that the length of starvation s i g n i f i c a n t l y affected the re s u l t s . Crangon crangon was observed to excrete more ammonium when starved than when fed only after four days of starvation: before that i t excreted less. Through calculations of the r a t i o of oxygen consumption to nitrogen excretion, Regnault (1981) inferred that carbohydrate stores were catabolized for 3-4 days, then l i p i d s and proteins, then proteins. This pattern is probably not universal as Regnault (1979) has reported that Crangon crangon has a high nitrogen requirement for growth and a correspondingly high rate of ammonium excretion. 73 The excretion rates for starved Pandalus platyceros reported in this study were measured after the animals had been held without food for seven days. Both ammonium and urea excretion were lower than for the fed animals. This decrease may be a function of the decrease in metabolism which often accompanies starvation (Lockwood, 1968, p. 153). Since only nitrogen excretion was measured, I cannot comment on the substrate of tissue catabolism. D. DISSOLVED ORGANIC NITROGEN As is discussed in the introduction to t h i s thesis, the contribution made by dissolved organic nitrogen (DON) to the nitrogenous excretion of marine invertebrates i s highly variable and poorly understood. Unfortunately the results of the present study do l i t t l e to elucidate the problem. What they do show i s that the excretion of DON by Pandalus platyceros i s not affected by temperature, s a l i n i t y , n u t r i t i o n , or body size (at least not within the ranges studied here). These results agree with those of S t i c k l e and Bayne (1982) who report that the release of organic nitrogen (free amino acids) by Thais l a p i l l u s is not related to temperature, s a l i n i t y or body s i z e . However, on occassion individual prawns excreted as much as 65% of their 'waste' nitrogen in forms other than urea. This suggests that DON i s released by a d i f f e r e n t mechanism and/or for different reasons than ammonium and urea. Burger (1957) determined that ammonium and urea were released by d i f f u s i o n across the g i l l s of the lobster, Homarus. Kormanik and Cameron (1981) also report that the excretion of 74 ammonium in the Seawater Blue Crab occurs by d i f f u s i o n across the g i l l s . The crustacean orders Amphipoda, Euphausiacea, and Decapoda a l l have paired segmental excretory organs which open on the t h i r d , antennal, segment (Parry, 1960). These glands function by u l t r a f i l t r a t i o n as well as by secretion and perhaps reabsorption (see G i l l e s , 1975b, for a review). Large molecules (some undoubtedly containing nitrogen) are f i l t e r e d by the antennary glands of the lobster (Burger, 1957). In some decapods the tubule of the antennary gland i s expanded d i s t a l l y to form a bladder (Lockwood, 1968, p.38). In addition, Gardner and M i l l e r (1981) report that the release of amino acids by Daphnia magna i s not continuous. These facts lead me to hypothesize that DON i s excreted by crustaceans via a bladder which is emptied p e r i o d i c a l l y . The more sensitive a n a l y t i c a l techniques being developed by workers such as Gardner and M i l l e r (1981) and Hawkins and Keizer (1982) w i l l enable the time pattern of release of DON to be studied and correlated to bladder contents. This approach w i l l permit the examination of factors which influence the functioning of the antennary glands and the release of DON, and w i l l determine whether the release of DON i s simply leakage of c e l l u l a r contents due to membrane changes under stress. 75 V. CONCLUSIONS It has been shown that metabolic changes in response to temperature are not simply rate changes in overall metabolism, but also result in changes in the composition of the nitrogenous excretory products of Pandalus platyceros. S i m i l a r l y , the observed l i f e stage dependency of the temperature e f f e c t i s evidence for q u a l i t a t i v e changes in metabolism with development. One may make the e c o l o g i c a l l y useful generalization that, in adults, the rate of excretion of t o t a l nitrogen increases with temperature, but, because of the ecological significance of the composition of the nitrogenous excretion, i t is of interest to the ecologist as well as to the physiologist to further investigate the complex molecular basis of temperature acclimation. The e f f e c t s of s a l i n i t y on the nitrogenous excretion of P.  platyceros are at least as complex as the ef f e c t s of temperature. The potential role of ammonium for sodium uptake, amino acids for osmoregulation, and urea retention for water balance may be dire c t effects of s a l i n i t y on nitrogen excretion. In addition, c e l l volume and ionic composition e f f e c t the functioning of enzymatic systems ( G i l l e s , 1975b) in turn aff e c t i n g nitrogen metablolism. These molecular e f f e c t s would not be s i g n i f i c a n t e c o l o g i c a l l y i f the net influence of s a l i n i t y on nitrogenous excretion could be generalized. In thi s study ammonium excretion and urea excretion were each affected d i f f e r e n t l y by changes in s a l i n i t y , and further, these effects were l i f e stage dependent. It i s not yet known whether .the 76 patterns observed here can be generalized to species other than P. platyceros. The protein content of diet was important in determining both composition and rates of excretion of nitrogenous waste by each of the species of Crustacea studied. Although there are c o n f l i c t i n g reports, the observed relationship between dietary protein content and t o t a l nitrogenous excretion may be a general one. The studies in disagreement with t h i s generalization either measured only ammonium (Nelson et a_l. , 1 977) or did not provide enough food to avoid the confounding effect of tissue metabolism (Diehl and Lawrence, 1979). The e f f e c t s of diet on the composition of nitrogenous waste are more complex, and possibly only important in animals lacking an active ornithine cycle and urease. Excluding juveniles, the weight-specific excretion of P.  platyceros decreased with increasing body s i z e . The rate of t h i s decrease was temperature and s a l i n i t y dependent. Similar i n t r a s p e c i f i c dependence of excretion rate on body size has been reported by other workers (for example, Ross, 1979, for E.  pac i f i c a , and Hawkins and Keizer, 1982, for a benthic amphipod). Unfortunately, the i n t e r s p e c i f i c application of t h i s r e l a t i o n s h i p (as suggested by Corner e_t a_l. , 1965, and Ikeda et a l . , 1982a) i s uncertain, even within the class Crustacea. The eualid studied here had weight-specific excretion rates much higher than the other species, yet they were larger that both the euphausiids and the amphipods. However, i t would be unwise to dismiss the p o s s i b i l i t y of a useful generalization on t h i s 77 basis because both the amphipod and euphausiid experiments were conducted prior to my awareness of the p o s s i b i l i t y of urease i n h i b i t i o n and therefore excretion rates may have been underest imated. The excretion of dissolved organic nitrogen (DON) is independent of the factors studied here. To determine whether or not DON is excreted via a bladder w i l l require detailed time series studies of individual animals. Urea was an important excretory product of each of the four marine crustaceans in this study. Ikeda (1977) states that "It is well known that ammonia i s the major form of nitrogen excreted by marine zooplankton ... ". As a consequence of the pervasiveness of thi s paradigm (which extends to a l l marine invertebrates) many interesting studies continue to report only ammonium excretion ( e.g. Hawkins ejt a l . , 1983). The results presented in thi s thesis are strong evidence against the assumption that ammonium is the only important nitrogenous excretory product of marine invertebrates. 78 BIBLIOGRAPHY Antia, N.J., B.R. Berland, D.J. Bonin and S.Y. Maestini. 1977. E f f e c t s of urea concentration in supporting growth of certain marine microplanktonic algae. Phycologia 16: 105-111. Antia, N.J., B.R. Berland, and D.J. Bonin. 1980. Proposal for an abridged nitrogen turnover cycle in certain marine planktonic systems. Mar. Ecol. Progress Series 2: 97-103. Armitage, M.E. and R.J. 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