ABSTRACT Mitchell E. Landen. EFFECTS OF FOOD AND TEMPERATURE ON SIPHON REGENERATION AND GROWTH IN THE ESTUARINE CLAM RANGIA CUNEATA (Under the direction of Dr. Carlton Heckrotte). Department of Biology, Autumn 1984. Rates of siphon regeneration in the mactrid clam Rangia cuneata(1831) were measured under varied temperature and food levels. The rates of siphon regrowth were found to be dependent on temperatures, but were food-independent. The temperatures involved were llo C, 21° C, and 280 c. The food source was Pseudomonas aeruginosa, a motile bacterium. The concentrations of bacteria maintained were a) 0/ml (starvation) b) 2.5 X 105 /ml , and c) 1.0 X 106 /ml. Each of the nine tanks used in the research contained a group of experimental clams (those having their siphons snipped) and a similar group of control clams. Total weights and lengths were measured for each subject at the beginning and the end of testing, as wel 1 as the siphon weights of the experimental clams. The data was compared and contrasted using an analysis of variance. Time necessary for clams to regenerate their snipped siphons increased at lower temperatures. Food levels had no effect on the time for regeneration. Total clam weight and length increases were attributed to the higher temperature levels, whereas the higher food levels showed no major effect, possibly because of the clam's ability to regulate food intake by regulating water intake. Mortalities occurred during the course of the research, but neither temperature level, food level, nor snipping appeared to have caused a notable rise in deaths among the subjects. Mortality in the clams remained below 14%, generally, with no statistical ly important differences between the experimental and control clams. EFFECTS OF FOOD AND TEMPERATURE ON SIPHON REGENERATION AND GROWTH IN THE ESTUARINE CLAM RANGIA CUNEATA A Thesis Presented to the Faculty of the Department of Biology East Carolina University In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology by Mitchell E. Landen Autumn 1984 9. r. JOYNRH LTBW'.fY CAKOLî^îA UNí ' EFFECTS OF FOOD AND TEMPERATURE ON SIPHON REGENERATION AND GROWTH IN THE ESTUARINE CLAM RANGIA CUNEATA by Mitchel 1 E. Landen Approved By Supervisor of Thesis Dr. Carlton Heckrotte ACKNOWLEDGEMENT My sincere thanks to Dr. Carl Heckrotte for his guidance and assistance in this project. Many thanks are also due Dr. Charlie O'Rear and Dr. Takeru Ito for their valuable critiques and other forms of help. To Dr. Kevin O'Brien, I offer my deepest gratitude for his statistical aid, encouragement, and general support through the thesis process. Sandy Tomlinson and Cindy Stack of the Institute of Coastal and Marine Resources deserve congratulations for their perseverance and thanks for their help while I learned the ropes of word-processing. I am indebted to Mr. Jerry Freeman and Mr. Laddie Crisp for their technical help during the experimentation period of this research. 10,000 thanks to all the people who helped with suggestions and constructive criticisms. Special thanks to Mrs. Dianne B. Norris for her assistance in the microbiological aspect of the thesis. Ms Lori Stewart deserves mention for her invaluable contributions in procuring subjects for the experimentation done in the research. Also, I recognize myself as owing much to the late Dr. Don Jeffreys, who served as chairman of my thesis committee untilili ness forced hi s retirement. 111 TABLE OF CONTENTS Page ACKNOWLEDGEMENT Ü LIST OF FIGURES iv LIST OF TABLES v LITERATURE REVIEW 1 Introduction 1 Natural History 1 Rangia Siphons: Functions and Ecological Value 3 Digestive Process 8 The Paml i CO Ri ver 10 Regeneration 11 Previous Bivalve Siphon Studies 12 MATERIALS 15 METHODS 20 RESULTS AND DISCUSSION 24 Effects of Temperature 37 Effects of Food Levels 40 Snipping Effects 43 BIBLIOGRAPHY 48 IV LIST OF FIGURES Page 1. Water circulation in Rangia cuneata 4 2. Anatomy of Rangia cuneata 9 3. Observations on stages in siphon regrowth ... 14 4. Map of North Carolina, showing the site of clam col 1 ecti on 17 5. Experimental design used in research 21 6. Graph on temperature-to-regeneration time relationship 25 7. Graph on total weight gain-to-temperature level relationship 26 8. Graph on clam 1ength-to-temperature level relationshi p 27 9. Graph on food 1 evel-to-regeneration time relationship 23 10. Graph on total clam weight-to-food level relationship 29 11. Graph on clam 1 ength-to-food level relationship 30 M LIST OF TABLES Page 1. Composition of Utility Marine Mix 16 2. Componants of the media used 19 3. Statistics on independent variable relationship to each other with regard to time for regeneration . . 31 4. Statistics on independent variable relationship to each other with regard to weight gain 32 5. Statistics on independent variable relationship to each other with regard to length gain 33 6. P values and correlation coefficients of variabl es used 34 7. Effects of food and temperature levels on days required for full siphon regeneration 35 8. Effects of food and temperature levels on weight and length increases for both experimentáis and control s 36 9. Weight, length, and mortality between snipped and control clams 38 10. Impact of snipping, alone and in conjunction with food and temperature levels 45 Introduction Regeneration, the redevelopment of normal functional tissue, is a phenomenon not seen on a major scale in humans or most vertebrates. With many invertebrates, however, it is commonplace. Regeneration of siphon tissue in the estuarine cl am Rangia cuneata is the subject cf this study. The chief goal of the study was to determine how siphon regeneration in the clam was affected by temperature and food levels. A secondary goal was to ascertain food and temperature effects on clam growth (weight and shel 1-1ength). Also investigated was the impact of siphon removal by snipping on clam growth and mortality. Natural History Rangia cuneata, of the class Pelecypoda and family Mactridae, is found from the mid-Atlantic states to the Gulf coast (Fairbanks, 1963; Grzimek, 1974). This clam is readily adaptable to temperature and salinity variations. Attesting to this is the record of its rapid increase in distribution. The first report of living, non-Pl eistocene Rangia found on the eastern United States coast was in 1955 in the Newport River of North Carolina. By 1960, the clam was reported in Virginia and Maryland (Hopkins and Andrews, 1970). Rangia inhabit low- salinity embayments. Though many can survive in fresh water, they cannot reproduce in it. In tests done with Rangia to determine tolerance to salinity, populations of the clam survived in salinities from 0 to 39 o/oo (Bedford and Anderson, 1972). In North Carolina's Pamlico River, 2 R a n g 1 a are most common in areas with salinities between 0.5 and 10.Q o/oo (Crump, 1971). Their ability to acclimate to temperature extremes has also been examined. Rangia have shown 100% mortality below O.Eo C and above 35o c, but can survive between those ranges (Naylor, 1965; Cain, 1973; Sample and Landy, 1978; and Landy, 1979). Another indicator of Rangia's hardiness is its constant densities in spite of substantial salinity and temperature changes in the Pamlico River (Tenore, 1972; Sutherland, 1982). The clam itself is an ecological asset to the Pamlico, since it converts detritus to meat, which is then used as food by birds, fish, and crustaceans. Molluscans form 45% of the benthic invertebrate species list of the Pamlico estuary (Tenore, 1972). In the food web of Lake Pontchartrain, Louisiana, Rangia densities place it among the three most important invertebrates, the others being the mud crab, Rithropanopeus harrisii, and the blue crab. Cal 1inectes sapidus (Green, 1968). This implies Rangia's ecological significance in some aquatic systems. The filtering activity of the clam is as high as 40 1/h according to some estimates (Potts, 1967; Duffy, 1980). Therefore, major decreases in the clam population could result in an acute ecological imbalance. 3 Rangia Siphons: Functions and Ecological Value R a n gia are fi1ter-feeders , subsisting on bacteria, dinof1 agel 1 ates, di atoms, dead organic matter, and other microscopic organisms (Hyman, 1967; Vernberg, 1977; Peterson and Quammen, 1982). Two types of bacteria, Serratia marcescens and Serratia 1iquefaciens, have been shown in experiments to be adequate food sources for Rangia (Brooks, 1978; Jeffreys, et al , 1980). Ingestion and egestion in Rangi a are performed by two siphons, the dorsal excurrent (exhalant) and the ventral incurrent (inhalant) seen in Figure 1. These are apertures controlling water intake and elimination (Jorgenson, 1966). The siphons are modifications of the posterior edges of the mantle. They are commonly retractable within the shell. This is performed by the siphonal retractor muscle, which is a local exaggeration of the palliai retractor muscle, located along the entire mantle edge (Pratt and Campbell, 1956). Some clams, such as the Mya arenaria, lack a totally retractable siphon and tend to remain so deeply buried that even a slight retraction brings the siphon safely under the sediment surface (Feder, 1972; Bernard, 1975). The Rangi a si phon, however, is totally retractable. Sensory tentacles line the opening of the incurrent siphon, allowing exclusion of undesirable particles without forcing the retraction of the entire siphon (Reid and Crosby, 1980). Where large populations of Rangia exist, their siphons may be of some importance to small and juvenile fish as a food source in eastern North Carolina estuaries. Small benthic-feeding fish, among them 4 Figure 1. Water circulation in the clam (Ellis, 1973) A) incurrent siphon B) foot C) mouth -- D) interior gill E) exterior gill F) excurrent siphon 5 juvenile flatfishes, feed heavily upon bivalve siphons and other soft parts of benthic fauna , as determined by examination of gut contents (Edwards and Steele, 1968). In studies by Peterson and Quammen (1982) off the southern California coast on the clam Prototheca staminea, it was noted that in addition to fish such as the California halibut (Paralichthys californicus), other predators feeding on the siphons were small cancer crabs. Cancer anthonyi, moon snails. Polinices reclusianus, staghorn seul pins, Leptocottus armatus, and diamond turbots, Hypsopsetta guttu1 ata. In research by Currin ( 1984) at Rose Bay, North Carolina, gut contents of spot and croaker were scrutinized. Among the contents were dipterans, small fish, amphipods, calanoids, whole bivalves, and bivalve siphons. The high number of siphons present suggests a significant role for them in the diet of the fish species under investigation. From work done at Loch Ewe, Scotland, Edwards and Steele (1968) found that in juvenile plaice gut contents examined, bivalve siphons constituted the highest percentages overall of individual structures present. Amphipods, cumoceans, and calanoid copepods were also found. Among common dabs, the numbers of siphons found followed in abundance that of amphipods, cumoceans, and harpacticoid copepods. The smaller fish (plaice and common dabs) tended to feed on siphons of the bivalve Tel 1ina tenuis and tentacles of the polychaete Nerine cirratulus. In a Dutch study (Kuipers, 1977), Macoma siphons were found in abundance in the gut contents of juvenile plaice. The nutritional value of the bivalve siphon has not been positively established, but the siphon is believed to impart a notable 6 measure of calories to the predator. Edwards, et ai (1969), discovered that bivalve siphons were a major component of the natural food of plaice in Loch Ewe during the first two months after metamorphosis. In their research, high growth rates for plaice were detected when the fish were allowed to feed on Tel 1ina siphons exclusively. Morowitz (1968) performed analyses of Macoma soft tissues to determine caloric values for them. The predominant components of the tissues were proteins, carbohydrates, and lipids, percentages of which varied according to the season (Beukema and de Bruin, 1977). The average caloric value for all seasons was 4.1 kcal-g*l for the carbohydrate, 5.5 kcal-g-1 for the protein, and 9.3 kcal-g-1 for the lipid. Percentages of carbohydrate in the soft tissues were somewhat higher than those for protein and much greater than those of lipids. The average caloric content for soft tissues was put between 5.5 and 6.0 kcal-g -1. In 1979, Beukema and de Bruin used Macoma balthica for an energy study involving a biochemical method (designated the "indirect method") and a method using a micro-bomb calorimeter (the "direct method"). From the indirect method, using conversion factors for various components to find calorific values, a figure of 5.47 kcalg-1 was derived for the tissue caloric content. Using the direct method, they got 5.59 kcal•g -1. Whether siphons may contribute significantly to energy needs of fish can be determined by investigation of fish requirements in general. For pike, 14 to 33 % of its caloric consumption was found to have been converted into new tissue, while between 50 to 70 % was metabolized in 7 such processes as respiration, digestion, excretion, and maintenance of existing tissue (Lagler, et al, 1977). Although energy efficiencies for younger fish are greater than those of older fish (45 % as opposed to 15%, general ly), metabolic rates of younger fi sh are al so higher. Therefore, food demand is higher. The daily maintenance ration of red hind (Epinephelus guttatus), for a 250 g specimen between 19 and 28oC, ranges between 1.7 and 5.8 percent of body weight. Carp yearlings (Cyprinus carpió) in ponds may have a daily maintenance ration of 16 % of their body weight at the beginning of the growing season. These rations naturally vary due to food availability, reproductive stage, and season. Blue gill (Lepomis macrochi rus), for example, may consume up to 5 % of its body weight daily in summer when the mean water temperature is 20OC, but the food intake in winter, when the mean water temperature is 30C, may only be 0.14 % per day. According to a report edited by Neuhaus and Hal ver (1969), 1000 ml of oxygen consumed by a fish (in this case, salmon) corresponded to 4.69 kcal expended. A 100 g fish requires 16.48 ml of oxygen per hour, equal 1 ing 395.5 ml per day, therefore the daily maintenance re qui rement for a 100 g fish would be 1.8 kcal. F rom this study, using adult Rangia averaging 40 mm in length and 39 g in weight, a mean dry siphon weight of 0.0020 g was calculated. Approximately 500 siphons, then, would be needed to constitute 1 g (dry weight) and 5.59 kcal. The 1.8 kcal required by a 100 g fish is 31 % of the number of calories found in 1 g of siphon tissue. Therefore, around 155 siphons would be needed to fill the daily maintenance requirement for a fish of this size. Flounder, croaker, and spot, known for their 8 predation of bivalve siphons, abound in the Pamlico estuary and probably rely to some extent on the sizeable Rangia and Macoma populations for sustenance. Other species of fish, such as striped bass, have not been found to feed heavily on bivalve siphons, although examination of their guts has revealed the presence of some siphons (Dr. T.J. Lawson, ECU, personal communication 1984). Gut contents of yellow perch, white perch, channel catfish and white catfish from the western Albemarle Sound yielded few siphons when examined. Whether the siphons found were from Macoma , Rangia, or some other bivalve species was not determined, since the purpose of that study was identification of predators of juvenile yellow perch. If regeneration of snipped siphons is the norm, an almost perpetual crop of siphon tissue would be available for the fish to feed upon. Digestive Process The food particles entering by the incurrent siphon are sorted out according to size by the labial palps, with the over-sized ones rejected (Bernard, 1975). Smaller particles are passed via cilia action (because the clam's alimentary canal is devoid of muscle) to the esophagus and thence to the stomach. The crystalline style, a gelatinous rod-like structure, is then rotated against the gastric shield, aiding gastric enzymes in gradually digesting the stomach contents (Barnes, 1968). The food is sent to ducts of the digestive gland (Figure 2 ), where cells ingest it. Following this, the nutrients are digested intracel 1 u 1 arly (Fretter and Graham, 1976). 9 Figure 2. Anatomy of Rangia cuneata (Fretter and Graham, 1976) A) incurrent siphon I) protractor muscle B) mantle J) retractor muscle C) foot (containing loops of K) umbo intestine and gonads) L) ligament D) palps M) visceral mass E) interior gill N) retractor muscle F) exterior gill O) adductor muscle G) digestive gland P) excurrent siphon H) adductor muscle L 10 Solid matter not absorbed by these cells in the diverticula enters the intestinal groove and passes into the intestine. More digestion occurs there, because the lumen of the intestine possesses amoeboid cells capable of phagocytosis (Morton,1960). Wastes are removed from the blood by the nephridial tubules, U-shaped kidneys below the heart (Hickman, 1967). The wastes are then excreted by the excurrent siphon. The siphons, therefore, serve necessary functions in the clam, with the sensory tentacles also having a role. The Pamlico Ri ver The Pamlico Ri ver has sal i ni ties ranging from 1 to 20 o/oo, with temperature variations between 0 and 3lo c (Jarrett, 1966; Tenore, 1972). The river is divided into three zones (using the Venice classification of estuarine waters): a) The oligohaline region, dominated by Rangia and Nereis succinea, with salinities from 0.5 to 5.0 o/oo, and having the highest concentration of Rangia, b) The mesohaline region, dominated by N. succinea, Macoma balthica, and Heteromastus filiformis, with salinities from 5.0 to 18.0 o/oo, and c) The polyhaline region, dominated by Macoma Phenax, Mu1inia 1 ateralis, and Glycera dibranchiata (Tenore, 1972). The Pamlico River, a wide and shallow estuary, extends from Washington, N.C., for 65 km to the Pamlico Sound. It has an average 11 depth in its central muddy areas of 2 to 3 m, and an average depth in the near-shore zones of 1 m. Rangia is most abundant in the latter zone. In 1967, the density of Rangia in some pi aces was as high as 275 clams/m (Tenore, 1972). Regeneration Regeneration occurs in many organisms, but the physiological prompting of regeneration is unknown. The ability of the planarian to regenerate is wel 1-documented, the first known experiments having been done in 1774 by Pallas (Brpnsted, 1969). In salamanders, legs and tails 'are regenerated fully, with normal motor function resulting (Brookbank, 1978). Other amphibians, like the newt, are also capable of limb regeneration (Ede, 1978). After amputation, the wound is closed by epithelium. This is accompanied by proliferation of subepithelial growth. The stump blastema formed is undifferentiated at first, but eventual 1 y becomes adu It tissue (Ful ton and Klein, 1976). According to one theory, nerve supply to the amputation site in the newt may be invol ved in tissue regeneration, because nerve supply is much greater relative to the total cross-sectional area of the limb itself as compared to that of an animal incapable of regeneration (McKenzie, 1976). In studies involving the cockroach Leucophaea, legs were regenerated but only if amputation were conducted, and not a mere wounding (Grant, 1978). Success has been noted in regeneration of severed or crushed optic nerves in adult frogs (Graham and Wareing, 12 1978). Although tests invol ving weak pulses of electricity through damaged tissue in humans have had some positive results, regeneration in post-embryonic humans occurs only in the liver, by normal replacement phenomena (e.g., uterine endometrium), and through wound-healing (Clark et al, 1980; Borgens, 1981). Previous Bival ve Siphon Studies Sutherland (1981) placed cages with two densities of bivalves (Macoma balthica and Macoma phenax) and three densities of fish (juvenile spot, Leiostomus undulatus, and juvenile croaker, Micropogon xanthurus) in Rose Bay, N.C., to find effects of siphon snipping by fish on growth of the clams. The cages were made of plastic restaurant bussing trays with a 1.8 cm pipe frame, covered by a 0.63 cm2 CONWED plastic mesh. The volume of each cage was 0.046 m3 , with a surface area of 0.177 m2 and a height of 0.26 m. Biweekly, fish were taken from the cages and placed in 10% formalin, and their stomach contents examined for siphons. The Macoma showed no difference in growth when subjected to siphon snipping by juvenile spot than did those serving as controls. Only those Macoma being snipped by juvenile croaker showed a decline in growth rates. Control clams initially 10 mm long grew 3.3 mm during 15 weeks in cages, whereas those in cages with croaker showed only 1.5 mm growth — a reduction of 45%. In a similar experiment done in California, a two-fold drop in growth of Prototheca staminea was attributed to siphon-snipping (Peterson and Quammen, 1982). In a siphon- 13 snipping experiment done in the Nether!ands with M. balthica, 1ittle growth reduction and little increase in mortality were seen after the bivalves were subjected to siphon snipping by juvenile plaice and flounder (de Vlas, 1981). In research done to establish general characteristics and results of siphon-snipping in Rangia, Landen (1983) used 30 Rangia in one tank. Salinity was maintained at 1 o/oo and temperature at 21o c (room temperature). The clams ranged in length from 50 to 55 mm and in weight from 70 to 77 g. Feeding was done once a week by addition of water (approximately 1 1) from Euglena and Parameeiurn cultures. Fifteen clams were snipped by hand and fifteen were used as controls. On the average, 7 days were required for new siphon tissue to be detected. After this period, the siphon length increased by increments of about 0.5 mm/day. Minute siphon sensory tentacles (~0.3 mm ) were observed by day 15, with siphon length at this point averaging 4 mm. By day 19, sensory tentacle length had increased to 1.3 mm, and by day 21, the maximum average of 1.5 mm was attained (Figure 3). By this time, siphon length was between 5 to 6 mm and remained at this length. After 21 days, no increase in siphon or tentacle length was noted. Mortality of 20% was seen among the snipped clams, but none occurred among the controls. 14 Figure 3. Observations on stages of siphon regrowth in Rangia A) Siphon Prior to Snipping (1.6 :r.:n ) 5-6 piia) sensory tentacles unseen incurrent siphon "1.5 ¡sín C) Siphon 15 Days After Snipping sensory tentacles ~0.3 mm incurrent siph.on ~4.0 i:i:n D) Siphon 19 Days After Snipping sensory tentacles “1.3 nm incurrent siphon “5.0 rrai E) Siphon 21 Days After Snipping sensory tentacles “1.5 r:im incurrent siphon “5.0 rani 15 Materials Nine plastic containers ("rat cages") which measured 0.17 m (width) X 0.29 m (length) X 0.13 m (height), providing a bottom area of 0.49 m2 and a total volume of 0.064 m3, were used to contain the clams. Into each container were placed four liters of water with a salinity of 1 o/oo . This water was made from disti 11 ed water to which "7 Seas" marina salts was added to get the necessary salinity (Table 1). This salinity level was chosen because the average salinity of Chocowinity Bay, the site of the clam collection, is between 0.5 o/oo and 1.0 o/oo (Figure 4). The salini ties were checked at 3-day intervals with a YSI Model 33 salinity meter. Oxygen concentrations were kept high by bubbling air through the water, and were checked twice weekly with a YSI Model 57 oxygen meter. For those tanks requiring a heat supply, Appco and Wil-Nes 77 thermoregulators were used. The food source used was the bacterium Pseudomonas aeruginosa (ATCC 10145), a motile bacteria commonly found in most natural waters. This bacterium was selected for several reasons: a) The relative ease and time-effectiveness in handling the cultures and growing the bacteria, b) The ubiquitous distribution of P. aeruginosa, leading one to assume that wherever Rangia are found, so are these bacteria, and c) the ability of P^ aerugi nosa to produce and release the antibiotic-like bacteriocins called pyocins (Pelczar and Reid, 1972). These substances are lethal to some forms of bacteria (via adsorption on specific receptors of host-cell walls) and should Table 1. Composition of Utility Marine Mix ( Utility Marine Bulletin #127 --? "7 Seas" sea salts) Compound Chemical Formula 1 Sodium Chloride (NaCl ) 37.0 Magnesium Chloride {MgCl2 • 6H2 0) 7.0 Magnesium Sul fate (MgS04 • 7H2 0) 9.3 Potassium Chloride (KCl) 0.9 Sodium Bicarbonate (NaHC03 ) 0.2 Strontium Chloride (SrCl2 ' 6H2 0) 27.0 Manganese Sulfate (MnS04 ? H2 0) 5.0 Disodium Phosphate {Na2 HPO4 • 7H2 0) 4.0 Lithium Chloride (LiCl) 1.3 Sodium Molybdate (Na2 M0O4 • 2H2 0) 1.3 Calcium Chloride (CaCl2 ) 1.7 Calcium Gluconate (CaCCgHii07)2) 0.8 Potassium Iodide (KI) 0.1 Potassium Bromide (KBr) Trace Aluminum Sulfate {AI2 {SO4 )3 ) 0.6 Cobalt Sulfate (C0SO4 ) Trace Rubidium Chloride (RbCl) 0.2 Copper Sulfate {CUSO4 • 5H2 0) 0.6 Zinc Sulfate (ZnS04 • 7H2 0) 0.1 17 Figure 4. Site of the collection of Rangia at Choccwinity Bay, North Carolina (Radford et al, 1968) 13 keep non-pseudomonad bacterial levels low or eliminate them altogether. Pseudomonads were cultured using tripticase soy agar (TSA) and were transferred to sterile 10-ml Pyrex test-tubes, which contained 8 ml each of trypticase soy broth (TSB). These are conventional media used for many bacterial experiments (Table 2). The inoculated tubes were incubated in an oven at 30o C for 24 hours to allow the bacterial populations to reach 1.0 X 109 cells/ml. The 24-hour period is the standard time requirement for such a population of Pseudomonas to be reached. Confirmation was attained by plate count. The cl ams, rangi ng from 37 to 45 mm i n 1 ength, were numbered 1 to 90 with red Maybelline fingernail polish. This polish proved the best of several tried, since it dried quickly (which reduced the time the clams were kept out of the water), peeled seldom, and was non-toxic. 19 Table 2. Componants of the media used (trypticase soy broth and trypticase soy agar) TSA typical formula (for 1 1 of distilled water): 15.0 g Peptone 140 (Pancreatic Digest of Casein) 5.0 g Peptone 110 (Papaic Digest of Soy Protein) 5.0 g Sodium Chloride 15.0 g Agar (pH 7.3 +/- 0.2 at 250 C) TSB typical formula (for 1 1 of distilled water): 17.0 g Peptone 140 (Pancreatic Digest of Casein) 3.0 g Peptone 110 (Papaic Digest of Soy Protein) 2.5 g Dextrose 5.0 g Sodium Chloride 2.5 g Potassium Phosphate Dibasic 20 Methods Ten clams were placed in each container, and randomly divided into two groups: 5 experimentáis were placed on one side and 5 controls on the other side of the container. The weight and length of the clams was measured (length defined as distance from umbo to base of siphon). They were then paired, experimental to control, according to length (intrapair comparisons, however, were not made). The experimental design in this research called for three levels of temperature and three levels of food to be used (Figure 5). The temperature levels chosen were llo C, 210 c, and 280 C, which are the approximate average temperatures found in Chocowinity Bay in March, May, and July, respecti vely. The three tanks designated 11° C were placed in cold rooms, which are maintained at 40 C. Heaters were attached to each tank and adjusted, over a period of two days, to raise the water temperature to lio c. The three containers used for 28o C water were placed in a room maintained at 21° C, and heaters were used to raise the water temperature to 28° C. The three tanks at 21o C were kept at the room temperature of 2lo C. These three were placed alongside the tanks holding the 28o c water. The three food levels chosen were: 0) 0 bacteria/ml (starvation level), 1) 2.5 X 105 bacteria/ml, and 2) 1 X 106 bacteria/ml The level of bacteria in natural waters, including the Pamlico River, is generally around 1 X 106/ml (Dr. R. Christian, ECU, personal communication 1982), hence the "base" amount used. To achieve this level Figure 5. Experiüiental design used in research ÍT“EMCPERATU)RE 2.5X10^ 1.0X10® FOOD{BáCTER¡ 22 of pseudomanal bacteria in the three high-food-group containers, four ml of the inoculate (containing 1 X 109 Pseudomonas/ml) were applied. This resulted in a nearly-immediate Pseudomonas population of 1 X 106/ml. For the second food-level tanks, one ml of the inoculate was added to the water, giving a Pseudomonas population of about 2.5 X 105/ml. No Pseudomonas were added to the tanks containing clams designated for starvation. After the clams were placed in the containers, the water temperature was si owly changed , over a 72 hour period, to al 1 ow time for the clams to acclimate to the temperature. Twenty four hours had been used initially, only to result in high and sometimes total mortality within 36 hours, particularly among those clams in 28o C water. For the clams designated for 28oc, the water was slowly heated. For those assigned the lioc temperature, the water was slowly cooled. A total water change was done every 48 hours. Care was taken to adjust the temperature of the new water to the experimental temperature to prevent possible shock to the clams by a quick change in temperature. Appropriate food levels were administered immediately following the water replacements. Snipping of the experimental subject's siphons was conducted at the beginning of the experiment, prior to the first feeding. This cutting was performed manually with scissors. The clams were observed individually on a daily basis to determine mortality and to ascertain when full siphon regeneration had occurred (i.e., when the sensory tentacles had reached pre-snipped size). The time needed for this 23 regeneration was recorded . At this time, the siphon was again snipped. After this second snipping, total clam weight and length measurements were recorded. An analysis of variance was done using the Statistical Analysis System (SAS) computer program (SAS Institute, Cary, North Carolina, 1982). The level of significance used in all comparisons of the analysis was the 0.05 level. Mortalities were noted as they occurred. 24 Results and Discussion Results from this experiment indicate variation in siphon regeneration rates (Figure 6, Table 7), variation in total weight gain (Figure 7, Table 8), and variation in total length increases (Figure 8, Table 8) as being dependent on varying temperature levels. The food levels used were found to have no notable impact on these variables (Figures 9, 10, and 11; Tables 7 and 8). It must be noted that with respect to the mean length and weight differences, statistical significance did not necessarily indicate biological significance, primarily because of the small differences in means. The degree of independence of the variables used (food and temperature) from each other is seen in Tables 3, 4, and 5. The correlation coefficients and p values of food level with the clam length and weight illustrate food's overall impact in the course of the research to be insignificant. The correlation values for temperature indicate a very significant effect on the two variables length and weight. The lack of significant interaction allowed the effects of temperature and food to be assessed seperately. 25 Figure 6 Relationship of temperature level to mean number of days required for full siphon regeneration (S.E. = standard error) 20^ (SAVQ) 16 -i L. 16 32 TEMPERATURE '?c) Figure 7, Relationship of clam weight gain to experimental temperature level (S.E. = standard error) (GRGWAAEMIGINSH)T TEMPERATURE (oc) 27 Figure 3. Relationship of clam length gain to temperature levels used (S.E. = standard error) FEPofFERATUREi^C) 28 figure 9. Relationship of food levels to days required for full siphon regeneration (S.E. = standard error) ( BACTERIA/ML) 29 Figure 10. Relationship of clam weight gain to varying food levels (S.E. = standard error) GAWÎNÎEGRJAGMSK) T 2.5x10^ Ix2lO