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L. , 5, 115,555,155'3'55: @1111, 1' v: \ I h-..” ,l" ‘gmtzx: 5S2. ..;- 1 c5. 2‘ 1% 4.. .—Z= Zima . 33:33: - 5- «25%;; I;_"Ef:_:;-‘_.Ifl’u~;a;go I E. . 5,? - i‘) 1w 5 I» i535." 553$ ‘35:! ‘ LIBRARY “lumen State University This is to certify that the thesis entitled The Effects of Food Availability and Temperature on the Specific Growth Rate of Dap_hnia Mg presented by Abdel M. Ab dal la has been accepted towards fulfillment of the requirements for Master of Science degree in Fisheries and Wildlife M N “9 W I“ Date 01 0-7639 MSU is an ‘fimmn'iw ‘ ' "1 ' A”. ', Institution MSU LIBRARIES .—_—. W"U\~ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. 3.12. i!!!3.i 8E. .:- may i 6 1994 5i::'- Elk?! i g jpg; _ .- Q.‘-".-'-;.. g L'r' -' t? .- THE EFFECTS OF FOOD AVAILABILITY AND ‘ TEMPERATURE ON THE SPECIFIC GROWTH RATE OF 2. PULEX Abdel Moez A. F. Abdalla Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1985 ABSTRACT THE EFFECTS OF FOOD AVAILABILITY AND TEMPERATURE ON THE SPECIFIC GROWTH RATE OF 2. PULEX by Abdel Moez A. F. Abdalla The population size and growth rate of Q. pulgg was observed at three temperatures (12, 20, 26°C) and five difference concentrations of torula yeast (cells/ml/day) at each temperature. .The population size and specific growth rate of Daphnia increased with increasing food concentration and temperature. Yeast concentrations had a greater effect on Daphnia population densities than temperature. The relationship between the specific growth rate of 2. pulg§ and the amount of torula yeast available per individual Daphnia per day followd the Michaelis-Menton equation with a correction for the threshold concentration. The predicted specific growth rate of 23 pulg§ increased with increasing food concentration (yeast cells/individual Daphnia/ day). The lowest and highest threshold yeast concentration (when growth rate is equal to zero) occurred at 200C and 12°C respectively. The lowest and highest concentrations of yeast (cells/individual/day) at maximum efficiency occurred at 260C and 12°C, respectively. ACKNOWLEDGMENTS I sincerely wish to thank Dr. Donald L. Carling, Jr. my major adviser for his help in constructing the equipment used and also for reviewing this manuscript and for solving all the problems which I faced during this study. Warmest thanks are expressed to Dr. Darrell L. King, my committee member for his guidance and assistance, without which this research would never have been possible. Heartfelt thanks are expressed to Dr. Alevin L. Rogers, another committte member, for his techinical assistance and understanding. Sincere thanks go to Dr. James D. Hall for all the advice and time which he gave me. I would also like to thank Dr. Niles R. Kevern for facilitating the use of his limnology lab. Thanks are extended to my fellow graduate students in the Aquaculture Laboratory, Anthony Ostrowski, Douglas Sweet and Ibrahim El Shishtawi, who made my days with them more enjoyable. Sincere thanks are expressed to my friend El-Eraki Mohamed for his statistical assistance. Special thanks are expresed to D. Hicks for the support and en- couragement. Finally, much appreciation is expressed to the American Agency for International Development (AID) for supporting me financially during my stay in the United States. ii Dedication This thesis is dedicated to the soul of my kind and lovely father (1923-1983). iii TABLE OF CONTENTS Page LIST OF TABLES ..................................................... V LIST OF FIGURES ............. ..... .................................. Vii INTRODUCTION ........... . . ..... . . . ............................ 1 LITERATURE REVIEW .................................................. 5 MATERIALS AND METHODS.. ............................................. 25 Experiment 1 .................................................. 28 Experiment 2 ..... . .............. . ............ ................. 32 Experiment 3 .................................................. 33 Statistical Analysis ................................. . ........ 33 RESULTS .................................. . ......................... 39 Experiment 1 ........................................... . ...... 39 Experiment 2 ................................... . .............. 48 Experiment 3 ...................................... . ........... 59 Comparative Results of the Three Experiments .................. 66 DISCUSSION ......................................................... 75 SUMMARY ............................ . ............................... 91 LITERATURE CITED ................................................... 93 APPENDIX . .......................................................... 103 iv Number LIST OF TABLES Page Total dissolved metal concentration in well water ......... 27 The relationship between the observed growth rate of 2. pulex and the number of torula yeast cells per individual 2. pulex per day at 12°C ....................... 41 The relationship between the number of torula yeast cells per individual_2. pulex per day (S/N), the observed growth rates of D. pulex and the efficiency in the 4 combined results of two concentrations of yeast (1x10 , 2.5xlO cells/ml/day) at 12°C ................ . ........... 44 The relationship between the observed growth rate of .2. pulex and the number of torula yeast per individual per day at 20°C . .................... . ............. .. ..... 50 The relationship between the number of torula yeast cells per individual 2. pulex per day (S/N), the observed growth rates of D. pulex and the efficiency in the combined results of yeast (0.5x104, 1x104, 1.5x104, 2x104 and 2.5x104 cells/ml/day) at 20°C ....... .... ................. 54 The relationship between the observed growth rate of 2, pulex and the number of torula yeast cells per individual 2. pulex per day at 26°C ...................... 61 The relationship between the number of torula yeast cells per individual_D. pulex per day (S/N), the observed growth rates of 2. pulex and the efficiency in the combined result. of fize concentra ions of yeast (0.5x104, 1x104, 1.5xlO , 2x10 and 2.5xlO cells/ml/day) at 26°C ......... 64 The relationship between different concentrations of yeast (cells/ml/day), temperature and the maximum growth rate (Umax day—l) for D. pulex ........... . ............... 67 Summary of the analysis of variance and multiple classification analysis of the population size of D, pulex at the three levels of temperature (120, 200, 260C) and the concentration of food (yeast cells/ml/day) ............ 7O Number A comparison between all the predicted parameters of the three experiments (120, 200, 260C) .................. Appendix Tables Daily numbers 0f.2- pulex living (L), dead (D) and replaced (R) concentrations of torula yeast cultured in duplicate (1,2) at 12°C ......... . ................. ... The average daily numbers of living Daphnia at 12°C cultured in five different duplicate concentrations of torula yeast .... ..................................... .. Daily number of Q. pulex living (L), dead (D), and replaced (R) cultured in duplicate (1,2) concentrations at 200C ......................................... . ...... The average daily numbers of living Daphnia at 20°C cultured in five different duplicate concentrations of torula yeast .................. ..... ........ . ..... . ..... Daily numbers of D. pulex living (L), dead (D) and re— placed (R) cultured in five different duplicate concentrations of torula yeaat at 26°C ............... ... The average daily numbers of living Daphnia at 26°C cultured in five different duplicate concentrations of torula yeast ........................................ vi 103 106 107 111 112 115 -..—w Number 11 LIST OF FIGURES The technique used to transfer Daphnia in the first experiment ................................................ The suction apparatus used to transfer_2. pulex in the second and third experiment ................... . ........... The system used to raise_2. pulex in the third experiment.. Population size of_Q. pulex at 12 degrees celsius with five different concentrations of torula yeast (cells/ml/day) ............................................ The relationship between the observed growth rates (*), the predicted specific growth rate and the amount of torula yeast available per individual 2. pulex per day at 12°C ............................................... The relationship between the efficiency and the amount of torula yeast available per individual_D. pulex per day at 12°C ................................................... Population size of D. pulex at 20 degrees celsius with five different concentrations of torula yeast (cells/ml/day) ............................................ The relationship between the observed growth rate (*), the predicted specific growth rate and the amount of torula yeast available per individual 2. pulex per day at 20°C ............................................... The relationship between the efficiency and the amount of torula yeast available per individual_D. pulex per day at 20°C ................................................... Population size of D. pulex at 26 degrees celsius with five different concentrations of torula yeast (cells/ml/day) ............................................ The relationship between the observed growth rate (*), the predicted specific growth rate and the amount of torula yeast available per individual 2, pulex per day at 26°C ... Page . 3O 31 35 40 46 47 49 S6 65 Number 3 12 The relationship between the efficiency and the amount of torula yeast available per individual E. pulex per day at 26°C ........................................... 58 13 The relationship between the predicted growth rate of 2. pulex with the amount of torula yeast available per individual 2. pulex per day at the three different temperatures used (12°, 20°, 26°C) ........................ 71 14 The relationship between the efficiency and the amount of torula yeast available per individual D. pulex per day at the three different temperatures usEd (12°, 20°, 26°C).. 73 viii INTRODUCTION One of the major problems to expanding pond fish culture in less developed countries (LDC‘s) is the lack of an adequate supply of fingerlings for stocking. Increasing the availability of fingerlings is a single factor that could result in a rapid increase in pond fish production in many LDC's. Increasing the fingerlings supply will depend on a better understanding of the reproductive biology, better techniques for the rearing of larvae and fry and better production hatcheries (Colt 1983, Torans 1983L Production of certain larval fishes in LDC's has been constrained by a limited understanding of the physical, chemical, and biological processes in pond, and the availability of appropriate sized and quality feed items in culture ponds throughout the rearing period (Yamada 1983, King and Garling 1983). However, in most LDC's, only supplemental feeding with locally available products will be economically feasible (Colt 1983, Lim 1983) since fish diets rely heavily on high quality protein meal which is not readily available in these countries (Mertiz 1972). LDC's should depend on natural productivity to solve their problem of the deficiency in high quality protein diet since the loss of edible protein with either fish or chickens fed protein rich food stuffs can lead to reductions in the protein level of the human diets in these countries in that the increased market cost of the higher grade protein is often beyond the means of a significant portion of the population (King and Garling 1983L Since larve need food items that are visible, suspended in the water, small enough to be ingested, high in protein and essential amino acid and are easily digested, it is no surprise that larvae of most fish feed initially on zooplankton (Sadykov 1975, Arnemo et a1. 1980, Lasker 1975L Fry normally consume 40-80% of their body weight daily (Gorbunova and Lipskaya 1975, Stephen 1976). However, as fish grow, they will usually select progressively larger prey items (Detwyler and Houde 1970, Stephen 1976, Tamas and Horvarth 1976L Daphnia (cladocera: Daphnidae) is a genus which is among the dominant consumers of primary producers in fresh water (Herbert 1978, Lampert 1977 III). It is found in oligotrophic and eutrophic lakes, in ponds and reservoirs where it forms a source of food for both invertebrate and vertebrate predators (Brooks 1959). However, the organisms within an ecosystem may be grouped into a series of more or less discrete trophic levels as primary producers, primary consumers, secondary consumers, etc., each successively dependent upon the preceding level as a source of energy, with the primary producers dependent upon the rate of incident solar radiation (Lindeman 1942L Generally, the importance of fertilizers {inorganic or organic) for enhancing pond production in modern fish culture is indisputable (Yamada 1983). Tang (1970) outlined three pathways through which the organic material entering the pond food web: Firstly, the materials enter as a source of nutritive substances (‘e.g. carbon, phosphorous) for photosynthesis in chlorophyll bearing plants, secondly as an organic substrate for micro organisms which, in turn, support a zooplankton population, and finally it may be consumed directly by fish, crustaceans or insects. However, overfertilization of ponds can result in low dissolved oxygen, high ammonia and high hydrogen sulfide at the same time (Boyd et a1. 1979, Hollerman and Boyd 1980L Walleye fry (7-8 mm) reared in the U.S. can serve as a model for developing extensive larval culture techniques in LDC's. They accept live feeds more readily than milled diets (Kraise and Meade 1982), therefore even the walleye fingerlings used in intensive culture still have to be raised extensively from the fry stage in rearing ponds with natural food. However, high rates of growth and survival in walleye fries were achieved by stimulating Daphnia production by weekly application of sheep or horse manure at 32 ppm plus torula and brewers yeast at 5.3 ppm (Beyerle 1979). Also, Nandy et a1. (1976) obtained high yield of Q; lumholtzi when feeding 0.1% uniform. Our research goal is to develop improved methods for extensive culture of walleye through natural feed production of _D_.pg_._l_g}_< which can serve as a model for larval rearing techniques in LDC's. The objective of this study was to determine the effects of food availability (torula yeast) at three different temperatures (12, 20, 26°C) on the specific growth rate of Daphnia pulex. LITERATURE REVIEW Daphnia Biology Daphnia species are well suited to laboratory studies. The life cycle is in the order of days. There are no free eggs or larval stages in the life cycle which would complicate the census of the population. Although males are produced, they are in such a smal l minority that sex ratio can be ignored in the analysis of the results. Except for possible mutations or position effects due to crossing over, the offspring of a single female are genetically identical (Slobodkin 1954L In favorable conditions, Daphnia species reproduce by parthenogensis. The eggs which are laid into the brood pouch develop into free swimming young in a few days. The young are liberated a few hours before the mother moults. Soon after moulting, another clutch of eggs is laid into the brood pouch. The young produced in this way are all females which mature and reproduce in the same manner. In unfavorable conditions, males appear among the offspring and some of the females produce eggs which require fertilization. In each instar, only one of these eggs is produced by each ovary. They pass into a modified brood pouch which darkens and eventually becomes black. This modified brood pouch is called an ephippium and the eggs in it can withstand being dried and frozen. The ephippium with its eggs is cast separately from the rest of the carapace when the female moults. Females which produce ephippia may return to parthenogenetic reproduction when conditions improve. Some arctic populations produce ephippia without any males appearing so that the ephippial eggs appear to be unfertilized. The ephippial eggs are often termed resting eggs since they may not develop for several months or even years (Green 1956, Brooks 1959L However, the egg size in Daphnia species may vary with the nutritional state of the mother, and the larger species have larger eggs and species with larger eggs have larger young (Green 1956). The average caloric value of newborn (0.7 mm), last pre-adult instar (1.3 mm) and actively reproducing Q; pglgfi is 4059, 4124 and 5075 cal/gm respectively. The increased amount of energy in the adults is due to increasing stored fat in the adults (Richman 1958L Daphnia species store energy for reproduction and survival in the form of triglycerides droplets. The amount of energy stored in adults is lowest just after egg production and increases during the intermolt period. Thus, based on the lipid index:(the number and size of droplets carried within cells in the cavity), each animal can be rank-scored from zero to three. Minimum lipid values occur during the peak and initial decline of population number. Growth, survival and reproductive success are paralleled with the magnitude of the lipid index in Q; pulgg (Holm and Shaprio 1984). In 2; catawba, the amount of lipid stored in the neonates was decreasing as the population was increasing, that is because when food was limited, the adults put less lipid into each egg or the neonates were using up that lipid more quickly under such conditions. However, lipid is stored in adult cladocerans whenever an animal achieves a positive net energy balance which means that the metabolic energy costs are less than the energy assimilated (Tessier and Gaulden 1982L Dissolved oxygen and pH are very important environmental factors which affect the Daphnia population. Lack of oxygen inhibits the growth and reproductive capability in Q; obtusa and Q; magna (Fox 1951, Green 1956). Oxygen was found to have a significant effect on the filtering rate of p_.pgl__e_:_< in that below a concentration of 3 mg/l, the rate decreased sharply (Kring and O'brien 1976). But, at the same time, hemoglobin can be produced at very low concentration of oxygen (0.6 mg/l) in Daphnia, thus enabling Daphnia to survive (Fox 1948, Fox et al. 1951, Kring and O'brien 1976, Landon and Stasiak 1983). The optimal pH for Q; pglgg and Q; magna is between 7-8 (O'brien 1976, Leonard and Lawrence, unpublished data). Changes in illumination did not appear to affect Daphnia (MacLaren 1963, McMahon 1965L Population Growth and Feeding Behavior The greatest growth increment (measured as carpace length) in seven species of Daphnia didn‘t always occur at the end of the adolescent instar, but it might occur at the end of the pre-adolescent instar or more rarely even earlier (Green 1956). As the initial size increased, the animals tended to become mature in earlier instars. A direct correlation was observed between the size of the females and the number of eggs carried in their brood pouches. A mature female of Q; maggg can assimilate enough material during each instar to produce eggs with a dry weight at least equal to that of her body after the eggs have been laid However, growth measured by total length, carpace length and height in Q; pglgg was sigmoid. The point of inflection in all curves came during the fourth instar. The increments increased up to the fourth instar, then decreased gradually to the eleventh instar, after which they remained low and relatively constant. However, the number of young released during the adult instars increases to a maximum at the tenth instar followed by a gradual decrease (Anderson et al. 1937, Richman 1958). Q; longispina which were starved throughout life, live about 40% longer than those well fed throughout life, but those starved for 11, 14 or 17 instars and then well fed until their death live significantly longer. However, Daphnia starved from birth to various periods after reproductive maturity and well fed for the remainder of life grew slowly during the period of starvation but markedly increased in body length after being given an abundance of food, even though the increased supply of food was provided as late as the eighteenth instar by which time all the well fed sisters have died. Also, when Daphnia were fed abundantly even after being starved, they promptly produced many more young in each brood and the frequency of moults increased and also the frequency of heart beats increased (Ingle 1937). Dunham (1938), Banta et al. (1939) also found that poorly fed animals in corresponding instars attained sizes below those of well fed individuals. Small 2; pulex have a higher production rate than large ones production is the accumulation of organic matter in body material and this relation is clear especially at high food concentration. The higher production rates of small animals are exclusively concerned with body growth, but after the animal reaches maturity 20-30 Ug 0), most of the produced substance is incorporated in the offspring (Lampert 1977 b). This can be explained also in terms of energy consumed as growth. The percent energy consumed as growth in preadult Daphnia was higher at any food concentration used than in the adult Daphnia. However, the adult Daphnia consumed more food than the preadult and most of the lO consumed energy went into the production of offspring (Richman 1958L 2; maggg population at 12°C persisted only for few weeks of faltering growth due to decreased metabolic activity which was not high enough to insure the reproductive and survival rates required for population growth and maintenance (Pratt 1943, MacArthur and Baillie 1929). Pratt (1943) stated that the population at 18° C showed a large initial peak followed by a relatively equiliberated phase which might be due to a series of overlapping generations. At 25°C the population continued to oscillate with no apparent approach to equilibrium and without any apparent environmental change. The source of oscillation is a lack of synchronization of a physiological state with the forces that provoke it. Also, he noticed that the life expectancy'of the animals at 25°C was so short that each population peak represented a separate generation. The effect of temperature on longevity of Daphnia was practically the opposite to the effect of temperature on the metabolic process (MacArthur and Baillie 1929, Pratt 1943, Mclaren 1963). In 2; magng, an increase in temperature up to 28°C increased the initial growth rate by shortening the duration of the instars (Brown 1927). Below 7°C the developmental time was too long to allow good population growth in D_. pulex and above 27°C the cultures died out after a few weeks (Lampert 1977b). At low temperature ll females of d9; magma increased in size more slowly, but reached a large final size than females kept at higher temperature (MacArthur and Baillie 1929, Smith 1963L However, Hall (1964) observed an increase in the size of Q; galeata mendota with increasing temperature up to 250C. The effects of temperature on egg production have been studied by many authors. Berg (1931) found that if the temperature remained below 3°C to 5°C for a long period, egg production by Q; mggga stopped, but if the temperature rose to 6°C to 100C, it started again. In 2; pulex temperature of 150 to 2500 were favorable for egg production, but above and below these .pa temperatures. there was a considerable reduction in the number of eggs produced (Tamson 1930). Daphnia species can graze on a broad range of high quality food particles and translate them into high rates of production and growth (Allen 1976). Rotifers and large cladocerans (Daphnia) and calanoids are all able to collect particles of the 1-15 U range. The competitive success of the larger plankton herbivores is probably due to the greater effectiveness of food collection and a relatively reduced metabolic demand per unit mass which permit the assimilation to go to egg production (Brooks and Dodson 1965). However, 2; pulex and Q; magng are able to select food of a certain size and the maximum particle size ingested by cladocerans, increases linearly with increasing carapace length (Berman and Richman 1974, Burn 1968, Gliwicz 1980). Log phase ghlgrgllg yulgarig didn't inhibit feeding, but senescent cells caused 24 magna to decrease the filtering rate and its maximum feeding rate (McMahon and Rigler 1965). However, Taub and Dollar (1968) concluded that Q; pglgx failed to reproduce normally when fed either ghlgggllg pyrenoidos or Chlamydomonas reinhardtii because these algae appeared to be deficient in meeting the nutritional requirements of Q; pulex especially with respct to reproduction. Also, Metz (1973) obtained a low assimilation rate when feeding yeast to Q; pglgx. However, there is some evidence that the animals used in his study were in insufficient condition because there was not any eggs at the beginning of the experiment, and also because of low carbon content and up to 75% mortality during the experiment (Lampert 1977 IIb). Although, it was believed that Q; pglgx never utilize the blue green algae, Holm'et al. (1983) observed that 2; pglgx can utilize the blue green at 1 gae Apbeaizemeso Elgg Aguag even if it was put in a mixture of green and blue green algae. Filtering rate of 9; Eggs; (measured in natural lake water and in pure culture of Rhodotorula glutinis yeast) increased with increasing body length and increasing temperature up to 20°C, also above a concentration of 0.25 x 105 yeast cells/ml, filtering rate decreased with increasing cell concentration and below this concentration, filtering rate was maximal (Burns and Rigler 1967L Similar results 13 were obtained for Q; pglgx (Burn 1969, Lynch 1977). These results coincide with Haney (1973) who stated that the grazing rate in the summer time for natural zooplankton communities exceeded 100% day“1 and became less than 10% day’1 during the winter. Peterson et al. (1978) calculated filtering rate for four different Daphnia species grazing on natural bacteria by counting bacteria before and after incubation (for 0.5—2 hours). He used the following formula to calculate the filtering rate: filtering rate (ml animal‘1 hr'l) = 1/t. ln (Co/Cl) ml/animal rate is independent of the concentration (McMahon and Rigler 1965). However, as the size of Q; maggg was increasing, both where: t = incubation time in hours Co = concentration of bacteria or yeast cells at the beginning of incubation CI = concentration of bacteria or yeast cells at the end of incubation. ml animal"1 = volume of water per experimental animal. Filtering rates for D; pulng Q; longermis and Q; middendorffiana ranged from 0.2 - 1.5 ml animal‘1 hr"l depending on the size of the animal and temperature. Feeding rate (cells, animal“1 hr‘l) is obtained by multiplying filtering rate (ml animal“1 hr‘l) by the concentration of cells in the food suspension (cells/ml) (MacMahon 1965, MacMahon and Rigler 1967L l4 Feeding rate of Q; magng measured on four different kinds of feed, showed that below a certain concentration of each feed, the feeding rate is proportional to the concentration of feed and above this concentration. feeding rate is independent of the concentration (McMahon and Rigler 1965). However, as the size of Q; magga was increasing, both maximum feeding rate and maximum filtering rate increased. The "incipient limiting level" (the external level above which there is no limiting effect of food supply) also increased as the size of Daphnia increased. Temperature also affected the feeding rate of Q; mggpg. The maximum feeding rate of Q; magna occurred at 24°C (McMahon 1965). Feeding rate was higher in females of Q; schoedlggi bearing an average of 10 eggs or embryos than in those bearing only two or no eggs apparently as a compensation for a greater metabolic demand of egg bearing females (Hayward and Gallup 1976). The can be explained by Slobodkin's (1954) observations on Q; obtusa. He noticed that the organism with a high filtering rate would show a higher reproductive rate than an organism with low filtering rate. The size dependence of the assimilation rate in Q; pglgx can be described by the power function of the relation A = a.Lb where L is the length of Daphnia in mm. The numerical value of the exponent "b" is mainly influenced by the ability of small and large animals to ingest a certain 15 diet. However, the curves relating assimilation rate to food concentration are very similar to those reported for feeding rates. At low food concentration, the assimilation rate is proportional to concentration, reaching a plateau above the ”incipient limiting level." Daphnids smaller than 3 mm showed a maximum assimilation rate at 20°C and in the animals 3 mm long, the maximum assimilation rate at 25°C (Lampert 1977aL Generally, the ingestion rate of 200 plankton increased significantly with increasing animal size, food concentration and temperature. The filtering rate also increases with the increase in animal size and temperature, but declines as the food concentration increases (Peters and Downing 1984, Frost 1972L Slobodkin (1954) in his study on the population dynamics of Q; obtusa demonstrated the absence of a direct density effect in Q; obtusa because the population size was linearly related to food supply. The population didn't follow a sigmoid population growth curve, thus the population oscillated due to the difference in ecology between different age-size categories of animals. However, this was in agreement with the results of Pratt (1943) and Frank (1952) who found the same oscillations under similar condition for Q; magpa and Q; pglicaria. Generally, time lags were of two types an individual lag which was the time required for an individual animal to adjust its physiological state to an environmental change, and a population lag which was the time required for the age and size distribution in a population to adjust to some environmental change affecting the entire population. However, at equilibrium, the populations maintained a constant size frequency distribution and reproductive rate while an oscillatory population showed a changing size frequency distribution (Slobodkin 1954L The rate of increase of the population in the field may be estimated if the birth, death, emmigration and immigration rates are known. Although no animals reproduce continuously, many do reproduce frequently enough so that, the instantaneous (observed) growth rate can be calculated from the equation: Nt = Noert' where No, Nt denote the initial and time (t) population size respectively, and r is the instantaneous rate of increase. However, if the effects of death, emigration and immigration are ignored, the growth equation becomes: Nt = Noebt, where "b" is the instantaneous birth rate (Hall 1964L Generally, under stable age condition, the instantaneous rate of increase remains constant (Lotka 1925), whereas under a continually changing age distribution, the instantaneous rate of increase will change accordingly (Hall 1964, Slobodkin 1954L The rate of increase "rs" measured by life table and fecundity table for Q; gglgaga mendota ranged from 0.07 to 0.51 day—1 depending on the temperature level used (UPC'20°Q,2§)and the food concentration used measured in Klett units (16K, 1K, l/4K) (Hall 1964). However, temperature influences were greater than food level under the conditions examined. Frequency of molting, duration of egg development and physiological life span were influenced principally by temperature (reproduction occurred every 2 days at 25°C, every 2.6 days at 20°C and every 8 days at 11° C). The growth per instar, maximum carpace length and brood size were influenced principally by food. Generally, there are three variants of rate of increase: the first one is the observed rate of increase "r" which is measured by regressing Loge of population size on time. The slope of this line estimates "r" which will be positive, zero, or negative depending on whether the population is increasing, stable or declining over the period it is observed (Cruchley and Birch 1977). The second one is the rate implied by the life table combinediaith the fecundity table "rs" (Brich 1948, Leslie and Park 1949, Evans and Smith 1952). Finally, the third one "rm" which is the intrinsic rate of natural increase. It is the rate of increase per head under specified physical conditions in an unlimited environment where the effects of increasing density don't need to be considered. It is the maximum rate at which a population with a stable age distribution can increase in a specified environment in the absence of predators. However, it can be calculated as a special case of "rs which is obtained when all the individuals have access to more food, shelter, water and other requirements than they need, or it can be calculated from the logistic curve as follows: L095 K-N/K = a - rmt where K = maximum population size N = population size at any time of the census a = constant rm = intrinsic rate of increase t = time (Birch 1948, Cruchly and Birch 1971). Buening (1978) proposed a model predicting population growth of laboratory cultures of Q; magng as a function of food availability. Combining food availability, initial density and time, the model estimated the finite birth rate, death rate, age of first reproduction, life expectancy, net reproductive rate and intrinsic rate of natural increase as a function of food supply per Daphnia per day. The intrinsic rate of increase appeared as a convex function of food availability. Wright (1965) proposed a model based on chlorophyll concentration, predator density and a constant death rate of Q; sphgglggi. A negative relation between density and the growth rate of Daphnia has been observed. Smith (1963) found a concave l9 relation between density and growth rate whether numbers or dry weights were used to measure density in Q; magaa. He proposed a new model for growth of Q; gagaa derived from the logistic curve. The Verhaulst—Pearl logistic dn/dt = rn(1— N/k) applies to ecology the principles that are used in the construction of rate models for chemical reaction and its appropriate for a single species population which have a single limiting factor in a constant environment. In order to compare the logistic model with any real system, either lags have to be introduced in the model or time free data have to be extracted from the system. However, Frank (1957) found that increasing density in Q; palag was accompanied by increased survival (over a wide range, decreased birth rate and lowered growth rate. The relation between numerical density and the intrinsic rate of natural increase was linear, thus, at maximum density the growth rate equalled zero. However, neither the exponential form of the growth rate nor the logistic formlof the growth rate relate the amount of food the organism used with the specific growth rate of the organism. Therefore, Monod (1949) related the specific activities rates of organisms to the substrate concentration as follows: U = Umax S/S+KS where U = specific rate of activity or growth at existing conditions of limiting substrate or factors. Umax = maximum specific rate of activity or growth ll Ks the condition or concentration of s at which u = 0.5 Umax- This equation is best studied linearly in the form: S/U = Ks/Umax + l/Umax (S) (King, unpublished data, Williams 1973, Wright and Hobbie 1966). There must be a minumum quantity of nutrients required to initiate the specific growth rate of the organism which is the threshold concentration. Young and King (1979) studied the interacting limits to algal growth: light, phosphorus and carbon dioxide availability to show the difference in the threshold concentration of carbon dioxide. They revealed that algae incubated at high light with ample phosphorus (915Lx, 580 Mg P 1‘1) grew faster, over a wide range of carbon dioxide concentrations, and to lower concentration of carbon dioxide than algae grow under other conditions. In contrast, algae incubated at low light with limited phosphorus (280 Lx, 53 Ug P 1‘1) grew at a lower rate, over a narrower range of concentrations of carbon dioxide, and ceased growth at a higher concentration of carbon dioxide compared to other conditions. However, algae yield (mg/day) decreased at high cell concentrations under ' .. I. .- ugha-F' ’ . - ”'13 Pirates: 2"- 'P In- , . .._ H.” i. '. :' .. :' I continuous sunlight irradiance since almost all the light is absorbed and further increase in cell concentration can add only to the overhead metabolism or respiration (Myers and Graham 1959). Generally, the threshold food concentration is necessary for an individual 11; gala; to keep its body mass constant or for a population to equalize all the biomass losses due to natural mortality and predation (Lampert 1977). She stated that from the balance equation (Production = Assimilation - Metabolic losses), it is clear that a high production, which would mean more fitness can be obtained by an increase in ingestion efficiency (higher assimilation rate and by a lower rate of metabolic expenditures. Moreover, if the food concentration is so low that the assimilation rate can not equalize the metabolic losses, a lower metabolic rate would mean better survival. The threshold was measured graphically by the point of intersection between assimilation rate and metabolic losses (when production is zero. There was a slight difference in the threshold concentration between 100 and 20° C, but the threshold increased at temperatures above 20°C. This increase is more profound for larger animals, so that the highest values occurred for animals 3 mm in length at 25°C. Thus, the threshold concentration were lower for small daphnids and it is also depended on the diet species. 22 However at very low concentrations of food, the collecting effort is reduced in copepods because more energy is lost than gained in the feeding process, thus, the maximum filtration effort may be predicted to occur at intermediate food concentration as copepod began to collect more food than they can physically or physiologically utilize efficiently (Lam and Frost 1976, Lehman 1976). This is in agreement with Beklenishev (1962) who believed that with increasing food, the ingestion rate increases and animals consumed food " in quantities far greater than could be utilizedfl' Field experiments on natural particles in sea water by Adams and Steel (1966) and Parsons et al. (1967) suggested that there is a threshold food concentration below which there is no feeding. Corner et al. (1972), Frost (1975) observed reduced grazing rates when copepods fed on very low concentrations of food cells. The copepod Acostia ggmaa dana had a maximum grazing rate at about 10 mg chlorophyll a/l) decreasing to zero below 1 mg chl a/l (Reeve and Walter 1977). If zooplankton cease feeding at a low food concentration in nature, then there is a ”refuge“ for phytoplankton in which they are free from mortality. The existence of such a "refuge" appearing as a positive x intercept of the curve relating ingestion to concentration “.— of food has been claimed by those using the modified Ivelv equation I = Im [(1teO(P_P ))], or those using the modified Michalis—Menton equation [I = Im(P—P"W/(K—P‘) + (P—P")) (Mullin and Fuglister 1975) where: I = the rate of ingestion Im= the maximum ingestion rate P = the concentration of phytoplankton o = constant P‘= the concentration at which ingestion ceases K = the concentration at which I = 0.5 Im. However, Porter et al. (1982) stated that Q; gagaa had no feeding threshold or reduced filtering activity at low concentrations as predicted by optimal foraging models. Generally, the advantage of fitting the hyperbolic ingestion curves to the Michaelis-Menton model in Anuran larvae (Genera: Hyla, Bufo and Rana) feeding on the blue green algae is that it allows calculating important aspects of the curve to be summarized simply with three quantitative parameters the threshold concentration, the half saturation constant and the maximum ingestion rate (Seale and Beckvar 1980). King and Garling (1983) used the same modified Michalis-Menton equation to relate the specific net carbon fixation rate of algae and submersed aquatic plants to the existing carbon dioxide concentration from the equation: U = U max (C — Cq)/ (Kc—Cq) + (C—Cq) where: u = specific net carbon fixation rate (time—1) c = existing carbon dioxide concentration (U moles C02/1) Kc = carbon dioxide concentration at which U = 0.5 Umax (U moles C02/l) Cq = threshold carbon dioxide concentration required to initiate net carbon fixation (U moles C02/l) And thus from this equation, one can relate the specific growth rate of any organism to the amount of food required plus the calculations of the maximum growth rate of the organism and also the calculation of the threshold concentrations which is the amount of food below which there is no growth. MATERIALS AND METHODS Stock Culture of Q; pulex g; 22l2§ were collected from ponds at Michigan State University, Aquaculture Laboratory with a simple conical tow net. 2; palag were identified under the microscope by the method of Brooks (1959). The Daphnia were then cultured in 9 and 30 l aquaria filled with well water. Temperature was controlled around 15—170 C. Daphnia were fed ground Purina Trout Chow every other day. Overcrowding of Daphnia in the aquaria was avoided by reducing the number of Daphnia whenever very high population densities occurred. Transferring the neonates to the experimental units: Individual adults were transferred from stock culture tanks to a depression slide under the microscope. Adults with eggs or embryos in the brood chamber were isolated by placing individual adult Daphnia in a separate 250 ml beaker fil led with well water. Then on the second day, new born neonates were transferred to the experimental units. 25 Experimental Units: All experiments were run in 1000 ml Erlenmeyer flasks using ten randomly chosen new born Daphnia. Three temperature levels (12 i 0.500, 20: 1 °C, 26 :0 C) were run separately with five different concentrations of torula (yeast supplied by the MDNR). Each yeast concentration was run in duplicate. Well water was used with all type experiments (Table 1). The light was continuously supplied by a fluorescent lamp. During each experimental run, additional five Erlenmeyer flasks stocked with 10 newborn Daphnia were maintained with the same yeast concentrations used in each experiment. The Daphnia in these flasks were used to replace any Daphnia which died during the first 48 hours of the experiment. Changing the medium and counting Daphnia: During the experimental runs, Daphnia populations were transferred daily. Any dead Daphnia were removed and counted. An appropriate volume of the new medium was placed in clean flasks. The medium was drawn down to few cc's by suction through silk bolting cloth. In experiment 1, the suction apparatus was made of fine mesh bolting silk which retained newborn Daphnia (134 U mesh nitex net), fastened over the large end of a small plastic funnel with an opening one inch in diameter. Hose from the 27 Table 1. Total dissolved metal concentration in well water. Sensitivity 2&1 Element Concentration 22m 0. 05 Aluminum AL NDA O. 05 Arsenic AS NDA Calcium Ca 85 O . Ol Cadmium Cd NDA 0 . 01 Chromium Cr NDA O. 005 Copper Cu NDA O. 1 Iron Fe NDA 0. 05 Mercury Hg NDA Potassium K 1 . 8 Magnesium Mg 28 0. 01 Manganese Mn NDA 0. 01 Molybdenum Mo NDA Sodium Na 7 . 1 0. 1 Phosphorus P NDA O . 05 Lead Pb NDA O . OS Selenium Se NDA O . 05 Thallium Tl NDA 0. 005 Zinc Zn NDA NDA = no detectable amount small end of the funnel lead to an aspirator which was connected from its other side by another hose attached to a tap. By operating the aspirator (opening the tap), the Daphnia were concentrated in a few cc's. Then the funnel was flushed with little water to get the attached Daphnia. No injuries or deaths for the Daphnia in the experiment were due to the suction system. Daphnia were transferred after this to white porcelain plates (12 cavities) by large mouth pipette with a rubber bulb and counted and transferred to the new medium (Figure 1). In experiments 2 and 3, the technique used to change the old medium (Figure 2) was modified. The suction apparatus used in experiments 2 and 3 was similar to that used in experiment 1. It was made of fine mesh bolting silk which retained newborn Daphnia (130 U mesh nitex net), fastened over the large end of a small plastic funnel with an opening one inch in diameter. Hose from the small end of the funnel was attached to a rubber bulb, and the hose was controlled by a valve to control the flow of water when it was being drawn down. Experiment 1: Experiment I was run from 3/3/84 to 4/3/84 (32 daysL The experiment was terminated after the population reached its maxima, then stabilized or declined. The average well water temperature was 12: O.5°C. The ten Erelemyer flasks 29 Figure l. The technique used to transfer Daphnia in the first experiment. Ezapua 3m: I .(:\J1.,. Amwfiua>mu may . woman cmoaooud AV Av Av umc woufic Hoccow ouuodfid LuzoEoquH .o lilo O \ OO 00 HOumdewm Figure l. Hose Figure 2. CU . > H 'U (U ad > :1' ..D I H 0.) ..D A 3 H l A l fl H 0 A G 54:! t: CDC :1 um I": HF! av .flu and) 0d 2‘. The.suction apparatus used to transfer 2: second and third experiment. Dulex in the 32 were placed on a tray which was fixed in a large aquarium. Very little air was supplied to each flask. Ten newborn Daphnia (1—5 days old) were placed in each flask and fed on one of the five concentrations of torula yeast. Suspensions of yeast (1000 ml) were made withiuell water and counted on a Levi—Hauser Hemocytometer to establish concentrations of leO4, 2 x 104, 0.5 x 10 5, 1 x 105 and 2.5 x 10 5 number of yeast cells pernfl.per day. Experiment 2: Experiment 2 was run from 6—21-84 to 7—30-84 (40 daysL The experiment was terminated after the population reached its maxima and then began to decline. The average temperature was 20: 1°C. The water which was used in this experiment was a well water which was heated in large aquarium (30 l) with a submersed heater (200 W) to 19°C and aerated with an air stone. The temperature increased 2°C every 24 hours and became 210(3at the day of changing the old medium. The ten Erlenmeyer flasks were placed on a tray which was fixed in a large aquarium. No air supply was used in this experiment because oxygen levels were never less than 7 mg 02/1 measured by Winkler method (Kring and O'Brien (1976) observed 3 mg 02/1 as the limiting concentration for Q; pgiaa). Ten newborn Daphnia (1—3 days old) were placed in each flask.and fed one of the five concentrations of torula yeast. To prepare the suspensions of yeast, a standard was performed relating the number of yeast cells in a known weight of the torula yeast. We found that 5 mg yeast contained 286 x 105 cells, thus 40 mg yeast contained 2.288 x 108 cells. Our original suspension was composed of ulmg yeast mixed very well with 300 ml well water. This solution had a concentration of 2.86 x 105 cells/ml. From this solution, the five concentrations of the yeast (0.5 x 104, 1 x 104, 1.5 x 104, 2 x 104 and 2.5 x 104 cells/ml/day) were prepared. Experiment 3: Experiment 3 was run from 8—9—84 to 9-10-84 (34 days). The experiment was terminated after the population reached its maxima and began to decline. The average temperature was 26: 1°C. All the procedures used were the same as the procedures used in the second experiment except temperature regulation concern over a potential decrease of temperature during the course of the experiment. A water bath was set up by filling the aquarium with water and controlled the temperature by a submersed heater (200 W) at 25°C. Water was circulated by an aquarium pump to insure even heat distribution throughout all the aquarium (Figure 3L Statistical analysis: A two—way analysis of variance (ANOVA) and multiple classification analysis were performed with the SPSS program Figure 3. The system used to raise _I_)_. pulex in the third experiment. 35 kl 4.20; 12:1 52:115. \\\\ xmpe ogxm.u OH x N. _mH xm.H on x a ca x m.o q Figure 3. 36 by the Computer Laboratory at Michigan State University to test for the effects of temperature and concentrations of food and the interaction between them on the population density of Q; paiaa. The 0.05 level of probability provides the basis for all statements concerning statistically significant differences. Outline of general procedures: - a. Count the average number of Q; pgiaa daily within each concentration of torula yeast. b. Calculate the specific growth rate of Q; Ragga by the formula: U = ln N2 - In N] At where U = specific growth rate of Daphnia (day-1). N+1 = number of organisms at time 1 N+2 = number of organisms at time 2 At = difference in time between t2, t1- c. Calculate the number of yeast cells per individual Daphnia per day by the formula: ' ll 1 d )x 1000(f1ask volume) cells/individual/day= concentration (Ge S/m / ay Number of Daphnia 37 Combine the information relating the number of cells per individual per day (S/N) with the observed specific growth rate of Q; Raga; from each concentrations used at the specified temperatures. Calculate the threshold concentration (sq) of Q; EBAEfi at this temperature by averaging all sq"s used with different concentrations. However, the threshold concentration (sq) is the value of S/N when the growth rate is equal to zero, and it is determined by dividing the amount of yeast cells available per day by the maximum number of Daphnia attained (Sq = S/K). Manipulate the main equation which predicts the threshold concentration as follows: U = umax (S-Sq) + (Ks—Sq) ‘ 1 or = ax S/N — S/Nq 2 (s/N-s/Nq) + (Ks/n-S/Nq) where U = specific growth rate of Q; pulex per day Umax = Maximum growth rate of Q; pulex per day S/N = number of yeast cells per individual Daphnia per day Ks/n = number of yeast cells per individual Daphnia per day when U is equal to 0.5 Umax. S/Nq = Threshold concentration or the number of yeast cells per individual Daphnia per day required to initiate the growth rate of Q; Ragga. 01' U _ Umax S/N - S/Nq ' (S/N-S/Nq) + (Ks — S/Nq) /n I. :b { .. . . . _ “~3- ._ . .. .fifi'b =Msm'dr an”: flushifil ' _- . .. - flit mes-..- w- - . . -. ' .. ~ . fut-ME; I 15'?" ad? Swen-Mn:- . . - -- . .- ..-,.. . Fl '1 Nil. _$."-I‘l|: .= .: 'I .._~-_.. ll" . ' - I _ L_1 I 'F"' "a 38 01' s/N - S/Nq, = Ks/n - S/qu + U Umax Equation (4) takes the form of ragressed between -S/_N_E_S_le9.__ l ) The with a slope equal to ( is equal to Umax). The Y intercept multiplied by the reciprocal of the - S/Nq). Thus, from this step, the 7H 1 (S/N - S/Nq) 4 Umax Y'= a + bx and can be and (S/N - S/Nq). reciprocal of the slope ( Ksln — S/Ng ) Umax slope (Umax) yields Ks/n parameters of the equation Umax, Ks/n and S/Nq can be calculated. RESULTS Three experiments were run at three different temperatures (12, 20, 26°C). Five different concentrations of torula yeast (yeast cells/ml/day) were used at each temperature level. Each concentration of yeast was fed in duplicate 1000 ml Erlenmyer flasks containing ten Q; pglex. Each experiment was ended when population size stabilized or began to decrease. Experiment one: Five concentrations of torula yeast (1 x 104, 2.5 x 104, 0.5 x 105, l x 105 and 2 x 105 yeast cells/ml/day) were maintained at 12:0.5°C in the first experiment (Appendix Tables 1,2 and Figure 4). The population densities of Q; pglag increased at yeast concentration of l x 104, 2.5 x 104 and 0.5 x 105 yeast cells/ml/day. At higher yeast concentrations (1 x 105, 2 x 105 yeast cells/ml/day, the population density of Q; palaa was less than that at lower concentrations. The threshold concentrations (S/Nq = the amount of torula yeast available per individual 2; palag per day when the growth rate is equal to zero) at 12°C were 2.63 x 105 WNHU) zam—(IH‘C‘UO’U 110 Figurt A . Population size of L3 Dule} a: differen: concentrations of DRYS 12 degrees celsius with five torula yeas: \cells/nd/aayg. Al Table 2. The relationship between the observed growth rate of 2. pulex and the number of torula yeast cells per individual 2. pulex per day at 120C. 3. for l x 104 cells/ml/day Number of (S/N)cells/ Day Dapggia U individual/day 12 8 0.229 12.5 x 105 16 20 0.056 3 x 105 20 25 0.069 4 x 105 24 33 0.035 3.03 x 105 28 38 0 2.63 x 105 3o 38 0 2.63 x 105 32 38 o 2.63 x 105 b. for 2.5 x 104 cells/ml/day Number of (S/N)cells/ Day Daphnia U individual/day 12 8 0.347 31.25 x 105 16 32 0.126 7.81 x 105 20 53 0.035 4.72 x 105 24 61 0.094 4.1 x 105 28 89 0.011 2.81 x 105 30 91 0 2.75 x 105 32 91 0 2.75 x 103 42 Table 2. (cont'd.) c. for 0.5 x 105 cells/ml/day Number of S/N/cells/ Day Daphnia U individual/day 8 9 O.l87 55.6 :4 l05 l2 l9 0.l86 26.3 x 105 16 ’40 0.075 [2.5 x l05 20 54 0.075 9.26 x 105 24 73 0.073 6.85 v 105 28 98 0.015 5.1.0 X 105 30 101 0 4.95 .6 105 43 yeast cells/individual/day at the 1 x 104 yeast cells/ml/day concentration and 2.75 x 105 yeast cells/individual at the 2.5 x 104 cells/ml/day concentration ’Table 2). The average 2.? x L05 yeast cells/indiVIdual day was considered as The threshold concentration at 120C. The max1mum growth rate 3f 3. I L) ulex ‘Umax) increased with increasing food concentrations (yeast cells/ml/day) (Table 8). Umax calculations were based on the Michaelis- Menton equation corrected for the threshold concentration at each concentration of food (yeast cells/ml/day). Consequently, the results of the three highest yeast concentrations (0.53:105, 1 x 105, 2 x 105 yeast cells/ml/day) were omitted from the sequence analysis of data regarding the predicted values from the Michaelis- Menton equation at 12°C. The combined results from both yeast concentrations (1 x 104, 2.5 x 104 yeast cells/ml/day) and the observed specific growth rate (U) are shown in Table 3. The observed growth rate (U) increased in most cases with increasing food concentration. The three constant parameters (Umax, Ks/n, S/Nq) of the Michaelis—Menton equation at 12°C corrected for the threshold concentration were Umax = 0.46 day—1, Ks/n = 13.28 X 105 yeast cells/individual/day, S/Nq = 2.7 x 105 yeast cells/individual/day. The equation relating the predicted specific growth rate of D; pulex at 12°C to the 44 Table 3. The relationship between the number of torula yeast cells per individual 2. pulex per day (S/N), the observed growth rates of D. pulex and the efficiency in the combined results of two concentrations of yeast (1 x 104, 2.5 x 104 cells/ml/ day) at 120. S/N Calculated Efficiency Observed Order cells/individual/dav u u/S/N growth rate 1 31 25 x 103 0.336 1.0752 x 10'7 0.347 2 12.5 x 105 0.224 1.792 x 10'7 0.229 3 7.81 x 105 0.15 1.92 x 10‘7 0.126 4 5 x 105 0.082 1.64 x 10‘7 0.056 5 4.72 x 105 0.074 1.57 x 10'7 0.035 6 4.1 x 105 0.054 1.32 x 10‘7 0 094 7 4 x 105 0.05 1.25 x 10'7 0.069 8 3.03 x 105 0.014 0.462 x 10’7 0.035 9 2.81 x 105 0.005 0.178 x 10'7 0.011 10. 2.7 x 105 0 o 0 45 amount of yeast available per individual Daphnia per day is: S/N - 270000 U = 0'46 S/N + 788000 From this equation, Table 3, and Figure 5, it is evident that the predicted specific growth rate of Q; puleg increased with increasing food concentrations S/N (yeast cells/individual/day). However, the effect of changing a unit of food concentration (S/N) on the growth rate of D; pglgg at high food concentrations was less effective than the changes at low food concentration. The predicted growth rate at a food concentration of 24 x 105 yeast cells/individual/day was 0.308 day'1 and at 25 x 105 yeast cells/individual/day was 0.312 day‘l, thus an increase of one unit of (S/N) caused a 0.004 day"1 increase in the growth rate. Also, at a food concentration of 3 x 105 yeast cells/individual/day, the growth rate was 0.013 day‘1 and at 4 x 105 yeast cells/individual/day. The growth rate was 0.050, thus an increase in one unit of yeast concentration (S/N) caused a 0.03? day‘1 increase in the growth rate. The maximum efficiency was predicted to occur at a concentration of 7.81 x 105 yeast cells/individual/day (Figure 6, Table 3). At high concentrations of torula yeast, the efficiency was low and increased with decreasing food concentration until the efficiency approached its apex at an intermediate food concentration, after which the 0.30:I 0.25 0.20 0.15 010 I 1 l 1 T 1 1 1 l 1 1 1 T j 0 2 4 5 8 10 12 14 16 18 2O 22 24 25 28 3O 32 (s/n) x 10**5 cells/individuol/doy Figure 5. The relationship between the observed growth rates (*), the predicted specific growth rate and the amount of torula yeast available per individual 2. pulex per day at 12°C. efficiency (U/S/n) X 10**—7 1.500- 1.000- 2 ‘5» ([5 55 1?) 1‘2 1T4 116 1E2r0 2'2 214- 216 218 35312 (s/n) x 10**5 cells/individuol/doy Figure 6. The relationship between the efficiency and the amount of torula yeast available per individual D. pulex per day at lZOC. 48 efficiency started to decrease again with decreasing food concentration (S/N). Experiment 2: Five concentrations of torula yeast (0.5 x 104 x 1 x 104, 1.5 x 104, 2 x 104 and 2.5 x 104) were maintained at 20t1°C in the second experiment (Appendix Tables 3, 4, Figure 7). The size of Q; pglgx population increased at any yeast concentration with increasing the culture age, than stabilized (0.5 x 104 x 1 x 1.04 yeast cells/ml/day). The population size of Q; pglgx increased with increasing food concentration (yeast cells/ml/dayL The population size increased slowly during the first 27—30 days of the experiment, after which the population increased very fast and reached its apex at day 36. After day 36, the population either stabilized or declined. However, the population size of Q; pglgx growing at a concentration of 0.5 x 104 yeast cells/ml/day reached its apex at day 23 then stabilized. The threshold concentrations (S/Nq) at 200 C were 1.42 x 105, 0.72 x 105, 0.62 x 105 and 0.66 x 105 yeast cells/individual/day for the concentrations of 0.5 x 104, 1 x 104,1.5 x 104, 2 x 104 and 2.5 x 104 yeast cells/ml/day (Table 4). The average 0.82 x 105 yeast cells/individual/day was considered as the threshold concentration at 20°C. The maximum growth rate (Umax) of g; pulex increased with increasing food concentration (yeast cells/ml/day) mNHU) ZOH—iDI—C'UOT) 400 300 250 200 150 100 11111 .5 l 10:14 .0 I 10:14 .5 I 101:4 .0 I 101-4 -5 ‘ 101-4 Figure 7. Population size of five different concentrations 0 day). D. pulex at 20 degrees celsius with f torula yeast (cells/ml/ 50 Table 4. The relationship between the observed growth rate of 2. pulex and the number of torula yeast per individual per day at 20°C. a. for 0.5 x 104 cells/ml/day Number of S/N cells/ Day Daphnia U individual/day 8 10 0.107 5 x 105 14 19 0.05 2.63 x 103 15 20 0.02 2 5 x 105 20 26 0.176 1 92 x 105 21 31 0.03 1 61 x 105 22 32 0.09 1 56 x 105 23 35 0 1.42 x 105 b. for l x 104 cells/ml/day Number of S/N cells/ Day Daphnia U individual/day 8 10 0.168 10 x 105 10 14 0.153 7.14 x 105 12 19 0.156 5.26 x 105 14 26 0.02 3.85 x 105 19 29 0.09 3 .5 x 105 21 35 0.099 2 85 x 105 25 52 0.052 1.92 x 105 31 71 0.18 1.4 x105 33 102 0.113 0.98 x '105 35 128 0.03 0.78 x 10 37 137 0 0.72 x 10 Table 4. (cont'd.) c. for 1.5 x 104 cells/ml/day Number of S/N cells/ Day Daphnia U individual/day 8 10 0.257 15 x 105 12 28 0.113 5.35 x 105 16 44 0.027 3.4 x 105 20 49 0.093 3.06 x 105 24 71 0.06 2.11 x 105 28 91 0.073 1.64 x 105 32 122 0.171 1.23 x 105 36 249 0.004 0.6024 x 105 37 250 0 0.6 x105 d. for 2 x 104 cells/ml/day Number of S/N cells/ Day Daphnia U individual/day 8 10 0.257 20 x 105 12 28 0.16 7.14 x 105 16 53 0.1 3.77 x 105 20 78 0.019 2.56 x 105 24 84 0.019 2 38 x 105 28 92 0.023 2 17 x 105 32 216 0.08 9 x 103 u \1 ca N L.) O O O O\ N x .... O 52 Table 4. (cont'd.) e. for 2.5 x 104 cells/ml/day Number of S/N cells/ Day Daphnia U individual/day 8 10 0.365 25 x 103 12 43 0.173 5.81 x 105 16 86 0.05 2.9 x 105 20 106 0.03 2.35 x 105 24 119 0.05 2.1 x 105 28 147 0.179 1.7 x 105 32 302 0.05 0.8 x 105 36 374 0 0.66 x 105 (Table 8). Umax's calculations were based on the linear transformation of the Michaelis—Menton equation corrected for the threshold at each concentration of yeast. (The Umax‘s of Q; pglex were 0.11, 0.176, 0.318, 0.53 and 0.55 day‘1 for the population growing at concentrations of 0.5 X 104 x 1 x 104, 1.5 x 104, 2 x 104 and 2.5 :r. 104 yeast cells/ml/dayrespectively. The combined results from the five concentrations of yeast (0.5 x 104 x 1 x 104, 1.5 x 104, 2 x 104 and 2.5 x 104 yeast cells/ml/day) and the observed specific growth rate are shown in Table 5. In general, the observed growth rate of Q; pglgx increased with increasing food concentration (S/N). However, the observed growth rate increased in some cases with decreasing food concentration (s/nL The three constant parameters (Umax, Ks/n, S/Nq) of the Michaelis-Menton equation at 20°C corrected for the threshold concentration were: Umax = 0.5 day‘l, Ks/n = 16.32 x 105 yeast cells/individual/day, S/Nq = 0.82 x 105 yeast cells/individual/day. The equation relating the predicted specific growth rate of g; pglgx at 20°C to the amount of yeast available per individual Daphnia per day is: S/N — 82000 U = 0-5 m From this equation, Table 5 and Figure 8, it is evident that the predicted specific growth rate of Q; pglex increased 54 Table 5 . The relationship between the number of torula yeast cells per individual 2. pulex per day (S/N), The observed growth rates of D. pulex and the efficiency in the combined 7 results of five concentration pt yeast (0.5 x 10 , 1 x 10*, 1.5 x 104 2 x 104 and 2.5 x 104 cells/ml/day) at 20°C. 5 Observed Calculated S/N x 10 growth growth Calculated Order rate (U) rate efficiegcyi 1 25 0.365 0.305 1.22 x 10'7 2 20 0.257 0.277 1.39 x 10'7 3 15 0.257 0.239 1.59 x 10‘7 4 10 0.16 0.186 1.86 x 10‘7 5 7.14 0.16 0.145 2.03 x 10'7 6 5.81 0.173 0.122 2 1 x 10‘7 7 5.35 0.113 0 113 2 11 x 10'7 8 5.26 0.09 0.111 2.11 x 10-7 9 5 0 107 0.106 2.12 x 10‘7 10 3.77 0.1 0.08 2.12 x 10'7 11 3.7 0.03 0.078 2.11 x 10‘7 12 3.4 0.027 0.071 2.08 x 10"7 13 3.3 0.14 0.069 2.09 x 10'7 14 3.06 0.093 0.063 2.06 x 10-7 15 2.9 0.05 0.059 2.03 x 10—7 16 2.63 0.05 0.052 1.98 x 10-7 17 2.56 0.019 0.05 1.95 x 10—7 18 2.5 0.02 0.049 1.96 x 10‘7 19 2.38 0.019 5 0.046 1.93 x 10‘7 20 2.35 0.03 0.045 1.91 x 10 55 ’Cable 5 . (cont'd.) 5 Observed Calculated S/N x 10 growth growth Calculated Order rate (U) rate efficiency 21 2.17 0.023 0.04 1.84 x 10 22 2.11 0.06 0.038 1.8 x 10 23 2.1 0.05 0.038 1.81 x 10 24 1.92 0.176 0.033 1.72 x 10 25 1.9 0.05 0.033 1.74 x 10 26 1.7 0.179 0.027 1.59 x 10 27 1.64 0.073 0.025 1.52 x 10 28 1.61 0.03 0.024 1.49 x 10 29 1.6 0.07 0.024 1.5 x 10 30 1.56 0.09 0.023 1.47 x 10 31 1.23 0.171 0.013 1.06 x 10 32 1.2 0.11 0.012 1 x 10 33 0.9 0.213 0.003 0.33 x 10 34 0.82 0 0 9 4: O U 9 o o N C) CD WWJJ 44.121.444.421 1 9 u 03 0.15 0.10 (105 O N (s/n) x 10**5 cells/Individuol/doy Figure 8. The relationship between the observed growth rate (*), the predicted specific growth rate and the amount of torula yeast available per individual 2. pulex per day at 20°C. 1 r r 1 1 I 1 I l T F 1 I 1 l 4 6 8 1O 12 14 16 18 20 22 24 28 28 30 32 57 with increasing food concentration (S/N). However, the effect of changing one unit of food concentration (S/N) at high food concentrations on the growth rate of Q; pglgx was less effective than that at low food concentrations. The predicted specific growth rate at a food concentration of 24 x 105 yeast cells/individual/day was 0.300 day‘l, and at a concentration of 25 x 105 yeast cells/individual/day was 0.305 day‘l, thus an increase of one unit of food (S/N) at high food concentration caused a 0.005 day‘l increase in the growth rate. At a food concentration of 3 x 105 yeast cells/individual/day, the growth was 0.062 day—1, and at food concentration of 4 x 105 yeast cells/individual/day was 0.085 day-1. Thus an increase of one unit of food (S/N) at low food concentration caused a 0.023 day‘l increase in the predicted specific growth rate. The maximum efficiency was predicted to occur between concentrations of 5 x 105 and 3.77 x 105 yeast cells/individual/day, therefore a concentration of 4.385 x 105 yeast cells/individual/day was considered the concentration at which maximum efficiency occurred (Table 5, Figure 9). At higher concentrations of torula yeast, the efficiency was low and increased with decreasing food concentration until the efficiency approached its apex at a concentration of 4.39 x 105 yeast cells/individual/day, after which the efficiency started to decrease again with decreasing food concentration (S/NL efficiency 2.00- ,\ I I u i 1.50- O . x - A 1.00- C d \\\ . 09 . § . . V 0.50- 0300 1 I I I i I I I 1 I I I I I 0 2 4 6 8 10‘12 14 16 18 20 22 24 26 (s/n) x10**5 celis/individuol/doy Figure 9. The relationship between the efficiency and the amount of torula yeast available per individual 2. pulex per day at 20°C. Experiment 3: Five concentrations of torula yeast (0.5 x 104 x 1 x 104, 1.5 x 104, 2 x 104 and 2.5 x 104 yeast cells/ml/day) were maintained at 25:100 in the third experiment (Appendix Tables 5, 6; Figure 10). The population size of Daphnia reached its maxima after 14 days in the three lowest concentrations of yeast (0.5 x 104 x 1 x 104, 1.5 x 104 yeast cells/ml/dayL However, at the two highest concentrations (2.0 x 104, 2.5 x 104 yeast cells/ml/day, the population reached its maxima after 14 days, attained short equilibrium for 6—7 days. After day 22, the population started to increase again until day 28, after which the population started to decline again. The threshold concentrations (S/Nq) were 1.04 x 105, 0.89 x 105, 0.76 x 105, 0.81 x 105 and 0.87 x 105 yeast cells/individual/day for the concentrations: 0.5 x 104 x 1 x 104, 1.5 x 104, 2 x 104 and 2.5 x 104 yeast cells/ml/day (Table 6L The average 0.87 x 105 yeast cells/individual/ day was considered as the threshold concentration for the population of g; pglgx growing at 26°C. The maximum growth rate (Umax) of Q; pglgx increased with increasing food concentrations (yeast cells/ml/day) (Table 8). Umax's calculations were based on the Michaelis— Menton equation corrected for the threshold concentration at each concentration of food (yeast cells/ml/day). The Umax‘s mNHm ZOHflDFCTOV 300 H05 I 10II4 M 1 o u A 250 - 1 5 . ZOI ZSI 200 _ 150 _ 100 _ so _ 5 10 15 213/ 25 30 35 40 oan Figure 10. Population size of Q. pulex at 26 degrees celsius with five different concentrations of torula yeast (cells/ml/day). 61 Table 6. The relationship between the observed growth rate of Q. pulex and the number of torula yeast cells per individual 2. pulex per day at 26°C. a. for 0.5 x 104 cells/ml/day Number of S/N cells/ Dav Daphnia U individual/day 6 10 0.168 5 x 105 8 14 0.269 3.57 x 105 10 24 0.159 2.08 x 105 12 33 0.187 1.52 x 105 14 48 0 1.04 x 105 16 48 0 1.04 x 105 b. for l x 104 cells/ml/day Number of S/N cells/ Day Daphnia U individual/day 5 6 10 0.668 10 x 10 8 38 0.333 2.63 x 105 10 74 0.278 1.35 x 105 12 129 0.011 0.78 x 105 14 132 o 0.76 x 105 c. for 1.5 x 10“ cells/ml/day Number of S/K cellsfl Dai Daphnia 1 individual/day 6 10 0.752 15 x 105 8 45 0.306 3.35 x 105 16 83 0.299 1.8 a 105 1" 13- 0.05- 6.99 x 105 14 168 0 0.89 1 105 15 168 0.89 x 105 62 Table 6. (cont'd.) d. for 2 x 104 cells/ml/day Number of S/N cells/ Day Daphnia U individual/day 6 10 0.763 20 x 105 8 46 0.357 4.35 x 105 10 94 0.253 2.13 x 105 12 156 0.066 1.2820 x 105 14 178 0.008 1.12 x 105 24 193 0.056 1.03 x 105 26 216 0.045 0.93 x 105 29 247 0 0.81 x 105 31 247 e. for 2.5 x 104 cells/ml/day Number of S/N cells/ Day Daphnia U individual/day 6 10 0.784 25 x 105 8 48 0.223 5.2 x 105 10 75 0.412 3.3 x 105 1: 171 0.058 1.46 x 105 14 192 0.002 1.3 x 105 24 195 0.056 1.2821 x 105 26 218 0.136 1.14 x 105 28 286 1 0.87 x 105 31 286 0 0.87 x 103 63 of g; pulex at 260C were 0.18, 0 772, 0.87, 0.92, and 0.98 day‘1 for the population growing at concentrations of 0.5 x 104 x 1 x 104, 1.5 x 104, 2 x 104 and 2.5 x 104 yeast cells/ml/dayrespectively. The combined results from the five concentrations of yeast (0.5 x 104, 1 x 104, 1.5 x 104, 2 x 104 and 2.5 x 104 yeast cells/ml/day) and the observed specific growth are shown in Table 7. Generally, the observed growth rate increased with increasing food concentration (yeast cells/ individual/day). The three constant parameters (Umax, Ks/n and S/Nq) of the Michaelis-Menton equation at 26°C corrected for the threshold concentration were: Umax = 0.91 day’l, Ks/n = 5.6 x 105 yeast cells/individual/day, S/Nq = 0.87 x 105 yeast cells/individual/day. The equation relating the predicted specific growth rate of Q; pglgx at 26° to the amount of yeast available per individual Daphnia per day is: S/N — 87000 U " 0'91 S/N + 386000 ‘ From this equation, Table 7 and Figure 11. it is evident that the predicted specific growrh rate of Q; pglgg increased with increasing food concentration (S/NL However, the effect of changing a unit of food concentration (S/N) on the growth rate of Q; pglgx at high food concentrations was less effective than the changes at low food concentrations. The predicted growth rate at a food Table 7. The relationship between the number of torula yeast cells per individual 2;_pglg§ per day (S/N), The observed growth rates Of 2. pulgx and the efficiency in the Eombined rgsults of 4 five concentrations of yeast (0.5 x 10 1 x 10 , 1.5 x 10 , 2 x 104 and 2.5 x 104 cells/ml/day) at 26°C. 5 Observed Calculated S/N x 10 growth growth Calculated Order , rate (U) rate efficiency 1 25 0.784 .0761 5.044 x 10'7 2 20 0.763 0.73 3.65 x 10'7 3 15 0.52 0.682 4.55 x 10'7 4 10 0.668 0.56 5.6 x 10’7 5 5.2 0.223 0.435 8.36 x 10'7 6 5 0 168 0.424 8.48 x 10‘7 7 4.35 0.357 0.386 8.87 x 10'7 8 3.57 0.269 0.331 9.27 x 10'7 9 3.33 0.306 0.311 9.34 x 10'7 10 3.3 0.412 0.301 9.12 x 10'7 11 2.63 0 333 0.247 9.39 x 10‘7 12 2.13 0.253 0.191 8.97 x 10'7 13 2.08 0.159 0.185 8.9 x 10'7 14 1.8 0.299 0.15 8.3 x 10‘7 15 1.52 0.187 0.11 7.24 x 10'7 16 1.46 0.058 0.1 6.85 x 10'7 17 1.35 0.278 0.084 6.22 x 10‘7 18 1.3 0.002 0.075 5.77 x 10’7 19 1.282] 0.056 0.073 5.7 x 10‘7 20 1.2820 0.066 0.073 5.7 x 10‘7 21 1.14 0.136 0.049 4.3 x 10‘7 22 1.12 0.008 0.046 4.11 x 10‘7 23 1.03 0.056 0.03 2.9 x 10'7 24 0.99 0.053 0.025 2 3 x 10’7 25 0.93 0.045 0.04 1.18 x 10'7 26 0.87 0 0 0 65 :3 n 1 I 1 I 1 l I l 0'00 2 4 6 8 1‘0 1'2 1'4 1‘6 18 20 22 24 28 28 30 52 (s/n) x 10**5 celis/individuoI/doy Figure 11. The relationship betweet the observec growth rate (*g. the predicted specific growth rate and the amount of torula yeast available per individual D. pulex per daV at 260C. 66 concentration of 24 x 105 yeast cells/individual/day was 0.756 day‘l, and at 25 x 105 yeast cells/individual/day was 0.761 day‘l, thus an increase of one unit of food (S/N) at high food concentration caused a 0.005 day'l increase in the growth rate. At a food concentration of 3 x 105 yeast cellS/individual/day. the growth rate was 0.283 day‘1 and at food concentration of 4 x 105 yeast celis7indiv;dual,day. the growth rate was 0.362 day‘l, thus an increase of one unit of food (S/N) at low food concentration caused a 0.079 day‘l increase in the growth rate. The maximum efficiency was predicted to occur at a concentration of 2.63 x 105 yeast cells/individual/day (Figure 12, Table 7). At higher concentrations of torula yeast, the efficiency was low and increased with decreasing food concentration until the efficiency approached its apex at intermediate food concentration, after which the efficiency started to decrease again with decreasing food concentration (S/NL Comparative results of the three experiments: The size of Q; pglgx population increased with increasing the food concentration (yeast cells/ml/day) at each temperature used (12, 20, 26°C). In general, the population size of Q; pglgx increased with increasing the temperature. However, up to day 25, the higher the temperature, the higher the population size of Q; pglgx at any food concentration used. After day 27, the population 67 Table 8. The relationship between different concentrations of yeast (yeast cells/ml/day), temperature and the maximum growth rate1 (Umax day‘l) for D. pulex. Concentration Eggperature 0.55.104 1x104 1.55.107 2x107 2.55.107 12°C 0.377 0.47 20°C 0.11 0.176 0.318 0.53 0.55 26°C 0.18 0.772 0.87 0.92 0.98 lUmax was calculated at each yeast concentration (yeast cells/ml/day) by using the Michaelis—Menton equation corrected for the threshold. efficiency (LI/s/n) x 10**—7 Figure 12. Is 5.2. 1 1 1 1 1 1 1 1 1 fii 1 2 4 5 8 10 12 14 16 18 2O 22 24 25 (s/n) x 10**5 cells/individuol/doy The relationship between the efficiency and the amount of torula yeast available per individual 2. pulex per day at 2 6°C. 69 size at 20°C started to increase wary fast and reached a larger final population size than that of populations growing at 12° and 26°C. The effect of food concentration (yeast cells/ml/day on the population size was proved to be more determined than the effect of temperature (Table 9). The food alone had a significant effect on the population size of Q; pulgg (P = 0.011). The temperature alone had a significant effect on the population size of 4Q; pulgg (P = 0.011). The interaction between food and temperature also had a significant effect on the population size of Q; pulgg (P = 0.011). It is worth noting that the effect of food concentration was more profound than the effect of temperature on population size of Q; pulgg since 21% of the variance of population size were due to the food concentration, while only 14% of the variance of population size were due to temperature. The predicted specific growth rates of Q; pulgg at 26°C was much higher than that at 12° or 20°C (Figure 13) at any concentration, while at high food concentration (S/N), the growth rates for the population growing at both temperatures (12, 20°C) were almost similar. The threshold concentration (S/Nq) was higher at 12°C (2.7 x 105 yeast cells/ individual/day) than at 26°C (0.87 X 105 yeast cells/ individual/day) or at 20°C (0.82 x 105 yeast cells/ individual/day). 70 Table 9. Summary of the analysis of variance and multiple classifi— cation analysis of the population size of 2. pulex at the three levels of temperature (12, 20, 36°C) and the con— centrations of food (yeast cells/ml/day). Source of Significance , variation of F Beta (R) R” Food* 0.011 O.+b 0.2ll6 Temperature 0.011 0.37 0.1369 Food and Temperature 0.011 *Two concentrations of yeast used at 12°C (1 x 104, 2.5 x 104 yeast cells/ml/day) and five different concentrat'ons of y ast used at 20°C and 26°C (0.5 x104, 1 x 104,1.5 x 10, 2 x 10 , and 2.5 x 10" yeast cells/ml/day). 000 0 Figure 13. I I I I I I I I' 2 4 6 8 1O 12 14 16 18 2b 22 £4 26 £8 50 £2 (s/n) x 10**5 cells/individuol/doy The relationship between the predicted growth rate of 2. pulex with the amount of torula yeast available per individua; L. pulex per day at the three different temperature; usec (12°. 200. 260C). Table LO. A comparison between all the predicted parameters of the three experiments (lZO. 100, 160C). Parameter LZOC 200C 360C -1 Umax Iday ~) .046 0.5 ).31 t: L“=I3 "zvj sw'j LS/N J.—8 0 LE)..) .. O .7 .0 (cells/individual/day) Maximum efficiency _7 _7 _7 (number of Daphnia/ l.92 x 10 2 l2 x LO 9.39 x l0 yeast concentration) S/N at maximum efficiency 5 S 5 (cells/individual/day) 7.81 x 10 4.39 x 10 2.63 x 10 U at maximum efficiency (day‘ ) 0.15 0.093 0.247 Doubling time at maximum efficiency (days ln /U at maximum efficiency 4.62 7.45 2.8 Doublind time at Umax (daYS) an/Umax 1.51 1.39 0.76 9.00- 8.50- 8.00- 7.50- 7.00- 6.50— 6.00- 5.50- 5.00- 4.50- 4.00- 3.50- 3.00- 2.50- 2.00- 1.50- 1.00- 0.50- 0‘00 ‘ I I I I I I I I I I I I I I I I O 2 4 6 810121416182022242628303: (s/n) x10**5 cells/individuoI/doy Figure 14. The relationship between the efficiency and the amount of torula yeast available per individual 2. pulex per day at the three different temperatures used (12°, 200, 260C). efficiency (u/s/n) x 10**—7 \I 41‘ At each temperature, the efficiency (u/s/n) was low at high food concentration (S/N) and increased with decreasing food concentration until the efficiency reached its maxima. The efficiency started to decrease after that with decreasing food concentrations. The efficiency was higher at 26°C than at 12°C or 2000 at any food concentration used (S/N) Figure 14. The efficiency was higher at 20°C than at 12°C. However, at high food concentration the efficiency was almost the same.at 12°C and 20°C. However, all the predicted parameters from the three experiments at 12, 20 and 26°C are summarized in Table 10. The amount of torula yeast required per individual Daphnia per day to achieve the maximum efficiency was higher at 12°C (7.81 x 105 yeast cells/individual/day than at 20°C (4.39 x 105 yeast cells/ individual/day) or at 26° c (2.63 x 105 yeast cells/ individual/day). The predicted specific growth rate at maximum efficiency was higher at 26°C (0.15853 day‘l) than at 12°C (0.15 day‘l) or at 20°C (0.093 day‘l). The doubling time (In 2/U) at maximum efficiency was 4.62 days for the population growing at 12°C, 7.45 days for the population growing at 20°C and 2.8 days for the population growing at 26°C. However, the doubling time at the maximum specific growth rate (Umax) was 1.51 days for the population growing at 12°C, 1.39 days for the population growing at 20° and 0.76 days for the population growing at 26°C. DISCUSSION farvae of most fish feed initially on zooplankton (Sadykov 1975, Arnemo et al. 1980, Lasker 1975). The role of zooplankton in fish culture is important. Zooplankton abundance and composition are clearly affected by fish standing stocks and they can influence the fish yield. A strong correlation has been noted between food conversion rate (Kg food supplied : Kg fish yield) and zooplankton standing stock at zooplankton densities of 0.1 to 1.1 mg dry weight/l. This correlation appeared to be weakest at the highest zooplankton densities (Schroeder 1973L Fertilizers (organic or inorganic) are very important in enhancing food production for natural feeds by increasing phytoplankton and zooplankton which, in turn, support fish populations. Organic fertilizers are very important in fish culture especially in LDC's where the high quality protein required for fish feeds cost more than these countries can afford. However, the estimation of required nutrients for a pond fertilization program depends on the pond's morphology, hydrology, bottom material, water quality, type of fish cultured and type of fertilizer employed (Yamada 1983). 111 11111. .1' 76 High rate of survival and growth of walleye fry were achieved by stimulating Daphnia production in ponds by applying sheep and horse manure plus torula or brewers yeast as fertilizers (Merna 1977, Beyerl 1979). Our observed and predicted growth rates of Q; gglex red different concentrations of :orula yeast at three temperatures 112°, 20°, 26°C) are discussed separately. The batch cultures: Three experiments were run at three levels of temperature (12°, 20°, 26°C) and five different concentrations of torula yeast at each temperature. Each experiment was terminated when the population stabilized for a short period of time, or when the population density (number of Q; pglgx per liter) began to decline. In other words, the experiment was terminated when the indirect density effect resulting from interaction between animals and their external environment began to appear (Birch 1948, Slobodkin 1954, Smith 1952L The number of dead animals was recorded (Appendix Tables 1, 3, 5) in the three experiments. Mortalities were rare in all the experiments until the populations approached the maximum densities, after which the mortalities increased due to a shortage in food supply or build up of metabolities. Since mortalities were minimal, the population growth rate was considered to be equal to the birth rate (Hall 1964). 77 The mean size of the population densities over time can not be used in comparisons in place of the daily values because mean values are subject to random error of sampling, therefore observed daily variation in Daphnia population density was compared at different concentrations of yeast and levels of temperature (12°, 20°, 26°C) (Slobodkin 1954% At each temperature level tested. Daphnia population densities increased with increasing food concentration (yeast cells/ml/day) (Appendix Tables 1, 2, 3, 4, 5, 6g Figures 4, 7, 10). However, at 12°C experiment, the population densities, unexpectedly, decreased at yeast concentrations above 0.5 x 105 cells/ml/day and the population growth rate decreased above concentration 2.5 x 104 cells/ml/day (Umax = 0.21 day’1 at 0.5 x 105 yeast cells/ml/day, Umax = 0.377 day'1 at 1 x 104 yeast cells/ml/day and Umax = 0.469 day‘1 at 2.5 x 104 yeast cells/ml/day. The reason for this decline might have been the result of decreased water quality with increasing food concentration. Generally, the higher the food concentration, the higher the reproductive rate (Ingle et al. 1937, Fox et al. 1949, Slobodkin 1954). Starvation decreases growth in two ways, it increases the duration of the instars and reduced the increment in each moult. The effect of changing food concentrations on population densities appeared to be more profound than the effect of changing temperatures based on multiple 78 classification analysis (Table 9). Twenty one percent of the variance of population size were due to yeast concentration and 14% of the variance in population size were due to temperature. This observation is in agreement with those of Ingle (1937), Richman (1958), Allen (1976) and Lampert (1977b) who stated that the food concentration is regarded as the most important environmental factor influencing the production of Q; pglgx. At 120C (Figure 4), the Daphnia populations grew slowly and the final population density was lower than that at 20° or 26°C possibly due to decreased metabolic activities of Daphnia at 12°C (MacArthur and Baillie 1929, McLaren 1963L At 20°C, the Daphnia population growth was unexpectedly slow during the first 25 days of the experiment. From day 25 to 36 the population increased very fast, followed by a decline (Figure 7). The slow growth at the beginning of this experiment decreased the final growth rate of the population at 20°. The reason for this decline might have been the age-size frequency distribution (Slobodkin 1954M The adults began to reproduce slowly at the beginning. By day 25, the population had a large number of reproducing Daphnia which caused the numerical maxima. Consequently, the food supply decreased which caused high mortalities. As a result, the growth rate at 20°C was almost the same as the growth rate at 12°C despite the large difference in final population sizes. 79 At 260C (Figure 10), the Daphnia populations approached its maxima after 14 days in the first three yeast concentrations (0.5 x 104, 1 x 104 and 1.5 x 104 cells/ml/day), then began to decrease possibly due to shortage in food supply. In higher yeast concentrations (2 x 104, 2.5 x 104 cells/ml/day), the populations increased up to day 14, attained a short equilibrium period from day 14 to day 22, after'which they started to increase again till a second maxima at day 28, then they started to decrease. The short equilibrium period might have resulted from the adult Daphnia exhausting their energy supply through massive reproduction during the first 14 days of the experiment (Richman 1958, Green 1956). The new generation produced prior to day 22 started to reproduce to form the second maxima of the population density, after which the populations started to decrease possibly due to the shortage in food supply. Pratt (1943), Slobodkin (1954) and Frank (1952) studied single species population of Daphnia under somewhat similar conditions and observed that a population after reaching an initial peak declined somewhat and then irregularly fluctuated without any apparent changes in environmental factors. Population lags (age—size frequency distribution) and individual lag play a very important role in the oscillation process in Daphnia population (Slobodkin 1954). However, the differences in the genetic composition 30 of experimental population may be responsible for the oscillation (Herbert 1978, Weider 1984L Seneraliy, population peaks :Oincided with the maximum proportion of small animals and the population troughs with the maximum proportion of Large animals (Slobodkin 1954, Pratt 1943, Frank 1952). However in this study in the early stages of population growth, the few adult animals had a higher reproductive rate, resulting in a size frequency distribution that was skewed towards the small and (personal observation). This early population increase eventually gave rise to a population which was sufficient to reduce the food supply of all the animals so that reproduction almost stopped. At this point the population was at the apex of its initial numerical peak. It consisted largely of small animals. The experiment at 12°C was started with 1-5 day old Daphnia and they began to reproduce after 10 days. The experiment at 20°C was started with 1-3 day old Daphnia and they began to reproduce after 9 days. At 26°C, the experiment was started with 1-2 day old Daphnia and they began to reproduce after 7 days. This suggested an inverse relationship between temperature and the development time of the organism as noted by (Pratt 1943, McLaren 1963, MacArthur and Baillie 1929, Allan 1976L A population with an infinitesimally low rate of increase may reach a greater asymptote than that developed in a shorter 31 time with a much higher rate of increase (Pratt 1943). We found that the final population size at 20°C was greater than that at 26°C despite the large difference in the maximum rate of population growth at 20°C and at 260C (Umax at 20°C = 0.5 day‘l, Umax at 26°C = 0.91 day‘l) (Table 10). However. the neonates from a slow grOWing population grew to larger Size and lived for a longer period of time than the neonates from fast growing populations (Smith 1963L The population increased more rapidly at 26°C and the population growing at the other temperatures (12°, 20°C). This may have been due to increased metabolic activities of Daphnia at 26°C. However, MacArthur and Baillie (1929) stated that at normal temperatures and in both sexes the product of length of life x heart rate was approximately a constant, that is nearly 15,400,000 heart beats will occur in a daphnid's life regardless of temperature or sex, therefore the duration of life varied inversely with the intensity of the metabolism in Q; magna. At each temperature level, 9; pulex developed increased reddish coloration which meant an increase in the lipid index with increasing food concentrations (Tessier and Goulden 1982, Holm and Shapiro 1984). The population growing at 20°C had the greatest reddish coloration which meant an increase in the lipid index which might be because of optimal fitness conditions compared to populations at 12° or 26°C as noted by Lynch (1977). [Fitness is equal to feeding 82 rate (energy ingested/animal/time) divided by the basal metabolic rate (energy expended/animal/time.] Generally, the optimal life history strategy of an organism is to allocate its resource to maintenance, growth and reproduction so as to maximize its contribution to the persistence of the population (Lynch 1977). For any age class, it becomes advantageous to devote a large share of resources to growth and survival when future reproductive output is significantly enhanced. Increasing reproductive effort at any age wil l augment fecundity at that age, but only at the expense of future growth (260C experimentL Thus, while cladocerans continue to grow throughout their life, the rate of growth declines with the onset of reproduction (Anderson et al. 1937, Green 1956, Richman 1958). Prediction for the continuous culture: The continuous culture technique is designed to put the population at a selected specific growth rate (Novick and Szilord 1950) and thus at a constant food level (number of yeast cells/individual Daphnia/dayL A modified Michaleis- Menton equation with a correction for the threshold concentration which predicts the relationship between food availability or resource availability and the specific growth rate of Q; pulex is: S/N — S/N U = umax S/N + Ks/n — ZS/Nq 83 where U = specific growth rate of Q; oulex (day-1) Umax = maximum growth rate ofD.pulex (day‘l) S/N = number of yeast cells per individual Daphnia per day Ks/n = number of yeast cells per individual Daphnia per day when U is equal to 0.5 Umax. S/Nq = number of yeast cells per individual Daphnia per day required to initiate the growth rate ofg; Egigg (the threshold concentration) The prediction of population densities depend on the equation: N2 = N1 eUAt where N1 = numbers of the population at time 1 N2 = numbers of the population at time 2 At = difference in time U = specific growth rate of D. pulex or , S/N — S/N . (Um ) At L = L " ‘ + - v2 v1 e c/N Ks/n ZS/Nq Thus, we can calculate the constant requirements of food available per organism per day at any value of the growth rate of the organism. In ecology, the Michaelis—Menton or Monod equation (Monod 1949) R = Rk * C/C + C1 84 where R = specific growth rate of organism or the rate of uptake of a substrate Rk = maximum growth rate of organism, or the maximum rate of uptake of a substrate C = substrate concentration Cl = concentration of C when R = 0.5 RK has been used to study complex phenomena as rate of uptake of organic and inorganic nutrients by natural population (Strickland 1962, Wright and Hobbie 1966, Dugdale 1967. MacIsaac and Dugdale 1969, Krembeck 1979). This sort of analysis is instructive if the equation is obeyed, but if the results don't appear to fit the equation, as has happened in some studies of heterotrophic processes (e.q. Vaccaro and Jannasch 1967, Hamilton and Presland 1970L there is a dilemma. Vaccaro and Jannasch (1967) suggested that the failure to fit the Michealis—Menton equation may have stemmed from the heterogenous nature of the indigenous marine population. At low substrate concentration, the observed rates were higher than the predicted ones. The discrepancy between observed and predicted values increased as the population became more diverse (Williams 1973L However, Fogg (1975) studied the relationship of relative growth rate of algae to nutrient concentration. He stated that this relation is more complicated than the simple hyperbolic expression of Monod equation (without correction for the threshold. The Michaelis—Menton kinetics may 85 accurately describe the uptake of nutrients but relative growth rate is dependent more directly on intracellular concentration rather than the rate at which the nutrients enter the cell. The maximum specific growth rate (Umax) of Q; gule_ at 2000 (0.5 day-1) was almost equal to the maximum specific growth rate at 12°C experiment (0.46 day‘l) (Figure 13, Table 19). The population at 20°C grew slowly at the beginning of the experiment which caused a decrease in the growth rate despite the fact that they grew very fast at the end of the experiment. The threshold concentration from the Michaelis—Menton equation is the amount of food required to maintain the organism (e.q. movements, locomotion, standard metabolism). It is the amount of food required to initiate the growth rate of the organism (King and Garling 1983). The threshold value calculated by dividing the concentration of the food (number of yeast cells/day) by the maximum number of Q; pglex (Sq = S/K) when the grOWth rate equaled zero based on the assumption that the growth rate equals zero after the population reaches the maximum population number. Beyond this point the growth rates became negative after the Q; pglgx reached the maximum population number in most of my experiments resulting from a decline in the population size. 86 The threshold concentration was higher at 12°C than at 20°C or 26°C (Figure 13, Table 10). This may have resulted from two reasons. Firstly, the population density of Daphnia was lower at 120C than in the other temperatures tested (20, 26°C) WhiCh increased the S/K value. Secondly, at 12°C there was a higher percentage of large animals presumably due to decreased reproductive ability at that temperature. Lampert (1977c) observed that larger Daphnia required a higher threshold concentration. The threshold concentration at 26°C was higher than at 20°C (Table 10, Figure 13). This might have been due to an increase in the metabolic activities and, consequently, energetic needs at 26°C compared to 20°C (Richman 1958L However, Lampert (1977c) found that the threshold concentration for Q; pglgx increased with increasing the temperature up to 25°C. The threshold concentration for Q; pglgx increased with increasing the temperature up to 25°C. The threshold concentration at 20°C was lower than at the other temperatures tested. The low threshold value was expected at 20°C since the largest maximum population size (K) occurred at 20°C or perhaps as a result of maximum feeding efficiency, which is defined as feeding rate/basal metabolic rate which was high at 20°C as stated by (Burns 1968, 1969; Lynch 1977). However, the relationships of assimilation efficiency to food quantity and quality, .. MW 1“": out: :1 M -- . .. I I“ __r_. v _ ._1 ., -_I. 'l.‘.- . r d... a.“ "-' - -- .- ' '- - ..f - ' - - ' g! I '-I , k , ..r ... ;. n4§w=anl .J - limb), .. '2' :-. 0.; i .I' 37 temperature and body size in Q; RE£E§ are not clear due to conflicting observations between experiments (Lynch 1977, Leonard and Lawrence 1981L The efficiency of a machine is the ratio of the output to the input (Slobodkin 1960). The term efficiency here (U/s/n) represented the food conversion rate as number of Q; pulex divided by the amount of food available per time as compared to the definition given by Reeve (1963) where food conversion rate was equated with the weight of animals produced/weight of the food conversion rate was equated with the weight of animals produced/weight of the food consumed. There was a definite peak efficiency at each temperature (Figure 14). The peak always occurred at low food concentrations. Reeve (1962) observed that the food remained in the gut of Artemia progressively longer at low food concentrations, presumably from less pressure in the absence of larger amounts of the incoming food to force it through the gut. The longer stay in the gut should result in greater digestive efficiency. At very low concentrations of food, the efficiency was low since there was greater effort involved in feeding process which was not compensated adequately by the food ingested. Lower efficiency at high feed concentrations may have been caused by an increase in the food rejection rate and respiration rate (Porter et al. 1982), or by an increase in food ingestion rate which exceeded the capacity of the gut 38 to digest food (Slobodkin 1960). However, growth efficiency is related to the physical and biological conditions of any particular laboratory experiment, and can not be directly related to observations from fluctuating natural habitats (Reeve 1963). Strict comparison should not even be made between growth efficiency estimates from different laboratory observations where units measured have included dry weight (Ivlev 1939, Ricker 1946), calorific value (Richman 1958, Slobodkin 1960) and radioactive carbon (Lasker 1960). The increase in size of a given animal associated with increasing age would be expected to increase the energy cost of its maintenance, and to reduce correspondingly the total efficiency of growth unless the increase in maintenance is compensated for by an increase in growth rate (Brody 1945). Efficiency increased with increasing temperatures from 12°C to 20°C to 26°C (Figure 14). This might have been due to an increased growth rate of with Daphnia at increasing temperatures at any level of food concentration used (Figure 13L The doubling time is the length of time necessary to double population size at any value of the specific growth rate (Hall 1964). It is calculated by dividing ln 2 by the growth rate. Thus, the higher the growth rate, the lower the doubling time of the population. The doubling time at maximum growth rate was longer at 12°C (1«51 days) than at 2000 (1.39 days) or at 260C (0.76 days). This occurred as a result of the increase in the maximum growth rate from 12QC to 20°C :0 26°C (Table 10). Also, the doubling time at maximum efficiency was longer at 200C (7.45 days) than at 1200 '4.62 days) or at 26°C (2.5 days). The increase of doubling time at naXLmum efficiency at 20°C was due to decreased growth rate at 20°C at maximum efficiency. Based on our experimental results, it appears that: 1. If torula yeast is at a reasonable cost, readily available in large quantities, high concentrations of yeast should be used to insure high growth rate. However, very high concentration of food may lead to depletion of oxygen, an increase in ammonia concentration and other disadvantages in the system. 2. If the yeast is not available in large quantities or expensive, it should be used at maximum efficiency L7.81 105, 4.39 x 105 and 2.53 x 105 yeast cells/individual Q; pulex per day for temperatures 12, 20 and 26°C respectively). However, the point of maximum dollar efficiency (maximum yield in terms of dollars) must be considered as the best concentration which can be used despite the fact that it might be higher than the concentration of food required at maximum efficiency. 90 Generally, the prolonged exposure of Q; pulex to any constant yeast concentration may result in unpredictable population changes and growth characteristics (Jannasch 1974)- Therefore, more research is needed to clearly establish the relationship between prolonged exposure to different concentrations of yeast and the population dynamics of Q; pulex. Finally, more research is needed to determine the long term relationship between zooplankton densities and fish fry requirements before generally recommendations can be made with confidence. . 3'". WP '— 1- - ' 'li‘ ..- ...- _ f: . "in. ' m fi-E‘fi-uo‘?w*i.¥' at“ "Ilidlllt '- (Q 01 SUMMARY As the age of the culture increased, the population size increased then stabilized or declined. As the food concentration increased, the population size and the growth rate increased. As the temperature increased, the growth rate of Q; pglgx increased and the population size increased for a short period of time then declined. The effect of food concentration and temperature and the interaction between them on population size of 2; pulex was significant. The effect of yeast concentration (cells/ml/day) on Daphnia population size was more profound than the effect of temperature. 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Estimating food limitation in Cladoceran population. Limnol. Oceanogr. £7 (4): 707—717. Torrens, E. L. 1983. Fish plankton interaction. 9. 77—88 In Lannon, J.E; Smitherman, R.O.; Tchobanoglous, G. (edsJ. Principles and Practices of Pond Aquaculture: A State of the Art Review. USAID Title XII CRSP in Pond Dynamics/Aquaculture Report (Grant No. AID/DSAN— G—0264). Oregon State University, Marine Science Center, Newport, OR. Vaccaro, R.F.,and H.W.Jannasch. 1967. Variations in uptake kinetics for glucose by natural populations in sea water. Limnol. Oceanogr. 12: 540-542. Weider, L. J. Spatial heterogeneity of Daphnia genotypes. Vertical migration and habitat partitioning. Limnol. Oceanogr. 9(2): 225—235. Williams, P. J. 1973. The validity of the application of simple kinetic analysis to heterogenous microbial populations. Limnol. Oceanogr. 18: 159-165. Wright. J. C. 1965. The population dynamics and production of Daphnia in Canyon Ferry Reservoir. Montana. Limnol. Oceanogr. 10: 583—590. Wright, R.T., and 1.8. Hobbie. 1965. The uptake of organic solutes in lake water. Limnol. Oceanogr. 10: 22—28. . and . 1966. Use of glucose and acetate by bacteria and algae in aquatic ecosystem. Ecology 47(3): 447~464. Yamad, R. 1983. Pond production system: Fertilization practices in warm water fish pond. pp. 103e115. In: Lannon, J.E.;Smitherman, R.O.;Tchobanoglous,G. (edsJ. Principles and Practices of Pond Aquaculture: A State of the Art Review. USAID Title XII CRSP in Pond Dynamics/Aquaculture Report (Grant No. AID/DSAN- G—0264). Oregon State University, Marine Science Center, Newport, OR. Youngy T. C” and D.Ih King. 1980. Interacting limits to algal growth: light, phosphorus and carbon dioxide availability. Water Research, Vol. 14: 409—412. APPENDICES 103 Appendix Table 1. Daily numbers of 2. pulex living (L), dead (D) and replaced (R)concentrations of torula yeast cultured in duplicate (1, 2) at 12°C. Concentration of torula yeast (ml/day) Day 1x 10“ 2.5 x104 0.5x 105 1x10 2 x103 L D R L D R L D R L D R L D R 1 10 10 1o 10 1o 1 2 1o 10 10 10 1o 1 9 1 1 9 1 1 10 o o 10 o o 8 2 2 2 2 9 1 1 8 2 2 1o 0 o 10 o o 10 o o 1 8 2 2 8 1 1 1o 0 0 1o 0 o 10 o o 3 2 9 1 1 7 3 3 9 1 1 91 1 10 o o 1 8 2 10 10 o 10 o 10 0 4 2 9 1 10 o 9 1 9 1 1o 0 1 8 o 9 1 10 o 10 0 1o 0 5 2 9 o 9 1 8 1 8 1 10 o 1 8 o 9 o 9 1 10 o 10 6 2 9 o 9 0 8 o 8 o 10 o 1 8 0 8 1 9 o 10 o 10 o 7 2 9 o 8 1 8 o 8 o 10 o 1 8 o 8 o 9 ‘o 9 1 1o 0 8 2 8 1 8 o 8 fo 8 o 10 o 1 8 o 8 o 9 go 9 0 1o 0 9 2 8 o 8 o 8 ‘0 8 o 9 1 1 8 o 8 o 20 )0 16 o 10 0 1o 1 2 8 o 8 o 9 To 12 0 9 o 1 8 0 8 0 18 :2 16 o 10 o 11 2 8 0 8 0 16 o 9 3 9 o 1 8 o 8 o 24 o 16 o 10 o 12 2 8 o 8 0 16 0 9 3 9 0 104 Appendix Table l. (cont'd.) Concentration of torula veaSt (ml/day) Day 1 . 10 2.2410“ 0.5::10J 1 x 103 2 . 10 1. D R 1. D 1. D L D 1. D 13 1 3 o 16 1) 25 0 17 13 9 1 2 13 o 21 o 17 o 9 o 9 o 1 15 o 22 o 39 o 17 0 9 o M 2 27 0 38 o. 23 o 10 0 20 o 1 17 0 29 0 46 o 17 o 9 o 15 2 25 2 41 o 28 o 10 o 20 o 1 17 o 25 4 49 o 21 o 9 o 16 2 23 2 37 4 31 o 12 o 16 4 1 31 0 41 o 53 0 3o 0 . 11 o 17 2 29 o 33 4 31 o 13 o 15 1 1 21 o 39 2 56 0 27 3 9 2 18 2 25 4 33 o 48 o 12 1 12 3 1 20 1 4o 0 56 o 27 o 9 0 19 2 26 o 38 o 48 o 12 o 12 o 1 23 o 49 o 62 0 28 o 9 o 9 “O 2 27 o 57 0 46 o 28 0 12 0 1 23 o 49. o 70 o 28 o 9 o 31 2 27 o 52 o 44 2 26 2 12 0 1 24 o 49 o 70 o 36 o 8 1 22 2 32 0 53 4 52 o 23 3 11 1 1 23 1 50 o 71 .3 36 o 8 o ‘3 2 33 o 53 o 53 o 23 o 11 0 1 36 o 69 o 82 o 47 o 10 o 2" 2 3o 3 53 o 64 o 30 o 12 o Appendix Table l. (cont'd.) Concentration of torula yeast (ml/day) Day 1 . 10‘ 2.5x 10* 0.5 x 103 1 10 x 10 L D L D R L D R L D L D 1 36 o 87 o 92 0 60 0 1o 6 “5 2 3o 0 63 0 68 o 50 o 12 o 1 42 o 91 o 101 o 66 o 10 o 26 2 30 o 79 0 83 o 80 o 18 o 1 42 o 95 o 101 o 85 0 10 o 27 2 34 o 79 o 89 o 92 o 18 o 1 4o 2 92 3 103 o 81 4 10 o 28 2 35 0 86 o 94 o 93 o 17 o 1 4o 0 96 0 109 o 77 4 9 1 29 2 25 o 86 o 93 o 81 12 15 2 1 4o 0 96 o 102 76 1 9 o 30 2 36 o 86 o 99 o 80 1 15 o 1 42 o 92 4 100 2 70 o 9 o 31 2 36 o 88 o 103 o 81 o 15 o 1 41 1 92 o 100 o 71 0 9 o 32 2 34 2 88 o 103 3 77 4 15 o 106 Appendix Table 2. The average daily number of living Daphnia at 120C cultured in five different duplicate concentrations of torula yeast. Concentration of torula yeast cells/ml/dav Day 1 x 10* 2.5 x 10* 0.5 x 10’ 1 x 10D 2 x 103 1 1o 10 10 10 1o 2 10 1o 10 1o 10 3 1o 10 1o 9 10 4 9 10 1o 9 1o 5 9 9 9 9 1o 6 9 9 9 9 1o 7 9 9 9 9 1o 8 8 8 9 8 10 9 8 8 9 8 9 10 8 8 15 14 9 11 8 8 17 14 9 12 8 8 20 14 9 13 11 19 21 14 9 14 21 3o 31 15 15 15 21 35 37 14 9 16 20 32 40 18 15 17 25 37 42 22 13 18 23 36 52 19 11 19 23 39 52 19 11 20 25 53 54 28 11 21 25 53 58 27 11 22 28 51 61 30 1o 23 78 51 62 30 1o 24 33 61 73 3 1o 25 33 75 8o 55 11 26 36 85 92 73 14 27 38 87 95 88 14 28 38 89 98 37 14 29 38 91 101 ‘9 12 30 3 91 61 78 12 31 3 91 101 76 12 _.—.. 'l '-_-!:-l." 9..- J4- -. I. .—-I-- -———--—v---‘ 107 Appendix Table 3. Daily numbers of D. pulex living (L), dead (D), and replaced (R) cultured in duplicate (1,2) concen- tractions of torula yeast at 20°C. Concentration of veast cells/ml/day / I )1 I y/ Dav 0.5 x 10* 1 x 10* 1.5 x 10‘ 2 x 10* 2.5 x 10* L D R L D R L D R L D R L R D 1 10 10 10 10 10 l 2 10 10 10 10 10 1 7 3 3 7 3 3 9 1 1 10 1o 0 0 2 3 2 9 1 1 9 1 1 10 0 o 10 o 0 10 o 0 1 1o 0 10 0 10 0 10 o 10 o 4 2 10 0 10 0 1o 0 10 0 1o 0 1 10 0 10 o 10 0 1o 0 10 o 5 2 10 o 10 0 10 0 10 0 1o 0 1 10 0 10 o 10 0 10 o 10 o 6 2 1o 0 10 o 10 0 1o 0 1o 0 1 10 0 10 o 10 0 10 o 10 0 7 2 1o 0 10 0 1o 0 10 o 10 0 1 10 0 10 0 10 0 1o 0 10 0 8 2 10 0 1o 0 10 0 1o 0 1o 0 1 10 0 1o 3 10 0 1o 0 27 0 9 2 10 0 10 9 10 0 15 J 18 o 1 10 0 14 0 15 0 12 0 26 1 l0 2 10 0 14 0 18 0 18 0 25 o 1 20 0 15 o 23 o :2 0 39 0 ll 2 10 o 15 0 22 0 34 0 44 o 1 1o 0 20 0 27 o 22 o 39 o 12 Appendix Table 3. (cont'd.) Concentration of jeast cells/ml/day / Day 0.5 x 10 1 x 10* 1.5 x 10* 2 x 10* 2. b1 L D R L D R L D R L D R L 1 ll 0 18 2 31 0 35 O 118 13 2 11 0 18 0 31 0 46 o 72 1 21 0 24 0 49 o 40 0 53 14 2 17 o 28 0 34 0 47 0 88 1 21 0 24 0 49 o 44 o 62 15 2 18 0 30 o 34 0 53 o 104 1 20 1 24 0 53 o 44 o 71 16 2 17 1 30 o 35 0 62 o 101 1 18 2 24 o 53 o 44 o 79 17 2 16 1 3o 0 35 o 62 0 101 1 18 o 23 1 57 o 44 0 79 18 2 17 o 30 o 34 1 75 o 102 1 18 o 27 o 54 3 50 o 93 19 2 15 2 30 o 35 1 80 2 108 1 33 0 3o 0 55 0 6o 0 94 2° 2 19 0 30 0 43 0 96 0 118 1 36 o 33 0 75 0 76 0 110 ll 2 26 0 36 0 52 0 99 0 118 1 37 0 49 0. 80 0 75 1 108 22 2 26 0 51 0 56 0 96 3 130 1 38 0 50 0 85 o 74 1 108 2° 2 31 0 50 1 54 1 94 2 133 1 36 2 51 89 0 76 0 108 24 Appendix Table 3. (cont'd.) 109 Concentration of yeast cells/ml/day / Day 0.5 x 104 1 x 104 1.5 x 10* 2 x 104 2.5 x 104 L D R L D L 0 R L 0 R L D R 1 38 0 51 0 90 0 79 o 108 0 7 “5 2 27 0 53 0 67 o 90 2 128 2 1 36 2 60 o 95 0 79 0 121 0 26 2 26 1 67 0 73 0 89 1 134 o 1 34 2 60 0 95 0 86 121 27 2 23 3 66 1 85 0 92 0 136 0 1 33 1 6O 0 98 o 86 o 133 o 28 2 20 3 65 1 84 1 97 o 161 1 3o 3 60 0 98 o 94 o 157 o 29 2 19 1 67 o 84 0 97 0 182 o 1 30 o 60 0 98 o 94 0 181 o 30 2 19 0 67 o 92 0 110 0 238 0 1 29 1 73 o 110 133 o 233 0 31 2 20 0 69 o 96 0 151 281 0 1 27 2 82 0 131 0 186 0 267 o 32 2 23 83 0 112 0 246 0 336 0 1 25 2 104 0 172 0 215 0 296 o 35 2 23 0 99 0 151 0 334 0 375 0 1 25 o 113 0 223 0 264 0 339 o 34 2 24 0 126 0 193 0 348 0 396 0 1 23 2 123 0 253 0 282 0 339 0 35 2 24 o 132 0 231 0 343 0 396 o 1 20 3 135 0 265 0 291 0 352 o 36 2 24 0 143 o 233 0 351 o 396 o 110 Appendix Table 3- (cont'd.) Concentration of veast cells/ml/day Day 0. 1 x 104 l." x 104 2 x 104 Z x L D L D R L D L D R L D 21 0 134 1 267 0 295 0 350 2 37 25 0 143 0 233 0 351 0 363 33 21 0 130 4 260 7 262 33 352 o 38 25 0 143 0 233 0 343 9 340 23 22 0 134 o 221 39 252 10 341 11 39 26 0 140 239 0 291 52 311 29 23 o 131 3 221 o 250 2 335 6 4° 26 o 142 o 233 6 291 o 315 0 111 Appendix Table 4. The average daily numbers of living Daphnia at 200C cultured in five difference duplicate concentrations of torula yeast. Concentration of yeast cells/ml/day Day 0.5 x 104 1 x 101+ 1.5 x 104 2 x 104 2.5 x 104 l 10 10 10 10 10 2 10 10 10 10 10 3 10 10 10 10 10 4 10 10 10 10 10 5 10 10 10 10 10 6 10 10 10 10 10 7 10 10 10 10 10 8 10 10 10 10 10 9 10 10 10 13 23 10 10 14 17 15 26 ll 10 15 23 28 42 12 11 19 28 28 43 13 ll 18 31 41 60 14 19 26 42 44 71 15 20 27 42 49 83 16 19 27 44 53 86 17 17 27 44 53 90 18 18 27 46 6O 91 19 17 29 45 65 101 20 26 30 49 78 106 21 31 35 64 88 114 22 32 50 68 86 119 23 35 50 70 84 121 24 32 52 71 84 119 25 33 52 79 85 118 26 31 64 84 84 128 27 29 63 90 89 129 28 27 63 91 92 147 29 25 64 91 96 170 30 25 64 95 102 210 31 25 71 103 142 257 32 25 83 122 216 302 33 24 102 162 275 336 34 25 120 208 306 368 35 24 128 242 313 368 36 22 139 249 321 374 37 23 139 250 323 357 38 23 137 247 303 346 39 24 137 230 272 326 40 24 137 227 271 325 Appendix Table 5 . 112 Daily numbers of 2. pulex living (L), dead (D) and replaced (R) concentrations of torula yeast cultured in duplicate (l, 2) at 120C. Concentration of torula cells/ml/day Day 0.5 x 104 1 x 104 1.5 x 104 2 x 104 2.5 x 104 L D R L D L D R L L D R 1 10 1o 10 10 1o 1 2 1o 10 10 1o 10 1 9 1 1 9 1 10 o o 10 10 o 2 2 9 1 1 10 o 10 o o 9 8 2 1 8 2 2 10 0 10 o o 10 1o 0 3 2 10 o 0 1o 0 1o 0 o 10 10 o 1 10 o o 10 o 10 0 1o 10 o 4 2 1o 0 0 1o. 0 10 o 10 1o 0 1 10 o 10 o 10 o 10 10 o 5 2 10 o 10 o 10 0 1o 10 o 1 1o 0 10 0 1o 0 10 1o 0 6 2 1o 0 1o 0 1o 0 10 1o 0 1 13 o 39 o 27 o 31 36 o 7 2 12 o 32 o 37 o 37 38 1 13 o 39 o 35 0 4o 42 o 8 2 14 o 36 o 54 o 52 54 o 1 29 o 53 o 56 o 66 65 o 9 2 21 0 51 o 65 o 70 72 o 20 o 66 o 72 o 83 70 o 10 2 27 o 82 o 93 o 105 80 o 1 26 o 109 o 101 o 147 122 o 11 2 36 o 94 o 129 o 139 126 o 1 28 o 150 o 152 o 157 168 o 12 2 37 o 107 o 150 o 154 173 o 113 Appendix Table 5. (cont'd.) Concentration of torula yeast cells/ml/day Day 0.5 x 104 l x 104 1.5 x 104 2 x 104 2.5 L D R L R D L D R L D R L 1 42 o 150 160 o 164 o 168 13 2 51 o 115 0 155 o 169 o 175 1 42 o 148 2 165 o 165 o 199 14 2 54 o 115 o 171 o 191 o 184 1 43 o 136 o 163 2 163 2 196 15 2 51 3 108 7 172 0 190 1 184 1 44 o 110 26 140 23 163 o 195 16 2 s1 0 99 9 110 62 189 1 182 1 46 o 110 o 134 6 '161 2 192 17 2 50 o 95 4 97 13 185 4 188 1 41 5 110 o 130 4 160 1 193 18 2 56 o 95 o 96 1 186 o 180 1 41 o 110 o 119 11 160 o 187 19 2 54 2 102 o 101 o 195 o 173 1 38 3 116 o 115 4 160 o 179 20 2 50 4 108 o 116 o 195 o 174 1 38 o 116 o 114 1 158 2 174 21 2 48 2 114 o 114 2 191 4 171 1 37 1 122 o 119 o 167 o 175 22 2 43 5 115 o 116 o 199 o 184 1 29 8 115 7 121 o 173 o 183 23 2 42 1 115 o 114 2 200 o 186 1 25 4 103 12 122 o 181 o 181 24 2 39 3 98 17 117 0 205 O 198 Appendix Table 5. (cont'd.) Concentration of torula yeast cells/ml/day Day 0.5 x 10 l X 104 1.5 X 104 2 x 104 2.5 ‘ 1 22 3 97 6 122 o 199 0 195 25 2 39 o 90 8 117 o 216 o 220 1 21 1 96 1 136 o 211 o 201 7 ‘6 2 35 4 93 0 116 1 220 o 23 1 20 1 103 o 150 o 239 o 271 27 2 31 4 106 0 117 o 270 o 283 1 16 4 104 o 155 o 259 o 281 28 2 27 4 99 7 132 0 284 o 290 1 16 o 70 34 121 34 241 18 270 ‘9 2 32 o 82 17 130 2 253 31 285 1 11 5 64 6 111 10 245 o 267 30 2 27 5 79 3 98 32 250 3 295 1 9 2 58 6 78 33 280 o 261 ’1 2 22 5 52 27 32 16 313 37 310 1 9 o 49 9 77 1 261 19 254 49 3 71 11 204 / 298 lg to C) O Appendix Table 6. The average daily numbers of living Daphnia at 260C cultured in five difference duplicate concentrations of torula yeast. Concentration of yeast cells/ml/day Dax 0.5 x 10 1 x 104 1.5 x 10” 2 x 10‘ x 1 10 10 10 10 10 2 10 10 10 10 10 3 10 10 10 10 10 4 10 10 10 10 10 5 10 10 10 10 10 6 10 10 10 10 10 7 13 36 32 34 37 8 14 38 45 46 48 9 20 52 61 68 69 10 24 74 83 94 75 11 31 102 115 143 124 12 33 129 151 156 171 13 47 133 158 167 172 14 48 132 168 178 192 15 47 122 168 177 190 16 48 105 125 176 189 17 48 103 116 173 190 18 49 103 113 173 187 19 48 106 110 178 180 20 44 112 116 178 177 21 43 115 114 175 173 22 40 119 118 183 180 23 36 115 118 187 185 24 32 101 120 193 195 25 31 94 120 208 208 26 28 395 126 216 218 27 26 105 134 255 277 28 22 102 144 272 286 29 24 76 126 247 278 30 19 72 105 248 281 31 16 55 80 247 286 3: 16 49 74 233 276 33 13 44 74 202 232 34 13 44 2 178 214 "1111111117111111111115 ‘