Temperature Eff ects on Growth, Maturation, and Lifes- pan of the California Sea Hare ( californica)

DUSTIN STOMMES, BLA, LYNNE A. FIEBER, PHD,* CHRISTINA BENO, ROBERT GERDES, MS, and THOMAS R. CAPO, BS

We conducted a hatchery growth study to describe the variability in growth rates, spawning, and mortality of Aplysia californica in regard to rearing temperature. were housed at a standard hatchery density of fi ve animals per cage, at temperatures of 13, 15, 18, and 21°C. Animals reared at 13 or 15°C grew as much as four times as large, lived twice as long, matured later, and spawned longer than did animals reared at 18 or 21°C. At age 170 to 205 days the fastest growth rates occurred at 18 and 21°C, and the slowest at 13°C. As animals at 18 and 21°C reached sexual maturity at ages 190 to 197 days, or ∼60% through their lifespans, their growth rates slowed such that by age 260 days, the fastest growth rate was at 13°C, and the slowest was at 21°C. Animals reared at 13 and 15°C reached sexual maturity at 242 and 208 days, respectively, or at ∼40% of their life spans. Lifespan and maximum average weight were signifi cantly inversely correlated with temperature (P ≤ 0.0001). However, there were no signifi cant diff erences at any temperature in the age at which maximum animal weight was reached when this age was expressed as a percentage of the life span: animals reached their maximum weight at ∼80% of their life span. Aging rate was highest for animals reared at 21°C, while the mortality rate doubling time was lowest at this temperature. Th is would be expected for the accelerated lifecycle observed at higher temperatures.

Aplysia californica has a natural lifespan of approximately 1 year Materials and Methods (3), that is preserved in hatchery animals reared at 15°C during their Rearing experiments under diff erent experimental temperatures post-metamorphic life (5). As is the case for all ectothermic organisms, were conducted at the University of Miami, National Institutes of the growth rate is temperature-dependent. Several laboratory stud- Health National Resource for Aplysia, from June 1996 through ies of A. californica have documented these rates at diff erent rearing September 1997. Th is facility is equipped with continuously fl ow- temperatures (5, 11, 15, 18). ing 50-μm fi ltered and chilled seawater, an indoor animal-rearing A. californica is laboratory-reared to provide experimental ani- laboratory, and an outdoor macro algae culture facility that provides mals for researchers working on this neurobiological model . food year round (5, 7). Recently we have been studying factors that infl uence growth in labo- Th e experimental set-up for rearing was as described in detail by ratory-reared A. californica maintained under controlled conditions. Capo and coworkers (5). Briefl y, 100 animals from a single egg mass In these studies, we varied one factor such as temperature, food or of wild-caught broodstock were used. At the start of the experiment stocking density, while keeping the others constant (5, 6, 11). Th ese ± (ta), the age of the animals was 170 days, and live weights (mean studies demonstrated that each factor had a strong eff ect on growth standard deviation) were 4.6 ± 0.22 g with the narrow weight range rate and size at sexual maturity. One factor that does not appear to of 4.22 to 5.10 g. Animals were distributed randomly among the four infl uence growth and maturation, however, is the time of year the temperature treatments. Feeding in these animals prior to the begin- life cycle begins (10, 14, 18). Th erefore it is possible to customize ning of the experiment was not ad libitum; feedings were four times animals for researchers’ needs throughout the year by manipulating per week at 430% of body weight per feeding in 3-g animals. Animals growth rate or size and age at sexual maturity. were housed in 16-liter polycarbonate cages submerged in fi berglass We hypothesized that rearing temperature would infl uence the troughs through which chilled, fi ltered seawater fl owed continuously growth rate and the age at which A. californica became sexually ma- at a rate of 2 liters/min. Rearing temperatures were 13, 15, 18, and ture in the laboratory as well as indices of sexual maturity, such as 21°C. Animal densities were fi ve animals per cage, with fi ve replicate the length of the reproductive period. It has been standard hatchery cages of animals reared at each temperature. One replicate at 13°C practice to maintain a post-metamorphosis rearing temperature of was lost from the experiment at age 317 days due to being shipped 15°C, with the objective to prevent the onset of sexual maturity to researchers. Photoperiod was a 14:10-h light:dark cycle. Animals before 9 months of age. Sexually immature animals provide a more were fed ad libitum daily fresh Gracilaria ferox in exponential growth consistent model in neurophysiological studies and ensure that the phase, after removal of uneaten food from the day before. animals do not approach senescence (13). Comparison of the study of Animals were monitored through maturity to senescence and Kriegstein and colleagues (15) at 22°C with our own studies at 15°C mortality. All animals were inspected each day for mortality, copula- (5, 6, 11) suggested survival and growth of A. californica were highly tion, and the presence of egg masses. Live weight of each animal was temperature-dependent. Th e purpose of this report is to describe determined weekly by using an electronic balance (Mettler, Toledo, growth, sexual maturation, and spawning, and lifespan of laboratory Ohio) after draining excess water from the animal’s parapodial cavity. animals held under diff erent rearing temperatures. Th is was accomplished by holding the animal tail down in air and shaking lightly three times. Sexual maturity for a cage of animals Division of Marine Biology and Fisheries, National Resource for Aplysia, University of was defi ned as the day the fi rst egg mass was laid. Animal densities Miami Rosenstiel School of Marine and Atmospheric Science, 4600 Rickenbacker Causeway, Miami, Florida 33149 were altered after the death of animals, such that from age 226 days, *Corresponding author the day the fi rst animal died, some cages had fewer than fi ve animals

Volume 44, No. 3 / May 2005 CONTEMPORARY TOPICS © 2005 by the American Association for Laboratory Animal Science 31 Figure 1. Live weight (mean ± 1 standard deviation) of Aplysia californica with age at each temperature from age 170 days to the death of individual animals. Th e smaller standard deviations for the last several points were caused by reduced sample size due to animal death. Arrow denotes age at which the fi rst egg mass was laid. (A) 13°C. (B) 15°C. (C) 18°C. (D) 21°C. per cage. Th e experiment concluded when the last animal died. An set values, except on one day when temperature in the 13°C cages ° actuarial analysis of mortality data was performed using the survival fell to 10.7 C, a 32% change. Salinity, pH, and O2 concentration program described in Wilson (19), which fi ts the Gompertz survival were also monitored and did not vary signifi cantly from established function to an observed survival curve by nonlinear regression analy- norms for the hatchery (salinity, 35.0 parts per thousand; pH, 8.4; = Gt sis. Th e Gompertz mortality rate function is defi ned as M Ae , O2, 7 mg/liter; 5). where M = mortality rate, A = initial mortality rate, G = Gompertz Th e average growth of A. californica maintained at each experimen- exponential parameter, which is considered the “aging rate,” and t = tal temperature is illustrated in Fig. 1, which shows the sigmoidal time (in days). Th e Gompertz function commonly is used to describe growth typical of this species (5, 11, 15, 18). Temperature strongly the exponential increase in the mortality rate with time that is typical aff ected growth rate, lifespan, and maximum size. Growth rates, de- of aging populations. Th e initial mortality rate, A, is the mortality rived from linear regression fi ts from the fi rst 35 and 90 days of the rate independent of senescence, which can vary between cohorts. G experiment (ages 205 and 260 days, respectively) show that growth is commonly expressed in terms of the mortality rate doubling time rates were highly dependent upon temperature (Table 1). Growth rates (MRDT), a constant, where MRDT = ln 2/G. to age 205 days show the fastest growth rates occurring at 18 and 21°C Statistical tests (analysis of variance [ANOVA], ANOVA with and the slowest growth rate at 13°C. Th e eff ect of temperature on Scheff é post-hoc pairwise comparison, and the Wilcoxen test) were growth was so strong that animal weights were signifi cantly diff erent done using Datadesk 6.2 for Macintosh (Ithaca, N.Y.). α = 0.05. (P ≤ 0.0001) after just 1 week of rearing at the diff erent temperatures. Th is trend continued for the fi rst 5 weeks, to age 205 days, but then Results began to invert. At 6 weeks, or age 212 days, the animals at 13 and ° ≤ Th e temperatures (mean ± 1 standard deviation) over the course 15 C began to gain signifi cantly (P 0.0001) more weight than those of the experiment were 12.7 ± 1.04°C, 14.9 ± 0.831°C, 18.1 ± reared at the two higher temperatures. By age 260 days, the inver- 0.917°C, and 21.4 ± 1.14°C in the cages designated as 13, 15, 18, sion was complete: growth rates to age 260 days showed the fastest ° ° and 21°C, respectively. Daily temperatures varied < 20% of their growth rate was at 13 C, and the slowest growth rate was at 21 C; Table 1. Average growth rates and growth trends of Aplysia californica during the fi rst 90 days of the experiment Rearing temperature g live weight/day Growth trend to g live weight/day Growth trend to (°C) (170–205 days) 205 days (205–260 days) 260 days 13 0.327 3.63 fastest 15 0.493 2.99 18 0.698 2.77 21 0.702 fastest 1.95 Growth rates derived from linear regression fi ts in which all R2 values were ≥ 0.850. Th e arrows indicate that animal weights refl ecting growth rate at rearing temperature were signifi cantly diff erent from weights at all other temperatures (P < 0.01; individual analyses of variance by Scheff é post hoc pairwise comparison of data in Fig. 1).

32 CONTEMPORARY TOPICS © 2005 by the American Association for Laboratory Animal Science Volume 44, No. 3 / May 2005 Figure 2. Maximum (mean ± 1 standard deviation) animal weight with age at the diff erent rearing temperatures. Figure 3. Age at which animals in replicate cages at experimental temperatures fi rst spawned. Symbols superimpose at ages where more than one of the fi ve animal weights at these temperatures were signifi cantly diff erent (P replicates had the same spawn date. ≤ 0.0001). Overall, the inversion of growth rates beginning at age 212 days dominates the growth rate calculated from the beginning spawned. Because the parents of any egg mass are unknown, the = of the experiment (ta age 170 days) to age 260 days. age of sexual maturation was designated by the fi rst egg mass found Rearing temperature aff ected lifespan. Animals reared at 13°C lived in each cage (Fig. 3). Age at fi rst spawning represents 39 to 67% of longest, to a maximum age of 618 days, whereas animals reared at the maximum lifespan of animals reared at 13 to 21°C, respectively. 21°C lived the shortest time, to a maximum of age 282 days (Fig. Because mating and spawning events often instigate spawning in 1). Lifespan was signifi cantly inversely correlated with temperature additional Aplysia housed together (6), these spawning ages likely are (ANOVA with Scheff é post hoc pairwise comparison, P ≤ 0.0001). close to the maximum age of fi rst spawning in those cages. Th e ages Rearing temperature also aff ected maximum size. Th e relationship at fi rst spawning at all temperatures were signifi cantly diff erent from between age and maximum weight at the diff erent temperatures one another (ANOVA with Scheff é post hoc pairwise comparison, is illustrated in Fig. 2. Animals reared at 13°C reached an average P ≤ 0.0001), with animals reared at higher temperatures spawning maximum weight of 813 g at age 471 days; those at 21°C reached an earlier. average maximum weight of 192 g at age 254 days. Maximum average Spawning data for the animals reared at the diff erent temperatures animal weight was signifi cantly inversely correlated with temperature demonstrated that animals reared at lower temperatures (13 and (ANOVA with Scheff é post hoc pairwise comparison, P ≤ 0.0001). 15°C) had a longer reproductive period than 18 and 21°C animals Although age at which maximum weight was achieved was signifi - (Table 2). Age at fi rst spawn, age at fi nal spawn, and spawning period cantly inversely correlated with temperature due to the diff erences all were signifi cantly inversely correlated with temperature (ANOVA in lifespan (ANOVA with Scheff é post hoc pairwise comparison, P with Scheff é post hoc test, P ≤ 0.0001). Animals reared at 15 or ≤ 0.0001), there were no signifi cant diff erences at any temperature 21°C had their fi nal spawn 36 to 45 days before the last animal died, in the age at which maximum animal weight was reached when this whereas animals at 13°C last spawned 96 days before the fi nal death. age was expressed as a percentage of the lifespan. Animals at 13°C Total spawn weight divided by the number of animals per cage was not reached maximum weight at 76% of their lifespan, whereas those at signifi cantly diff erent at any rearing temperature, however, when just 21°C reached maximum weight at 89% of their lifespan. the fi rst 84 reproductive days at each rearing temperature were con- Although 13 of 95 mortalities occurred before senescence, reducing sidered, total weight of spawn at each temperature was signifi cantly the animal density in the aff ected cages density by 1, reduced cage diff erent (ANOVA with Scheff é post hoc test; P ≤ 0.0001). Th e total density did not aff ect mean weight of the animals. Th ere were no spawning period for animals reared at 21°C was 84 days, but it was signifi cant diff erences between the weights of animals in reduced- only 27% of the spawning lifetime of animals at 13°C. density cages and normal density cages at any temperature, regardless Th e survival data for animals at each temperature, illustrating the of whether the mortality occurred during growth or senescence. observed fraction of animals in the cohort surviving over time, are Rearing temperature affected the age at which animals first shown in Fig. 4. For each temperature, mean and median lifespans

Table 2. Spawning history 13°C 15°C 18°C 21°C Average age at fi rst spawn (days) 244 ± 1.64 208 ± 3.19 196 ± 0.894 190 ± 0a Average age at last spawn (days) 491 ± 40.3 421 ± 15.4 316 ± 12.1 221 ± 101b Maximum spawning period (days) 308 237 133 84 Spawning period as % of total lifetime 63 56 42 38 Cumulative spawn divided by number of animals per cage (kg) 0.389 ± 0.118 0.262 ± 0.0535 0.205 ± 0.0217 0.167 ± 0.0391 Spawn weight divided by animal number during fi rst 84 spawning days (kg) 0.077 ± 0.0174 0.133 ± 0.0249 0.153 ± 0.0459 0.167 ± 0.0592c Data presented as mean ± 1 standard deviation of four (13°C) or fi ve (15, 18 and 21°C ) cages at each temperature. aAge at fi rst spawn in each cage at each temperature was signifi cantly diff erent from age of fi rst spawn at all other temperatures (P < 0.0001; individual analyses of variance by Scheff é post hoc test). bAge at last spawn at each temperature was signifi cantly diff erent from age at last spawn at all other temperatures (P < 0.0001; individual analyses of vari- ance by Scheff é post hoc tests). cTotal spawn weight for the fi rst 84 spawning days at each temperature was signifi cantly diff erent from spawn weight at all other temperatures (P < 0.0001; individual analyses of variance by Scheff é post hoc test).

Volume 44, No. 3 / May 2005 CONTEMPORARY TOPICS © 2005 by the American Association for Laboratory Animal Science 33 Table 3. Life history and aging determinations 13°C 15°C 18°C 21°C Mean lifespan (days) 533 386 298 265 SD 53.1 58.0 36.4 14.5 SEM 11.9 11.6 7.29 2.90 Median lifespan (days) 537 391 307 267

the plasticity of these features of the animal’s life cycle. Th e results show that it is possible to accelerate the life cycle with increased temperature and to leave some aspects of maturation, such as lifetime fecundity, unchanged. One casualty of raising the rearing temperature, however, is maximum animal size, which decreases signifi cantly as the temperature is raised. From the observed results, rearing temperature had a strong eff ect on the growth and maturation of A. californica in a hatchery set- ting. Animals reared at lower temperatures (13 and 15°C) grew as Figure 4. Survival curves for Aplysia californica at each temperature. Observa- much as four times as large, lived twice as long, matured later, and tions expressed as a Gompertz survival function, s = exp [(A/G) ( 1 - eGt)] derived from the Gompertz mortality rate function M = AeGt as defi ned in spawned longer than animals reared at higher temperatures (18 and the Methods (13°C, n = 20; 15, 18, and 21°C, n = 25 each). 21°C). At any rearing temperature, maximum average weight was achieved considerably later than onset of sexual maturity, suggesting that either egg production contributes signifi cantly to animal weight (when survival was 50%) are similar (Table 3), indicating mortali- or that somatic growth continues during egg production in this her- ties were continuous and that the mean was not skewed by outliers maphroditic species. Fieber and colleagues (11) found that weight that died either very early or very late. Mortality curves formalize gains in hatchery-reared, sexually immature A. californica after age the observation made above that temperature strongly aff ected life 200 days were not accompanied by appreciable gains in total body span. Mean lifespan was signifi cantly diff erent at each temperature length, which suggested that weight gains were due to egg production. ≤ (Wilcoxen tests as pairwise comparisons, P 0.0002). Mortalities However, those animals were fed regimented dietary quantities and ° at 21 C occurred over a 161-day span, from ages 126 to 287 days. may have had diff erent growth patterns than those we observed. It is ° In contrast, mortalities at 13 C occurred over a 224-day span, from not unusual for aplysiids reared in the laboratory to reach maximum ° ages 395 to 619 days. Th us, the fi rst mortality at 13 C occurred 108 live weight long after fi rst spawning; this propensity was noted by ° days after the last animal reared at 21 C had died. Switzer-Dunlap and Hadfi eld (20) for four species of Hawaiian aply- Th e survival curves were best fi t by the Gompertz survival function siids fed ad libitum. Despite longer life and larger size, animals reared that is shown plotted with the survival curves in Fig. 4. Th e initial at lower temperatures in the present study did not spawn signifi cantly mortality rate, A, was extremely small for all treatments (Table 4). more than those reared at higher temperatures. Th erefore, the major Th e Gompertz parameter, or aging rate, was highest for animals advantage of lower rearing temperatures for hatchery-raised Aplysia ° reared at 21 C, whereas MRDT was lowest at this temperature, as destined for research use is that they produce animals of larger size would be expected for the accelerated lifecycle observed at higher that are immature for a longer proportion of the animal’s life. temperatures. Conversely, the aging rate is lowest and MRDT highest Maturation and growth in Aplysia also are aff ected by variations for animals reared at lower temperatures that had a comparatively in food quantity and nutritional quality (6, 8). Sexual maturation decelerated life cycle. occurred at 7 months at 15°C in this study, the same age observed by Capo and colleagues (6) for A. californica raised at 15°C with ad Discussion libitum feeding. In contrast maturation occurred at 8.25 to 10.8 Th e use of wild animals as experimental organisms in regimented months at 15°C under standard hatchery rations (10), which are 90% protocols under circumstances in which their age and growth his- of the body weight of 90-g animals per week. For animals raised at tory is unknown can lead to inconsistencies in experimental results. a constant temperature, growth rates are fastest when feeding is ad Laboratory rearing, in contrast, reduces the variability of experimental libitum but can vary with dietary composition. In the present study, subjects and enhances the validity of each experiment (17). Wild animals reared at 15°C and fed solely G. ferox ad libitum grew at and laboratory-reared animals of similar size may not be of similar 0.493 g/day for ages 170 to 205 days, whereas Capo and cowork- age because of diff erent histories of nutrition, disease, intraspecifi c ers (6) reported a rate of 1.88 g/day between 104 and 200 days of interactions, or life stage (4-6, 18). Th e simplest and most eff ective age for animals reared on an ad libitum, mixed algal diet at 15°C. way to reduce experimental variability is to use siblings of known In contrast, animals studied by Fieber and colleagues (11) achieved age that have been reared together under defi ned conditions and weight gains of 1.04 ± 0.055 g/day at 15°C between 104 and 200 days thus have greater uniformity with respect to size and condition. For of age on a mixed diet but at standard hatchery rations as described the past 15 years, we have been rearing spawn from wild-caught (i.e., calorie-restricted). Aplysia in our hatchery. Rearing begins with the eggs maintained at Growth was initially faster at higher temperatures, but these higher 22°C until shortly after the animals undergo metamorphosis, then temperatures stimulated the onset of sexual maturation, which in turn the rearing is completed at 15°C (5). Our assumption has been that slowed the growth rate. Th us at age 170 days, when the study began, 15°C maintains the 12-month live cycle these animals have in the animals reared at 21°C grew fastest, but by age 260 days, animals wild, yet delays sexual maturity triggered by warm water (3), thereby reared at 13°C grew fastest. Th is pattern most likely was caused by extending the animals’ useful experimental life. In the present study, preferential investment of energy into reproduction (gametogenesis) we examined in detail the variability in growth rates, spawning, and rather than in somatic growth as the animals reared at higher tem- mortality of A. californica with rearing temperature to understand peratures approached sexual maturity. For animals reared at 18 and

34 CONTEMPORARY TOPICS © 2005 by the American Association for Laboratory Animal Science Volume 44, No. 3 / May 2005 Table 4. Parameters of Gompertz mortality rate function 13°C 15°C 18°C 21°C Initial mortality rate (A) 6.43 × 10-8 5.52 × 10-6 1.47 × 10-6 1.41 × 10-11 Gompertz parameter (G) 0.0231 0.0200 0.0316 0.0829 Mortality rate doubling time (days) 29.9 34.7 21.9 8.36 n = 20 for 13°C; n = 25 each for 15, 18, and 21°C.

21°C, onset of sexual maturity occurred between 190 to 197 days. 6. Capo, T. R., L. A. Fieber, D. L. Stommes, and P. J. Walsh. 2003. Re- Meanwhile at 15 and 13°C, maturity was delayed until approximately productive output in the hatchery-reared California sea hare at diff erent ages 208 and 245 days, respectively. Gametogenesis in mollusks is stocking densities. Contemp. Top. Lab. Anim. Sci. 42(5):31-35. characterized by a slowing of somatic growth (9, 12, 16). 7. Capo, T. R., J. C. Jaramillo, A. E. Boyd, B. E. Lapointe, and J. E. Serafy. 1999. Sustained high yields of Gracilaria (Rhodopyhyta) grown Th e general rectangular shape of the survival curve at each rearing in intensive large-scale culture. J. Appl. Phycol. 11:143-147. temperature is typical for a population exhibiting age-related mortal- 8. Capo, T. R., D. L. Stommes, and J. E. Serafy. Eff ects of diet on feed- ity, where the likelihood of death increases with advancing age (1). As ing, growth, and survival of juvenile california brown sea hare, Aplysia expected, temperature had a strong eff ect on total lifespan. However, californica (: ), unpublished data. temperature eff ects on age at fi rst spawning also led to interesting 9. Davis, J. P. 1990. Costs of reproduction in Pacifi c oysters. 1990 Ann. results on the percentage of the animals’ lifespan that was reproduc- Meet. of the National Shellfi sheries Association, Williamsburg, Va. tive. Animals at 13°C had a spawning period that was approximately (USA), 1-5 Apr 1990 J. Shellfi sh Res. 8:431-432. 3.5-times longer than animals reared at 21°C, although as noted 10. Fieber, L. A. 2000. Th e development of excitatory capability in Aplysia californica bag cells observed in cohorts. Devel. Brain Res. 122:47- previously, there was no diff erence in total spawn weight over the 58. reproductive period at any temperature. 11. Fieber, L. A., M. C. Schmale, N. Jordi, E. Orbesen, G. Diaz and T. R. Our fi ndings corroborate those of Audesirk (2), who reported that Capo. 2005. Growth in hatchery-reared A. californica is variable but can the lowest temperature at which gametogenesis occurred in wild popu- be fi t to a von Bertalanff y growth model. Bull. Mar. Sci. 76:95-104. lations of A. californica was 13°C. Temperatures above this threshold 12. Franz, D. R. 1997. Resource allocation in the intertidal salt-marsh resulted in a condensed pre-reproductive phase, an earlier onset of mussel Geukensia demissa in relation to shore level. Estuaries 20:134- maturation and spawning, and an overall shorter lifespan. 148. 13. Goldberg, D. J. and S. Schacher. 1998. Culturing the large neurons of aplysia, p. 213-236. In G. Banker, K. Goslin (ed.), Culturing nerve Acknowledgments cells. MIT Press, Cambridge, Mass. Th is study was funded by grants RR10294 and ES05705 from the National 14. Kandel, P. and T. R. Capo. 1979. Th e packaging of ova in the egg cases Institutes of Health. Th is project required the expertise of coworkers to whom of Aplysia californica. Veliger 22:194-198 we are extremely grateful: Ana Bardales, for larval rearing of the animals, and 15. Kriegstein, A. R., V. Castellucci, and E. R. Kandel. 1974. Metamor- Albert Boyd, for culturing the seaweed. phosis of Aplysia californica in laboratory culture. Proc. Nat. Acad. Sci. 71:3654-3658. References 16. MacDonald, B. A. and R. J. Th ompson. 1986. Infl uence of temperature 1. Arking, R. 1998. Biology of aging, 2nd ed. Sinauer Associates, Sunder- and food availability on the ecological energetics of the giant scallop land, Mass. Placopecten magellanicus. 3. Physiological ecology, the gametogenic 2. Audesirk, T. E. 1976. Th e role of seasonal periodicity, chemical com- cycle and scope for growth. Mar. Biol. 93:37-48. munication, and chemoreceptive organs in reproduction in Aplysia 17. March, B. E., C. Macmillan, and F. W. Ming. 1985. Techniques for californica cooper. Ph.D. dissertation, University of Southern California, evaluation of dietary Protein quality for the rainbow trout (Salmo Los Angeles. gairdneri). Aquaculture 47:275-292. 3. Audesirk, T. E. 1979. A fi eld study of growth and reproduction in 18. Peretz, B. and L. Adkins. 1982. An index of age when birthdate is Aplysia californica. Biol. Bull. 157:407-421. unknown in Aplysia californica: shell size and growth in long-term 4. Barile, P. J., B. E. Lapointe, and T. R. Capo. Dietary nitrogen avail- maricultured animals. Biol. Bull. 162:333-344. ability in macroalgae enhances growth of the sea hare Aplysia californica 19. Wilson, D. L. 1994. Th e analysis of survival (mortality) data: fi tting (: ). J Exp. Mar. Biol and Ecol., in press. Gompertz, Weibull, and logistic functions. Mech. Ageing Dev. 74:15- 5. Capo, T. R., L. A. Fieber, D. L. Stommes and P. J. Walsh. 2002. 33. Th e eff ect of stocking density on growth rate and maturation time in 20. Switzer-Dunlap, M. and M. G. Hadfi eld. 1984. Reproductive patterns laboratory-reared California sea hares. Contemp. Top. Lab. Anim. Sci. of Hawaiian aplysiid gastropods, p. 199-210. In S. E. Stancyk (ed.), 41(6):25-30. Reproductive ecology of marine invertebrates. University of South Carolina Press, Columbia, S.C.

Volume 44, No. 3 / May 2005 CONTEMPORARY TOPICS © 2005 by the American Association for Laboratory Animal Science 35