work was supported by National Science Foundation grant Huntley, M.E., and M.D.A. Lopez. 1992. Temperature-dependent OPP 92-22715. growth of marine copepods: A global synthesis. American Natural- ist, 140(2), 201-242. Huntley, M.E., M. Zhou, and M.D.A. Lopez. 1994. Calanoides acutus References in Gerlache Strait, Antarctica II. Solving an inverse problem in population dynamics. Deep-Sea Research II, 41(1), 209-227. Andrews, K.J.H. 1966. The distribution and life history of Calanoides Kieppel, G.S. 1992. Environmental regulation of feeding and egg pro- acutus (Giesbrecht). Discovery Reports, 34, 117-162. duction by Acartia tonsa off southern California. Marine Biology, Dagg, M. 1977. Some effects of patchy food environments on cope- 112(1), 57-65. pods. Limnology and Oceanography, 22(1), 99-107. Lopez, M.D.G., M.E. Huntley, and J.T. Lovette. 1993. Calanoides acu- Huntley, M.E., and F. Escritor. 1991. Dynamics of Calanoides acutus tus in Gerlache Strait, Antarctica. I. Distribution of late copepodite (Copepoda: Calanoida) in antarctic coastal waters. Deep-Sea stages and reproduction during spring. Marine Progress Research, 38(8/9), 1145-1168. Series, 100(1/2),153-165.

Energy metabolism during development of the antarctic neumayeri ROBERT D. PODOLSKY, Department of Zoology, University of Washington, Seattle, Washington 98195 PAT VIRTUE, Institute ofAntarctic and Studies, University of Tasmania at Hobart, Hobart, Tasmania 7001, Australia TRACY HAMILTON, JAY VAvRA, and DONAI. T. MANAHAN, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0371

ittle is known about the developmental of lar- Changes in activity of citrate synthase during develop- al forms of antarctic marine . At tempera- ment tures typical of sea water in McMurdo Sound (-1.8°C), rates of revious research has shown that certain enzymes of energy development are slow (Pearse, McClintock, and Bosch 1991). pmetabolism can be accurate predictors of overall metabol- The feeding larval stage (pluteus) of the antarctic echinoid ic rate. Activity of citrate synthase (a citric acid cycle indicator) Sterechinus neumayeri develops approximately 20 days after has been used to estimate the aerobic metabolic capacity of fertilization; the juvenile stage is reached by 115 days (Bosch (e.g., Hand and Somero 1983) and vertebrate tis- et al. 1987). sues (e.g., Crockett and Sidell 1990; Yang and Somero 1993). We investigated the biochemical and physiological The technique (described in citations above) is versatile changes that occurred during the early development of this because it can be applied to frozen tissue (cf the difficulty of . Our approach was to making metabolic measurements on live organisms). • measure the changes in respiration during development to We measured no significant change in total activity of cit- the feeding larval stage; rate synthase during the period of development studied (figure • measure the changes in enzymes involved in aerobic 1B; yR of slope = 0.09flS; total degrees of freedom = 31). Our metabolism; and finding that enzyme activity does not correlate with increasing • measure the changes in total protein, total lipid, and spe- respiration rate suggests that attempts to establish quantita- cific lipid classes during development. tive relationships between enzymic activity and metabolic rate Respiration during development should proceed with caution for antarctic larvae. mbryos and larvae were removed from the culture vessels Total protein, total lipid, and specific lipid classes Eat various times during the 38-day period of development otal protein of homogenates was measured with the Brad- studied. The respiration of a known number of individuals Tford assay (1976). Protein is not used as an energy source was measured at -1°C in a microrespiration chamber (see by developing embryos of S. neumayeri (figure 1 Q. No signifi- Jaeckle and Manahan, 1992, for description of instrumenta- cant decrease occurred in the protein content from eggs (day tion used). Controls (same respiration chambers without ani- 0) to early pluteus stage (day 22) (VR=0.17 ns; total degrees of mals) were run before and after each measurement with ani- freedom = 42). mals present and were used to correct for any background For lipid analysis, samples were extracted quantitatively amounts of oxygen consumption. Figure 1A shows that the by the Bligh and Dyer method (1959) (see White et al. 1979) metabolic rate increased during development, from newly and stored at -20°C. A portion of the total lipid extract was hatched stages to the feeding larvae stage. A least-squares lin- analyzed for total lipid composition with an Iatroscan MK III ear regression was used to plot the line describing the rate of TH10 TLC-FID analyzer (latron Laboratories, Japan) (yolk- increase in metabolism with time of development (variance man and Nichols 1991). The response of the detector was cali- ratio, VR=38.59, F0001 [1,301_13.3). brated using external standards covering the concentration

ANTARCTIC JOURNAL - REVIEW 1994 157 A. Figure 1. A. The rates of oxygen consumption during the early develop- ment of the sea urchin S. neumayeri. Each data point represents a single 14 • • S measurement corrected for background respiration (50-100 individuals in 12 . . S a 75-microliter respiration chamber were used for each determination). • . 10 Slope of regression line shown = 0.2626±0.423 (standard error of the : slope); intercept = 3.5931; value for r2 8 =0.56. B. Changes in activity of the • enzyme citrate synthase. Activity is expressed as Ux10-6 • I per individual, 16C6 S •S • where 1 U = 1 micromole (tmol) of substrate converted to product per I.4 • I minute. Rates presented are activities at -1°C. Actual measurements were done at 5.2°C and corrected for temperature difference using a Q10 value of 1.82 (measured over a temperature range of -1°C to 12.2°C). C. :1 10 20 30 40 Changes in total protein content. Error bars are 1 standard error of the Time after fertilization (days) mean. f I - range found in the samples. Individual class-specific calibra- tions curves were used for each lipid class measured. Total lipid content dropped substantially from the egg to the 4-day-old hatching embryos (figure 2A); the later stages 0 lb io 3b 40 (feeding larvae) had the lowest lipid content. We measured no Time after fertilization (days) major changes in the composition of the three lipid classes 500 during the first 11 days of development (figure 2B). After 11 days (gastrulae), the neutral triacyiglycerols decreased as a percentage of total lipid, and the polar lipid class increased. This presumably reflects the increase in the number of cells 200 as larval development proceeds (phospholipids are major 100 components of cell membrane lipids). We thank Marc Slattery for collecting the sea urchins and Adam Marsh for advice and assistance. These data were Time after fertilization (days) obtained from some of the projects conducted during the McMurdo Biology Course of 1994. Supported by National Sci- ence Foundation grant OPP 93-17696 to the University of Southern California.

280 A. References 260 240 Bosch, I., K.A. Beauchamp, M.E. Steele, and J.S. Pearse. 1987. Devel - 220 opment, metamorphosis, and seasonal abundance of embryos 200 and larvae of the antarctic sea urchin Sterechinus neumayeri. Bio- 180 logical Bulletin, 173(1), 126-135. Bradford, M.M. 1976. A rapid and sensitive method for the quantita- 160 tion of microgram quantities of protein utilizing the principle of 140 protein-dye binding. Analytical Biochemistry, 72, 248-254. I 120 Crockett, E.L., and B.D. Sidell. 1990. Some pathways of energy metab- 100 olism are cold adapted in antarctic fishes. Physiological Zoology, 80 63(3), 472-488. Hand, S.C., and G.N. Somero. 1983. Energy metabolism pathways of 0 10 20 30 40 Time after fertilization (days) deep-sea hydrothermal vent : Adaptation to a food-rich and sulfide-rich habitat. Biological Bulletin, 165, 167-181. 80 R Jaeckle, W.B., and D.T. Manahan. 1992. Experimental manipulations of the organic chemistry of seawater: Implications for studies of 70 energy budgets in marine invertebrate larvae. Journal of Experi- mental Marine Biology and Ecology, 156, 273-284. 60 Pearse, J.S., J.B. McClintock, and I. Bosch. 1991. Reproduction of 50 antarctic benthic marine invertebrates: Tempos, modes, and tim- ing. American Zoologist, 31(l), 65-80. 40 30 i Figure 2. A. Changes in total lipid content of developing sea urchins, S. 20 neumayeri. Each point is the mean of triplicate analysis. Error bars are 1 10 standard error of the mean. (ng denotes nanograms.) B. Changes in lipid classes during development. TG, triacyiglycerols; PL, polar lipids; ST, 0 4 11 18 22 28 38 sterols. Errors (not shown) were small and ranged from 0.1 to 3.3 percent Time after fertilization (days) for all histograms shown.

ANTARCTIC JOURNAL - REVIEW 1994 158 Volkman, J.K., and P.D. Nichols. 1991. Applications of thin layer chro- Yang, T.H., and G.N. Somero. 1993. Effects of feeding and food-depri- matography-flame ionization detection to the analysis of lipids vation on oxygen consumption, muscle protein concentration and and pollutants in marine and environmental samples. Journal of activities of energy metabolism enzymes in muscle and brain of Planer Chromatography, 4, 19-26. shallow-living (Scorpaena guttata) and deep-living (Sebastolobus White, D.C., W.M. Davis, J.S. Nickels, J.D. King, and R.J. Bobbie. 1979. alascanus) scorpaenid fishes. Journal of Experimental Biology, Determination of the sedimentary microbial biomass by 181,213-232. extractable lipid phosphate. Oecologia, 40, 51-62.

Cytochrome b gene sequences from two eelpouts (perciformes, zoarcidae) from McMurdo Sound (Antarctica): Implications on the antifreeze gene structure GIACOMO BERNARDI, Department of Biological Sciences, Hopkins Marine Station, Stanford University, Pacific Grove, California 93950 ARTHUR L. DEVRIEs, Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois 61801

Present address: Department of Biology, University of California, Santa Cruz, California 95064.

he fish family Zoarcidae (eelpouts) comprises approxi- postulate that the extreme compositional and size hetero- Tmately 200 species, living mainly in the Northern Hemi- geneity present in a species like L. dearborni has resulted sphere (Nelson 1984). Nine genera with 22 species occur in from gene amplification and modification of the amplified the southern oceans (Anderson 1990, pp. 256-276). It is the genes and represents a relatively old antifreeze gene family. second largest group of antarctic ocean fishes (Nothoteniidae The gene family of P. brachycephalum, on the other hand, are represented by at least 32 species). All zoarcid species are would appear to be young because it is largely made up of benthic except Lycodapus and Melanostigma, which are ben- copies of a single 7 KDa protein and only a few of a very simi- tho- or mesopelagic. lar minor one. This major APP accounts for approximately 95 To avoid freezing, several eelpout species that live at sub- percent of the circulationg AFP (Cheng and DeVries 1989). zero temperatures synthesize antifreeze peptides (AFPs). This gene family organization suggests a recent origin Eelpout AFPs were characterized from Macrozoarces amen- because there has been insufficient time for recognizable canus, Pachycara brachycephalum, Lycodichthys dearborni, modification of the many copies present in the family. Thus, and Lycodes polaris (Li et al., 1985; Schrag et al, 1987; Cheng in an attempt to further infer phylogenetic relationships and DeVries, 1989). Thus far, the AFPs that have been charac- between these two species and the possible relationship of terized have been found to be encoded within large gene fam- the age of the APP gene families, we accessed an independent ilies and although the gene products are heterogeneous with locus, the mitochondrially encoded cytochrome b (Cyt b) respect to composition, they are approximately the same size gene of both McMurdo Sound species. (7 kilodalton) with the exception of an additional 14 KDa vari- P.brachycephalum and L. dearborni were collected using ant found in L. dearborni (Wang et al., in press). fish traps in 600 meters of water in McMurdo Sound, Antarcti- The sequences of the 7 KDa AFPs characterized from dif- ca. DNA was extracted from liver tissue according to a method ferent eelpout species can be both similar and quite different already described (Bernardi and Bernardi 1990). The poiy- depending upon the species. In the case of L. dearborni, the merase chain reaction (Saiki et al. 1988) was used to amplify a 14 KDa variant is a unique major component, which is species 836-base-pair (bp) region of the Cyt b gene. Primers and pro- specific and appears to be made up by joining of two 7 KDa tocols were from Kocher et al. (1989) and Palumbi et al. AFPs with a nine amino acid residue linker. The carboxyl 7 (1991). Approximately 100 nanograms of DNA was used as KDa portion is nearly identical to the minor AFP found in template for 100-milliliter PCR reactions that contained 10 another eelpout, P. brachycephalum, and quite unlike the 7 millimolar (mM) Tris, hydrogen chloride (pH 8.3), 50 mM KDa AFPs found within its own family. Because orthologous potassium chloride, 1.5 mM magnesium chloride, 0.01 per- proteins cannot be found in other species, and because the cent (weight-to-volume) gelatin, 200 mM of each dNTP, 2.5 sequence identities between species is often extremely vari- units of Taq DNA polymerase (Perkin-Elmer, Cetus), and 1 able, it is difficult to infer phylogenies based on antifreeze mM of each amplification primer. PCR products were genes. For these reasons, antifreeze gene trees and organism sequenced directly using the femtomole sequencing kit trees are not always concordant. Nevertheless, some phyloge- (Promega) and dATP33 (NEN) as the radiolabeled nucleotide. netic inferences can be drawn based on other traits such as Sequencing primers used were CB3-H, CB2-H, CB1-L, and the genomic organization of the antifreeze gene family. We GLUDG-L (Palumbi et al. 1991). Cyt b sequences were aligned

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