ADAM G. MARSH and DONAL T. MANAHAN, Department of Biological Sciences, University of Southern California, Los Angeles,California 90089-0371
Currently , a debate is underway concerning why antarctic invertebrate embryos and larvae have such protracted developmental times in comparison to their temperate counterparts. Thorson (1950) suggests that cold polar temperatures slow metabolism and, hence, developmental rates in antarctic invertebrates whereas Clark (1983) argues that the limited nutritional resources in polar environments restrict metabolic rates of developing embryos and larvae. Despite almost a half century of debate, we still know very little about how larvae survive and metamorphose in extreme polar environments. As a first step toward empirically determining the relative importance of temperature or nutritional controls, our research measures the specific costs of early development and the relative impact of feeding on those energetic costs in the larvae of a polar invertebrate, the antarctic sea urchin Sterechinus neumayeri .
The energetic cost of early development in S. neumayeri was measured by culturing embryos to the four-arm pluteus larval stage (day 22 of development at -1.5°C) and then raising the larvae to the beginning of the six-arm pluteus stage (day 60). For this study, larvae were then raised in two treatments: one group was fed ad libidum (a mixture of the red alga Rhodomonas sp. and the green alga Dunaliella tertiolecta ) and the other group was starved (all culturing details described in Leong and Manahan, Antarctic Journal , in this issue).
Over the course of development, the biochemical composition of different embryonic and larval stages was measured in terms of
Oxygen (O2) consumption rates were quantified using small (<1-milliliter volume) biological oxygen demand vials (mBOD), a new method developed in our laboratory. Between 50 and 500 individual embryos or larvae are incubated in a mBOD vial for 8-10 hours (at -1.5°C). A gas-tight syringe is used to inject an aliquot of the BOD seawater into a polarographic oxygen sensor (POS), providing a direct measure of the O2 concentration in the BOD vial.
During early development, the biomass of embryos does not decline as expected due to the oxidation of energy reserves (figure 1 A ). The lack of a significant decline in mass during early development has been previously documented for S. neumayeri (Marsh and Manahan 1996) and for another antarctic echinoderm, the asteroid Odontaster validus (Shilling and Manahan 1994). From figure 1 A , embryos of S. neumayeri reach the four-arm pluteus stage (day 22) with approximately the same total mass as the initial eggs. Early development, however, does have a total metabolic cost of 2.2 millijoules per individual (mJ individual-1) for the first 22 days of development (figure 2 B ). With no detectable change in mass, the energy source fueling development must be derived from an external origin. These findings suggest that embryos of S. neumayeri may exhibit a far greater ability for nutrient uptake than has been found in temperate sea urchin embryos.
Once larvae begin feeding at day 22, individual biomass increases steadily relative to starved larvae. At day 50, the third pair of larval arms begins to form, and larval biomass in the fed treatment increases rapidly between days 50 and 60. Figure 1 B shows that the cellular protein content does not change during development between the fed and starved treatments, indicating the change in biomass does not occur as a change in cell volume or size. This finding is substantiated in figure 1 C where the individual DNA content is higher in fed than starved larvae indicating that fed larvae have a higher cell number count. Thus, changes in larval biomass occur at the level of changes in cell number and not in terms of changes in cell size.
During embryonic development, respiration rates steadily rise to a maximum value of 16 picomoles of oxygen per embryo per hour (pmol O2 embryo-1 h-1) at day 22 (figure 2 A ). From day 22 to day 32, respiration rates are equivalent between fed and starved treatments. After day 32, fed plutei evidence a large increase in respiration rates, which continue to increase until day 60. By day 60, respiration in the fed plutei has more than doubled to 37 pmol O2 larva-1 h--1 from the initial pre-feeding level.
When the four-arm plutei begin to feed ad libidum at day 22, the total metabolic costs from fertilization to day 60 increase from 7.42 mJ individual-1 in the starved group to 12.67 mJ individual-1 in the fed group (figure 2 B ). Feeding results in a 71 percent increase in metabolic energy expenditure. A regression of the cumulative cost of development against time (between day 30 and day 60) results in a daily energy expenditure for fed larvae of 299.1 microjoules per individual per day (J individual-1 d-1) (r2=0.9980) while starved larvae expended only 135.7 J individual-1 d-1 (r2=0.9986) (figure 2 B ). The absolute difference in cumulative energy expended between fed and starved larvae at day 60 was 5.3 mJ individual-1 (assuming 484 kilojoules per mole of O2), whereas the mass difference was 24.0 mJ individual-1 [in energy equivalents, 31.75 kilojoules per gram for 50 percent protein and 50 percent lipid (Shilling and Manahan 1994)]. For fed S. neumayeri plutei, the energy acquired relative to energy expended during feeding increased by 457 percent between day 22 and day 60. This comparison indicates that feeding by this echinoplutei is highly energy efficient under the culturing conditions used here.
Feeding by S. neumayeri plutei did not alter the timing of development for the progression from the four-arm tothe six-arm stage. The morphometric events associated with the formation of the third pair of larval arms evidenced an identicatime of appearance between fed and starved plutei despite the large biochemical differences between them. In these experiments, the rate of early larval development was not limited by endogenous resources and the availability of exogenous food did not affect the timing of these events. In summary, the net effect of feeding was an increase in cell number but not in apparent cell size or function. Fed and starved larvae possessed similar energetic and developmental rate processes; the primary difference in metabolic rate between the two treatments was likely the total number of cells performing those activities.
We thank Patrick Leong and Tracy Hamilton (University of Southern California) for help with culturing and feeding the larvae; Rob Robbins (Antarctic Support Associates) assisted in diving operations required to collect adult sea urchins. This research was supported by National Science Foundation grant OPP 94-20803 to D.T. Manahan.
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