Stephen A. Watts

The effects of photoperiod on gametogenesis in the tropical sea urchin Eucidaris tribuloides (Lamarck) (Echinodermata: Echinoidea)

Abstract The tropical sea urchin Eucidaris tribuloides (Lamarck) was placed under two variable and two fixed photoperiod regimes at a constant temperature of 22 °C for a period of 1 yr. Gametogenic condition was ascertained at time zero (June 1988) and then every 3 months thereafter by scoring the ripeness of the gonads (presence of gametes oozing from gonad, microscopic examination of egg sizes and sperm activity from smears), measuring the gonadal index, and histologically analyzing subsamples of the ovaries and testes. Individuals held under ambient in-phase photoperiod regimes (14L : 10D summer; 10L : 14D winter) developed mature gonads in September 1988 in synchrony with the known annual reproductive cycle of field populations. Individuals held under photoperiod regimes 6 months out-of-phase with ambient photoperiod did not become reproductively mature until March 1989, indicating that gametogenesis had been delayed until the days shortened or nights lengthened during the artificial “winter” photoperiod. Sea urchins held under fixed long days (15L : 9D) delayed gametogenesis over the entire year, developing mature gonads in ≈ 20% of the individuals. Sea urchins held under fixed short days (9L: 15D) were stimulated to produce mature gametes throughout the year, and mature gonads were found in ≈ 60% of the individuals. These observations suggest that short days or long nights enhance and entrain gametic development in E. tribuloides . This is the first documentation of photoperiodic control of gametogenesis in a tropical echinoderm, and indicates that marine invertebrates may be able to cue reproductive events using relatively moderate, but highly predictable, incremental changes in annual photoperiod.

Biochemical and morphometric study of growth in the stomach and intestine of the echinoid Lytechinus variegatus (Echinodermata)

In June 1990, previously starved Lytechnius variegatus were fed a 5% fish meal-agar diet for 32 d at 20°C and 32‰. Stomach and intestine indices increased by 139 and 66%, respectively, with the most growth occurring by Day 16. The stomach had a faster growth rate than the intestine, as indicated by an increase in the stomach: intestine ratio. Gonadal growth did not begin until Day 16, after substantial gut growth. DNA and morphometric analysis revealed that cellular growth in the stomach and intestine was first hyperplastic and then hypertrophic. DNA concentration and histological examination indicated that the stomach was composed of large voluminous cells, and the intestine of small cells. The stomach of starved individuals contained numerous depressions that significantly decreased in number during growth. The number of depressions significantly increased in the intestine. Lipid concentration and amount were higher in the stomach, and increased during growth by 43 and 258%, respectively, by Day 32; little change in lipid occurred in the intestine. The stomach lipid:DNA ratio increased by 66% by Day 16, with little change in the intestine. The concentration of soluble protein remained constant in the stomach, but had decreased in the intestine by Day 16. The total energy stored in the stomach and the intestine increased by 152 and 66%, respectively, by Day 32. The stomach mucosa increased in thickness and appeared to decrease its surface area. These data suggest that the stomach constitutes the primary storage organ of the gut. The intestinal mucosa also inreased in thickness but remained thinner than the stomich while apparently increasing in surface area, suggesting an enhanced role in residual nutrient absorption and ranslocation, respiration or water ion-transport.

Biochemical Responses During Starvation and Subsequent Recovery in Postlarval Pacific White Shrimp, Penaeus vannamei

Postlarval shrimp, Penaeus vannamei Boone, 1931, were held individually in cages and exposed to two feeding regimes. One group was starved for 12 d and then fed during the following 12 d. A second group was fed throughout the 24 d study. Four individuals were sampled from each of the two groups on Days 0, 1, 2, 4, 8, 12, 13, 14, 16, 20, and 24. Molting and growth among the starved-fed postlarvae stopped after 2 d starvation, while fed postlarvae increased significantly in size throughout the 24 d study. Among the starved-fed postlarvae, water content increased rapidly in response to starvation. DNA and sterol concentrations increased significantly during starvation due to selective catabolism of cellular components. After 12 d, RNA concentration was not significantly different between the fed and starved-fed postlarvae, but became significantly higher in the starved-fed postlarvae 48 h after feeding resumed. Triacylglycerol reserves were severely depleted during the first day of starvation, while protein concentrations began to decrease after the second day of starvation. RNA, protein, and the polyamines spermidine and spermine, when expressed as a ratio to DNA, decreased in response to starvation. Concentrations of all measured parameters in the starved-fed postlarvae returned to levels similar to those in the fed group 8 to 12 d after feeding resumed. Results of this study suggest that triacylglycerol provides energy during short periods of starvation, while protein is utilized during prolonged starvation. The ratios of RNA:DNA, protein:DNA, spermidine:DNA, spermine:DNA, two unidentified amine compounds, and percent water content are all useful indicators of prolonged nutritional stress in postlarval P. vannamei.

Sex steroid levels in the testes, ovaries, and pyloric caeca during gametogenesis in the sea star Asterias vulgaris

The concentrations of progesterone, testosterone, and estradiol were determined via radioimmunoassay in testes, ovaries, and pyloric caeca of the sea star Asterias vulgaris during one complete and two partial gametogenic cycles. These compounds were found in all tissues examined and were present in quantities similar to those reported previously in other echinoderms and in vertebrates. Testes and ovaries exhibited annual growth cycles during which testicular and ovarian mass increased up to 100-fold as gametes were produced and stored until spawning. Pyloric caecal mass varied during the annual reproductive season; however, no seasonal trends were apparent. In the testes, sex steroid levels were highest at the onset of spermatogenesis. Transient increases in the levels of estradiol coincided with spermatogonial mitotic proliferation. Transient increases in the levels of testosterone and progesterone in the testes coincided with spermatogenic column formation and with spermiogenesis, respectively. In the ovaries, estradiol and testosterone levels were highest at the onset of oogenesis while progesterone levels did not change significantly throughout the annual gametogenic cycle. Male and female pyloric caeca exhibited similar seasonal variations in levels of sex steroids as compared with the gonads. It is hypothesized that transient increases in the levels of sex steroids during gametogenesis may serve as endogenous modulators of reproduction.

Combined effects of formalin fixation and tissue processing on immunorecognition.

Abstract It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.

The ecology of Lytechinus variegatus

This chapter summarizes about the ecology of Lytechinus variegates, a dominant species in many of the seagrass meadows throughout its regions. Lytechinus variegatus is the largest species, reaching 92 mm diameter in the Florida Keys, 87 mm in the West Indies, and 75 mm in southern Brazil. As a ruderal species, Lytechinus variegates has rapid growth, early reproductive maturity, and short longevity. These characteristics make Lytechinus variegatus a potential candidate for fisheries. An adult Lytechinus variegatus usually consume formulated diets and produce large gonads. This chapter also gives an additional insight such as genus, habitats, abundance, distribution, food and nutrition habits, growth and survival, reproduction, larval ecology, population, and community ecology needed to understand the characteristics of Lytechinus variegates. However, there is minimal information on the postmetamorphic, early juvenile life history stage, knowledge about nutrition, environmental influences, natural predators, and community interactions.

The use of feather meal as a replacement for fish meal in the diet of Oreochromis niloticus fry

Abstract Four diets were developed in which equal weights of hydrolyzed feather meal (F) replaced equal weights of fish and meat and bone (FMB) meal in the following percentages: 0/100, 33/66, 66/33 and 100/0 (F/FMB). Each diet was proffered ab libitum to two replicate groups of 60 fry (mean wet weight 12·3 ± 0·3 mg) for 42 days. Survival was not significantly different among treatments (> 93%). The individuals fed 0/100, 33/66 and 66/33 (F/FMB) did not differ significantly in median or mean weight gain, however, individuals fed 100/0 (F/FMB) were significantly smaller than those fed all other diets at days 21 and 42. The growth of Oreochromis niloticus fry was not compromised by replacement of up to 66% of fish meal and meat and bone meal (9·9% of the total diet) with feather meal. Feeding feather meal as a sole source of animal protein, however, appears to result in a decrease in weight gain and associated growth parameters.

Changes in polyamine levels in response to acclimation salinity in gills of the blue crab Callinectes sapidus rathbun

Abstract The concentration of putrescine in gills 3, 4, 6 and 7 was approximately two-fold higher in gills acclimated to 35 ppt salinity than in those acclimated to 10 ppt salinity for 28 days ( c . 90 nmol/g wet weight vs 40 nmol/g wet weight, respectively). Spermine concentration was significantly higher in gills 6 and 7 than in gills 3 and 4 at 10 ppt salinity, and also was significantly higher than in gills 6 and 7 of crabs acclimated to 35 ppt salinity. Putrescine concentrations varied inversely and spermine concentrations varied directly with specific activity of Na + , K + -ATPase in all gills. On the basis of these data and other recent reports, we suggest that polyamines are involved in the regulation of osmotic and ionic homeostasis by interacting directly with the Na + , K+-ATPase enzyme.

HYPOOSMOTIC STIMULATION OF ORNITHINE DECARBOXYLASE ACTIVITY IN THE BRINE SHRIMP ARTEMIA FRANCISCANA

In the brine shrimp, long-term (several days) osmotic and ionic regulation is controlled principally by the activity of Na+K+ATPase. However, short-term cell volume regulation in Artemia and most euryhaline crustaceans is not understood. In this study, Artemia stage 1 nauplii reared at 32 ppt salinity and exposed to hypoosmotic conditions (12 or 4 ppt) exhibited an immediate increase in the activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis. ODC activity increased within 15 min relative to control and reached a peak within 2 hr of hypoosmotic exposure. The level of increased enzyme activity was directly related to the degree of hypoosmotic exposure (a three- and sevenfold increase in activity at 12 and 4 ppt salinity, respectively). This increase in activity was apparently due to the synthesis of a new enzyme, based on cycloheximide administration. Activity decreased significantly by 4 hr but remained above that of the 32 ppt control throughout the 24-hr exposure period. Levels of putrescine increased significantly within 2 hr in organisms exposed to 4 ppt; spermidine and spermine levels were not affected. When nauplii were exposed to altered ion and/or osmotic concentrations it was determined that decreased total osmotic concentration was the proximate factor responsible for ODC induction. However, reduced ion concentration resulted in the induction/activation of DFMO-insensitive ODC activity, suggesting the production of a novel enzyme in polyamine metabolism during hypotonic exposure. Changes in ODC activity and polyamines preceded acclimatory changes normally associated with salinity acclimation, further suggesting that modulation of ODC activity and subsequent polyamines synthesis, degradation, and/or transport are mechanistically related to short-term regulation of osmotic and ionic balance.

EFFECT OF TEMPERATURE AND SALINITY ON LARVAL DEVELOPMENT OF SIBLING SPECIES OF ECHINASTER (ECHINODERMATA: ASTEROIDEA) AND THEIR HYBRIDS

Adult Echinaster Type 1 and Type 2 were collected along the west coast of Florida (25° C, 32‰S) and induced to spawn in the laboratory. Two-day old larvae of Type 1, Type 2, and their hybrids were subjected to temperature (T) and salinity (5) combinations (20, 25, 30°C; 25, 32, 39‰S). Response surface isopleths indicate that Type 1 and Type 2 larvae exhibit different developmental and growth rates in response to T/S combinations. Salinity was a dominant factor affecting development and growth. High and low salinities inhibited spine development. Developmental rates were directly related to temperature. Type 2 larvae exhibited a greater tolerance to temperature changes than did Type 1. Hybrids showed intermediate development and growth responses at the apparent optimal conditions and exhibited maternal characteristics.