Genetic studies in marine fishes

Nine polymorphic loci were found among 42 presumptive protein-coding gene loci surveyed among 474 red drum (Sciaenops ocellatus) sampled in 1987 from 13 nearshore and 1 offshore localities from the Atlantic coast of the southeastern USA and the northern Gulf of Mexico. The mean number of alleles over the polymorphic loci was 3.8, and the average heterozygosity over all loci examined was estimated as 0.047. These data indicate that red drum have normal levels of genetic variability. Wright'sF1 _ST values (the standardized variance of allele frequencies between samples) over all polymorphic loci ranged from 0.009 to 0.027 (meanF1 _ST =0.019), and estimates of the effective number of migrants (N _e m) per generation using Wright's island model ranged from 9.0 to 27.5. High levels of gene flow among the red drum samples were also indicated by Slatkin's qualitative analysis using conditional average allele frequencies. Nei's estimates of genetic distance between pairs of samples ranged from 0.000 to 0.009, indicating a high degree of nuclear gene similarity among all samples. Highly significant heterogeneity in allele frequencies at the locus for adenosine deaminase was detected between red drum sampled from the Atlantic and those sampled from the Gulf and among red drum sampled from the Gulf.     

Chitinolytic enzymes in the integument and midgut-gland of the shrimpPalaemon serratus during the moulting cycle

Chitinase andN-acetyl--D-glucosaminidase activity were quantified inPalaemon serratus (Pennant) integument and midgut gland during the moulting cycle. Studies were performed on specimens collected near Concarneau, France, in July 1989. The changes in specific activity are different in the two organs: in the midgut gland enzymatic activity is high throughout the whole moulting cycle with a weak peak at the early premoult Stage D1, whereas in the integument the activity of both enzymes is very low throughout post- and intermoult stages and rises only after premoult Stage D1. The highest specific activity is reached in D1 for chitinase and somewhat earlier (D1) forN-acetyl--D-glucosaminidase. The increase in specific chitinolytic activity coincides with an increase in ecdysteroids.     

Preliminary report on the digestive enzymes present in the digestive gland ofPerna viridis

Quantitative data on digestive enzymes of bivalves are very limited and so this study was performed to obtain such information, and to find out whether some of these enzymes are well adapted to conditions in the digestive gland. The green musselPerna viridis was obtained from Clementi West Market in 1985. The results show that various carbohydrases are present in the digestive gland. The activities of-amylase, cellulase and-glucosidase are higher than that of other carbohydrases, whilst the activities of both lipase and protease, especially the former, are low. The pH optima of the following enzymes are also given:-amylase = 5.8; laminarinase = 5.5;-glucosidase = 5.8; trehalase = 4.8;-glucosidase = 5.5. The only enzyme which was not well adapted to conditions in the digestive gland (pH 6.2) was trehalase. The wide spectrum of enzymes present in this bivalve indicates that it has the ability to utilize a wide range of nutrients efficiently.     

Species-specific patterns of hyperostosis in marine teleost fishes

The occurrence of swollen or hyperostotic bones in skeletal preparations, preserved museum material or whole fresh specimens of marine teleost fishes was identified in 92 species belonging to 22 families. Patterns of hyperostotic skeletal growth were typically consistent and often species-specific in all individuals larger than a certain size. The taxonomic distribution of hyperostosis in diverse phylogenetic groups suggests that it has arisen independently many times. Selected bones from two species of the family Carangidae, horse-eye jack Caranx latus Agassiz and crevalle jack Caranx hippos (Linnaeus), were examined in detail by light and electron microscopy. Nonhyperostotic bone contained osteoid-producing osteoblasts, resorbing osteoclasts, occasional osteocytes, and a rich vascular network, all characteristics of cellular bone. Thus, these fishes have a spatial juxtaposition of cellular and acellular bone tissues in adjacent and often serially homologous bone sites. The functional significance of hyperostosis is unknown, but it is a predictable manifestation of bone growth and development for the many taxa in which it occurs.     

Antioxidant enzymes in the digestive gland of the common musselMytilus edulis

Antioxidant enzymes function to remove deleterious reactive oxygen species, including the superoxide anion radical and H₂O₂. Subcellular distributions and optimal and other properties of catalase (EC. 1.11.1.6), superoxide dismutase (SOD; EC. 1.15.1.1), selenium-dependent glutathione peroxidase (Se-GPX; EC. 1.11.1.9) and total glutathione peroxidase (GPX) activities were determined in the digestive gland of the common musselMytilus edulis L. by spectrophotometric and cytochemical/electron microscopic (catalase) techniques. Assay conditions for Se-GPX and total GPX activities were determined which optimized the difference between the non-enzymic and enzymic rates of reaction. General peroxidase activity (guaiacol as substrate) (EC. 1.11.1.7) was not detectable in any subcellular fraction. Catalase was largely, if not totally, peroxisomal, whereas SOD and GPX activities were mainly cytosolic. Distinct mitochondrial (Mn-SOD) and cytosolic (CuZn-SOD) SOD forms were indicated. Catalase properties were consistent with a catalase, rather than a catalase-peroxidase. The pH-dependence and temperature-dependence of GPX activity were different with H₂O₂ or CHP as substrate, and these and other observations indicate the existence of a distinct Se-GPX. Under saturating or optimal (GPX) assay conditions, the apparent Michaelis constantsK _m (mM) were: catalase, 48 to 68 (substrate, H₂O₂); Se-GPX, 0.11 (H₂O₂) and 2.0 (glutathione); and total GPX, 2.2 (eumene hydroperoxide) and 1.2 (glutathione). Calculated catalase activity was 2 to 4 orders of magnitude greater than Se-GPX activity over an [H₂O₂] of 1 to 1000 M. The results are discussed in relation to theoretical calculations of in vivo oxyradical production and phylogenetic differences in antioxidant enzyme activities.     

Sulfide tolerance and detoxification in shallow-water marine fishes

Hydrogen sulfide is a potent inhibitor of aerobic respiration. Sulfide is produced in sediments, and many species of fish live in association with the bottom. Tolerance tests, enzyme assays, and chromatography of sulfur compounds in thirteen species of shallow-water marine fishes (collected in San Diego, California, USA in 1987–1988) indicate adaptations to sulfide that vary with habitat and lifestyle. Tidal-marsh inhabitants, like Gillichthys mirabilis and Fundulus parvipinnis, have higher tolerance to sulfide (96 h LC₅₀ at 525 to 700 M) relative to outer-bay and open-coast inhabitants (surviving <12 h at much lower concentrations). The cytochrome c oxidase of all species shows high activity and susceptibility to sulfide poisoning, with 50% inhibition at 30 to 500 nM in various tissues. The two marsh species are able to survive at sulfide concentrations already inhibitory to their cytochrome c oxidase and fatal to other species. All species detoxify sulfide by oxidizing it to thiosulfate. All have sulfide-oxidizing activity in the blood, spleen, kidney, liver and gills, which correlates significantly with heme content. Thiosulfate appears in the tissues of sulfide-exposed fish and builds up to high concentrations (up to 2 mM) with stronger and longer exposure. Unexposed fish contain little or no thiosulfate. Sulfide is barely detectable in the tissues, even in high-sulfide exposure tests. We suggest that fish blood, in having high sulfide-oxidizing activity and no cytochrome c oxidase, can act as a short-term first line of defense against sulfide, and thus minimize the amount that reaches the vital organs. The results of this study indicate that sulfide is a significant environmental factor influencing the ecological distribution of marine fishes.     

Comparative studies on the metabolism of shallow-water and deep-sea marine fishes. I. White-muscle metabolism in shallow-water fishes

Maximal rates of oxygen consumption in vitro have been measured under standardized conditions at three test temperatures (5°, 15°, and 25°C) on minced preparations of white muscle from 39 species of shallow-water marine teleost fishes. These fishes came from four different geographic areas, two with cool average water-temperatures (near 15°C: coastal southern California, Galápagos Islands) and two with warm average water-temperatures (near 25°C: Hawaiian Islands; Bermuda). The group includes species covering much of the range of variation to be found among the teleosts with respect to five additional variables: phylogenetic position, type of environment, body weight, activity level, and growth stage. The purpose of the work is to provide part of a base line of tissue-metabolism data on shallow-water fishes for comparison with similar results from deep-sea species. Major conclusions from statistical analyses of the results are: four groups of shapes of oxygen-uptake rate versus temperature curves exist: normal, flat, dipped and peaked. Over 50% of curves are normal. Intra-group differences, contributing significantly to the total variance of the results at given test temperatures, are: cool versus warm average environmental temperatures primarily for epipelagic species; epipelagic versus non-epipelagic environments; very active species versus all others; juvenile stages versus adults. In each case, the subgroup first mentioned shows higher muscle oxygen-uptake rates than the other subgroup. Variables not contributing significantly to the total variance are phylogenetic position and body weight. Physiological and ecological implications of these results are discussed.     

Comparative studies on the metabolism of shallow-water and deep-sea marine fishes. II. Red-muscle metabolism in shallow-water fishes

Maximal rates of oxygen consumption in vitro have been measured under standardized conditions at three test temperatures (5°, 15°, 25°C) on minced preparations of red muscle from 10 species of shallow-water marine teleost fishes. These fishes came from three different geographic areas, two with cool average water temperatures (near 15°C: coastal southern California, Galápagos Islands) and one with warm average water temperatures (near 25°C: Hawaiian Islands). The group is made up of post-juvenile or adult epipelagic fishes, which are moderately or very active in terms of their locomotor activities. A large part of the range of phylogenetic diversity among the teleosts is represented, as is the body weight range from a few grams to several kilograms. The purpose of the work is to provide part of a set of tissue-metabolism data on shallow-water fishes for future comparison with similar results from deep-sea species. Of 8 complete curves for oxygen uptake rate versus temperature (R-T curves), 6 are normal in shape (Q₁₀1.5), 1 is normal but with a low Q₁₀, and 1 is partly flat, partly normal. The differences between the species in terms of both absolute positions and slopes of the R-T curves are not related in any consistent way to any of the three testable variables: phylogenetic position, long-term adaptation temperature, and body size. The red muscles of a variety of adult epipelagic fishes, at ecologically realistic temperatures, are shown to be exceptions to the general rule that tissues of ectothermous lower vertebrates have lower metabolic rates than comparable tissues of non-torpid endothermous higher vertebrates. This circumstance probably is a major factor in the great capacities for sustained high-speed swimming shown by most epipelagic fishes. Other physiological and ecological implications of the results are discussed.     

High pressure effects on marine invertebrates and fishes

A survey of literature and of new information from the author's laboratory is presented concerning the comparative pressure physiology of marine invertebrates and fishes. Short term experiments on littoral marine animals have revealed that the taxonomic groups exhibiting the greatest resistance to high pressures are those with the greatest vertical distributions in the deep sea, namely, echinoderms, molluscs, amphipods, isopods and polychaetes. Shallow water species which possess high thermal and osmotic resistance also show an exceptionally high degree of pressure resistance. The relative differences in genetic pressure resistance of lower marine invertebrates are the same in whole, intact animals and in isolated, surviving tissue pieces. Adaptation of nonregulating euryoecous invertebrates to higher temperatures, higher osmotic concentrations and higher calcium contents of the tissues results in increased pressure resistance. Under pressure, the optimum cellular pH shifts downward to a lower pH range.Lecture presented on October 23, 1967 at Duke University, Marine Laboratory, Beaufort (USA); on November 6, 1967 at the Instituto di Biologia Marinha of the University of São Paulo (Brasil); and on November 8, 1967 at the Institute of Marine Science of the University of Miami (USA).     

A re-evaluation of the diel feeding hypothesis for marine herbivorous fishes

We re-evaluated the "diel feeding hypothesis" by measuring diel variation in starch, protein, and floridoside in three algal "types" collected from a fringing coral reef at Lizard Island, Great Barrier Reef, Australia. Samples of two species of rhodophyte algae, Gracilaria arcuata and Acanthophora spicifera, and the turf assemblage from the territories of the herbivorous pomacentrid Stegastes nigricans were collected at four time periods through the day: 0630-0730, 1000-1100, 1330-1430, and 1630-1730 hours. We also measured the ability of several species of marine fish (the herbivores Acanthurus nigricans, A. lineatus, A. olivaceus, and Parma alboscapularis and the detritivore Ctenochaetus striatus) to hydrolyse floridoside by estimating !-galactosidase activity in tissue from the anterior intestine. We detected no diel pattern in protein content of the algae but found a significant steady increase in starch content throughout the day. Floridoside content increased in the morning and decreased in the afternoon, a pattern that may be driven by midday photoinhibition of the algae. All the fishes tested could utilise floridoside. Our results support the diel feeding hypothesis. Although floridoside content decreased in the afternoon, our results suggest floridoside was used during the day by the algae to synthesise starch. Thus the algae increased in nutritional value until photoinhibition occurred at midday then subsequently maintained their nutritional value during the afternoon. This pattern of algal nutrients increasing to a midday peak and remaining relatively constant throughout the afternoon correlates well with the diel feeding pattern in many species of marine herbivorous fish.     

Acid-base responses to lethal aquatic hypercapnia in three marine fishes

Ocean sequestration of CO₂ is proposed as a possible measure to mitigate environmental changes due to the increasing atmospheric concentration of the gas. However, toxic effects of CO₂ on marine organisms are poorly understood. We therefore studied acid–base responses and mortality during exposure to fatal levels of CO₂ in three marine fishes (Japanese flounder, Paralichthys olivaceus; yellowtail, Seriola quinqueradiata; and starspotted dogfish, Mustelus manazo). The teleosts died during exposure to seawater equilibrated with a gas mixture containing 5% CO₂ (water PCO₂ 4.95 kPa); 100% mortality occurred within 8 h for yellowtail and within 48 h for flounder. Only 20% mortality was recorded at 72 h for the dogfish during exposure to 7% CO₂ (water PCO₂ 6.96 kPa). Arterial pH (pH_a) initially decreased, but completely recovered within 1–24 h for the teleosts at 1% and 3%, although the recovery was slower and complete only at 1% (water PCO₂ 0.99 kPa) for the dogfish. During exposure to 5%, the flounder died after the pH_a had been completely restored, suggesting that the mortality was not due to plasma acidosis. During exposure to 1% hypercapnia, plasma [Cl^–] appeared to be the main counter ion to balance increases in plasma [HCO₃^-]. There was a 1:1 stoichiometry for the rise in [HCO₃^-] and the fall in [Cl^–] for the teleosts, whereas the ratio was 1:0.4 for the dogfish at 1% CO₂. At the higher levels of hypercapnia, the rise in [HCO₃^-] consistently exceeded the fall in [Cl^–], and plasma [Na⁺] significantly increased.These results do not agree with the generally accepted model for acid–base regulation in marine fish in which Na⁺/H⁺ exchangers are assumed to play a predominant role, and indicate that an acid–base regulatory mechanism differs between teleost and elasmobranch fishes, as well as the intensity of acidic stress.Communicated by T. Ikeda, Hakodate     

Determinants of habitat association in a sympatric clade of marine fishes

Triplefin fishes reach their greatest diversity in New Zealand with 26 endemic species, and habitat diversification has been implicated as a key factor in the divergence of this group. Despite this, it is unknown whether species-specific habitat patterns in these sympatric fishes are established by passive processes (e.g. differential mortality) or by habitat selection during settlement. We investigate this question by comparing the habitat associations of new recruits with those of conspecific adults in five species. In addition, the amount of variation in habitat use of conspecific recruits and adults was calculated to identify ontogenetic shifts in habitat association. The results indicated that while there were some differences between recruit and adult habitats, these differences were small in magnitude and habitat use of new recruits was similar to that of adult conspecifics. This finding was further supported by the small difference in variation of habitat use between conspecific recruits and adults. The study suggests that new recruits are actively involved in the selection of habitats at settlement and maintain the use of these throughout demersal life. Habitat use in these territorial species has a large influence on mate choice, thus habitat selection by new recruits would provide a powerful mechanism for pre-zygotic isolation between individuals with different habitat preferences. Together these findings support the notion that habitat diversification has been a major component in the radiation of this sympatric group.     

The production and secreton of digestive enzymes in the purple seastar Pisaster ochraceus

The purple seastar Pisaster ochraceus contains clearly measurable protease and amylase activity. Centrifuged supernatants of pyloric caeca homogenates undergo a spontaneous threefold increase in protease activity when incubated under toluene for 50 h at 25°C. Amylase activity remains nearly constant over this period. Bovine trypsin at a 1 to 100 ratio (trypsin to supernatant protein) induces a twofold increase in protease activity over that of the control supernatant while having virtually no effect on amylase activity over the control. The data indicate a specific interaction of trypsin with a protease zymogen rather than a conspecific hydrolysis of membrane components or vesicles by trypsin. Two percent Triton X-100 used as a diluent in place of distilled, deionized water causes a sevenfold increase in protease activity and a twofold increase in amylase activity in pyloric caeca supernatants. The use of Triton as a diluent in the preparation of a stomach-tissue supernatant allows quantitative measurement of both protease and amylase activity in that tissue.     

Interacting pressure and temperature effects on enzymes of marine poikilotherms: Catalytic and regulatory properties of FDPase from deep and shallow-water fishes

At low temperature (3°C), and in the absence of substrate and cofactor, the enzyme, fructose diphosphatase (FDPase), extracted from liver of the benthic fishCoryphaenoides acrolepis, is reversibly inactivated by exposure to relatively low pressures (5,000 psi). At pressures of 20,000 psi at 3°C the native enzyme is irreversibly denatured. When the cofactor and the substrate are present, the enzyme is protected against pressure denaturation; hence, catalysis is insensitive to at least 10,000 psi at 3°C. Similarly, interactions between FDPase and its negative modulator adenosine monophosphate (AMP), are largely pressure insensitive. Pressure sensitivity of the native enzyme and of the catalytic process is much reduced at temperatures above about 9°C. The homologous enzyme from a surface fish,Pimelometopon pulchrum, is strikingly more sensitive to pressure, particularly at low temperatures.     

Plasma nonesterified fatty acids of marine teleost and elasmobranch fishes

The plasma nonesterified fatty acids (NEFA) of five species of temperate zone marine teleost fishes (Northwestern Atlantic), one Arctic marine teleost (Eastern Canadian Arctic), and four species of marine elasmobranchs (Northwestern Atlantic) are reported. Four fatty acids, 16:0, 18:1n9, 20:5n3 and 22:6n3, comprised 70 to 83% of total NEFA in the plasma of all temperate teleost species examined. With the sole exception of 18:1, these differed from the predominant fatty acids, 14:1, 16:1, 18:1 and 20:5n3, in the Arctic species. The predominant fatty acids in elasmobranch NEFA were 16:0, 22:6n3 and 18:1n9 in all species but saturated fatty acids (14:0, 16:0 and 18:0) accounted for 40% of all NEFA in all elasmobranchs examined. Monoenes represented a greater, and polyenes a smaller, percentage of the fatty acids in the Arctic sculpin compared to the temperate sculpin. Fatty acids of the n3 series were lower in the plasma of the Arctic sculpin compared to the temperate sculpin. Comparisons with published values for freshwater fish using the same method indicate the n3:n6 ratio is higher in the marine teleost fishes. Ratios of n3:n6 fatty acids ranged from 1.80 to 7.94 for the elasmobranchs and were on average lower than those for the marine teleosts. Total levels of NEFA for the elasmobranchs were between 193 and 399 nmol ml^-1, lower than the values reported here for teleosts but within the range reported by others for some teleost fishes.     

Geographical gradients of marine herbivorous fishes: patterns and processes

We present new data and the first rigorous analysis of latitudinal and thermal gradients of diversity, density and biomass of marine herbivorous fishes and review proposed explanatory mechanisms. Consistently, negative relationships between latitude, and positive relationships between sea surface temperature (SST), and relative richness and relative abundance of herbivorous fishes were found worldwide. Significant differences in the strength of gradients of richness and abundance with latitude and SST between tropical and extratropical zones were found consistently across ocean basins. Standardized sampling along the western Atlantic also showed negative relationships between latitude and total density and biomass. The trends, however, are driven by different components of the fish assemblages (i.e., scarids in the Caribbean and acanthurids in Brazil). Patterns of abundance along thermal gradients, generally associated with extensive latitudinal gradients, also were found at the local scale. Feeding rate of the ocean surgeonfish Acanthurus bahianus decreases with temperature more rapidly than the mean metabolic rate of teleost fishes. This relationship suggests a temperature-related physiological constraint. From the new standardized and comparative data presented and the review of the explanatory hypotheses, we conclude that temperature-related feeding and digestive processes are most likely involved in the distribution patterns of herbivorous fishes. Electronic Supplementary Material Supplementary material is available for this article at     

Diel changes in gut-cell morphology and digestive activity of the marine copepod Acartia tonsa

Morphometric measurements of the gut cells of the marine calanoid copepod Acartia tonsa were made over a 24 h diel cycle to test the hypothesis that feeding rhythms in this copepod are limited by the cycles of blishter-like cells (B-cells) in the gut. Copepods were collected from Long Island Sound, New York, USA, in June 1991. Experimental treatments included low (1 g chlorophyll al^-1) and high (10 g chlorophyll al^-1) food concentrations. Copepods were fixed and embedded in Epon at six time intervals over the 24 h period, and semi-thin sections through the length of Midgut Zone II were analyzed for gut-cell area and vacuole area, with area measurements integrated to yield estimates of gut-cell volume and vacuole volume. Concurrent measurements of gut fluorescence, a measure of gut fullness, and digestive enzyme activities also were made. A diel cycle in gut fluorescence was observed, most notably in the low-food treatment, which exhibited a 3-fold increase in gut pigments. There was little consistent change in digestive enzyme activities over this time span. Gaps in the gut wall indicative of spent B-cells were observed in 17 of 39 copepods, with no trend over time or food treatment. Generally, only a single gap was seen in any one copepod. B-cells were vacuolated throughout the 24 h period. Morphometric analyses revealed a correlation between gut fluorescence and both maximum vacuole area and vacuole volume, as well as percent vacuolated cell volume, in the low-food treatment. The high-food treatment, which had a relatively smaller increase in gut fluorescence (1.5-fold) during the night cycle, showed no significant increase in vacuole size. B-cells appear to have a life-cycle greater than the diel feeding period, and while B-cell vacuoles respond to the ingested food during the diel cycle, production of B-cells does not appear to be a limiting factor in A. tonsa's feeding cycle.     

Bacterial chitinase in the stomachs of marine fishes from Yaquina Bay,Oregon, USA

The occurrence of chitinase in the stomach contents of Enophrys bison and Platichthys stellatus was investigated. The highest chitinase activity and the greatest percentage of chitinoclastic bacteria in relation to the total bacterial flora were associated with fish whose stomach content was composed primarily of chitinous animals. Stomach contents lacking visible chitin possessed low or no detectable levels of chitinase activity and few chitinoclastic bacteria. Juvenile E. bison treated with chloramphenicol to remove their indigenous bacterial flora had no detectable level of chitinase in their stomach contents while non-treated juveniles showed inducible chitinase activity, indicating the bacterial origin for the chitinase.Published as Technical Paper No. 4435, Oregon Agricultural Experiment Station. This research was supported by NIH training grant 5TO1 GM-00704 and NSF grant DES73-06611 AO2.