Rates of oxygen consumption were measured for embryos, larvae and juveniles of the seastar Mediaster aequalis for 76 days post-fertilization. The rate increased from 0.65 nmol O2 ind−1 h−1 at 6 h after fertilization to 2.8 nmol O2 ind−1 h−1 at day 35. Larvae became competent to metamorphose around day 35 post-fertilization and began to decrease their metabolic rate after this time. Metamorphosed juveniles consumed 0.74 nmol O2 ind−1 h−1. Eggs contained 138.6 µg lipid ind−1 and 12.1 µg protein ind−1. Lipid levels decreased in concentration throughout development while protein levels increased slightly. The lipid levels decreased by 88.5 µg from eggs to day 76 larvae, accounting for 3.5 J of energy. Total oxygen consumption to this point was 3.74 µmol O2 ind−1, accounting for 1.84 J. The energetic demand up to day 76 was met completely through the use of lipid reserves. Metamorphosed juveniles expended 0.5 J more than larvae at the same age. Tubes of the polychaete Phyllochaetopterus prolifica were able to induce metamorphosis in M. aequalis larvae and a non-polar extract of these tubes also triggered metamorphosis. Larvae that are delayed to metamorphose can sustain their metabolic rate with lipid reserves for a limited, yet undetermined, period.

The present study investigated aspects of the antifoulant properties of three sympatric species of ascidians found in seagrass habitats of the Gulf of Mexico, Southern Atlantic Ocean, and Caribbean. Field observations in Saint Joseph Bay, Florida indicate that all three species are common and that the tunic of the solitary ascidian Molgula occidentalis is often heavily fouled, while the outer surfaces of both the colonial ascidians Amaroucium stellatum and Botryllus planus are free of fouling organisms. Antifoulant activities of a suite of increasing hydrophilic organic extracts prepared from the tunic of M. occidentalis and whole colonies of A. stellatum and B. planus were measured using both sympatric microbial (bacteria) and macroinvertebrate (cyprid larvae of Balanus amphitrite) fouling organisms in laboratory bioassays. In addition, field antifoulant assays were conducted by combining organic extracts with controlled-release resin and subsequently coating this material on to acrylic rods deployed in the field for a 72 h period. Extracts of the tunic of M. occidentalis generally did not inhibit bacterial growth. The exception was the methanol extract, which inhibited growth in one of the six marine bacteria tested. Moreover, only the highest concentrations of hexane and methanol tunic extracts tested prevented attachment of cyprid larvae. Field assays revealed no antifoulant activity on rods coated with resin containing extracts of M. occidentalis. Inhibition of both microbial growth and cyprid settlement were much more pronounced in whole-organism extracts of the two colonial ascidians. Most potent were the aqueous methanol extracts of colonies of B. planus and A. stellatum which inhibited growth in five of the six marine bacteria tested. In addition, hydrophilic and lipophilic extracts of the colonial ascidians significantly inhibited attachment of cyprid larvae, in many instances across a wide range of extract concentrations. Field antifoulant assays indicated that extracts of both colonial ascidians inhibited settlement of bryozoans and barnacles. The findings indicate that the colonial ascidians B. planus and A. stellatum possess chemical antifoulant properties. In contrast, the solitary ascidian M. occidentalis appears to either tolerate fouling or possess other non-chemical mechanisms to cope with the risks associated with epibiont overgrowth.

The circumpolar nudibranch Tritoniella belli Eliot occurs in abundance in shallow-water benthic communities of McMurdo Sound, Antarctica. Density estimates based on belt transects averaged collectively 0.46 individuals m−2 at three study sites between depths of 6 and 30 m in November 1996. At two of the sites, population densities increased linearly between 18 and 30 m depth (up to 0.7 and 1.15 individuals m−2 at 30 m depth). Individuals at all sites were rare or absent at depths shallower than 12 m. Size frequencies of individuals at the sites were similar, and a pooled analysis revealed a unimodal distribution skewed highly towards juvenile size classes. This suggests both recent recruitment and constant rates of mortality across size classes. The relationship between foot length and wet weight best fits an exponential growth equation, indicative of an allometric growth pattern. Distribution of T. belli in the field suggests that it is a habitat and diet generalist. Potential invertebrate predators include sea anemones and seastars, both of which co-occur in abundance in McMurdo Sound. Laboratory experiments indicate that the sea anemone Isotealia antarctica can capture and ingest T. belli. However, 70% of T. belli that are captured escape from the tentacles or, following ingestion, are rejected from the gastrovascular cavity. The seastars Odontaster validus, Perknaster fuscus, and Acodontaster conspicuus, avoid contact with T. belli, but if forced into contact with mantle tissues, retract their tube-feet. Mucus secreted from the mantle tissues, coated on to the tips of glass rods, and presented to seastar tube-feet, causes significantly longer tube-foot retraction times than control rods. Moreover, pieces of freeze-dried krill coated with mantle mucus are consumed significantly less often than untreated control pieces of krill by a benthic scavenging fish (Pseudotrematomas bernacchi). Employing seastar tube-foot retractions as a bioassay, we found the bioactive compound(s) are soluble in ethyl acetate, indicating they are lipophilic or moderately hydrophilic in nature. Chemical defenses in the mucus of T. belli probably contribute to its high abundance in Antarctic benthic communities.

The tube building polychaete Hydroides elegans Haswell was found living attached to colonies of the arborescent bryozoan Bugula neritina (L.) in Port Shelter, Hong Kong. Field data collected during the period of January through May 1996, showed that H. elegans density reached 77.6 individuals of H. elegans per g wet weight of B. neritina. Density of H. elegans on B. neritina at depths from the surface to 0.5 m was lower than that at depths below 1 m. In January–March, when there were no H. elegans settling on PVC plates or found on natural substrata, numbers on B. neritina were ca. 5 per g wet weight. H. elegans settled on B. neritina and grew rapidly as mean diameter of tubes increased from 605 μm in February to 936 μm in March. In laboratory experiments, larvae of H. elegans settled and metamorphosed on branches of B. neritina and on the bottom of dishes containing B. neritina leachate. Compounds extracted from the leachate of B. neritina induced 74% of H. elegans larvae to metamorphose at a concentration of 16 μg/ml seawater, compared to 5% in dishes containing only filtered seawater (controls). Metabolites from the leachate of B. neritina which were bound to amberlite XAD-2, indicating they are lipophilic in nature, induced over 70% metamorphosis in H. elegans larvae at 56 μg/ml seawater. A biofilm from one of four strains of bacterial isolates associated with the surface of B. neritina induced low levels of metamorphosis in H. elegans larvae, while other bacterial isolates were detrimental to the survival of juvenile H. elegans. Field experiments further demonstrated that H. elegans settled preferentially on Phytagel discs embedded with whole extracts of B. neritina over control Phytagel discs. Metabolites from B. neritina deterred feeding on alginate pellets by assemblages of local fishes in field assays. Metabolites originating from B. neritina, bacteria colonizing B. neritina, and the complex structure of B. neritina contributed to the recruitment of H. elegans to B. neritina surfaces. Hydroides elegans may gain a refuge from predation by associating with B. neritina colonies both from its structural and chemical attributes.

The attachment and metamorphic responses of veliger larvae of the abalone, Haliotis diversicolor (Reeve) to potassium chloride (KCl), gamma-aminobutyric acid (GABA) and natural cues were determined. Attachment and metamorphosis of H. diversicolor were two distinctly different responses. Attachment was characterized by larval contact with a substratum followed by crawling with the larval foot firmly attached to the surface of the substratum. This behavior began approximately 48 h post-fertilization at 22°C. Larvae could detach from the substratum and continue to swim for over 96 h after attachment behavior began. Metamorphosis was irreversible and could occur in larvae that were 96 h post-fertilization. The neurotransmitter GABA, stimulated attachment behavior and induced low levels of normal metamorphosis at a concentration of 10−5 M, but was toxic to larvae at concentrations of 10−3 and 10−4 M. KCl at 10, 20 and 30 mM concentrations above normal seawater salinity stimulated attachment behavior, but became toxic at 30 and 40 mM. Low levels of metamorphosis were induced at excess KCl concentrations of 20 and 30 mM. Single films of mucus, diatoms and three species of bacteria were not as stimulatory as a combination film of diatoms and mucus in dish assays. Survival of larvae settled in dishes coated with a film of diatoms and mucus was higher than that in dishes containing either diatom or mucus films alone. The combination of diatom film and conspecific mucus may produce a novel cue that is not present in either film alone. Observations suggest that the condition of the adults may influence cues present in mucus. Moreover, when given a choice of vertical attachment sites, H. diversicolor larvae attached more frequently to slides filmed with both diatoms and mucus than to clean or diatom-filmed slides. A combination film may increase numbers of attached larvae and reduce mortality in commercial aquaculture facilities.

Six strains of marine bacteria were isolated from benthic algal films and subtidal rocks. Films and aqueous leachates of isolates were tested for effects on larval attachment of Bugula neritina (Linnaeus) in three experiments. Confluent films of four bacterial isolates significantly inhibited larval attachment of B. neritina. A fifth isolate inhibited B. neritina attachment in two of three experiments, while the sixth isolate had no effect on attachment of B. neritina larvae. Results of additional experiments employing Petri dishes filmed by bacteria on one half of the dish bottom suggest both soluble and surface film factors are involved in mediating larval settlement. Aqueous extracts of two bacterial strains inhibited bryozoan settlement in each of the three replicate assays and were toxic to larvae. A. final series of experiments tested paired bacterial films and leachates for direct comparisons. For certain isolates, the film inhibited settlement and the water soluble-leachate had no effect on settlement. Other isolates displayed opposite results. These results suggest both surface properties and water-soluble compounds released from bacterial films are responsible for settlement inhibition.

The gastropod Calliostoma canaliculatum displays a series of aggressive escape behaviors upon contact with tube feet of the predatory seastars Pycnopodia helianthoides and Pisaster giganteus. Escape behaviors are predator specific. Calliostoma canaliculatum moves away from contact with P. giganteus more frequently than P. helianthoides, clamping down with the foot or retracting the head and foot into the shell when exposed to P. helianthoides. If escape from the grasp of either seastar fails, C. canaliculatum releases a yellow-colored exudate from the hypobranchial gland and subsequently retracts both the head and foot fully into the shell. This exudate contains noxious compound(s) as evidenced by retraction of tube feet and arms away from the exudate in both seastars. Tube-foot retraction responses to dilutions of the exudate indicates that both species of seastars are able to detect the exudate at a concentration of 3.2 × 10−3 mg exudate/ml seawater. Pisaster giganteus is more responsive to the exudate than Pycnopodia helianthoides, moving away from the source as well as retracting the tube feet and arm. Snails spread the exudate over their shells with their foot, perhaps to ensure defense from predators for some time period after exudate release. The exudate was collected and extracted in chloroform–ethyl acetate (1:1), then fractionated using flash chromatography. The most bioactive fraction, as evidenced by tube-foot retraction, was soluble in ethyl acetate and appeared to contain two major compounds.

The feeding deterrent effects of echinoderm body-wall tissues and ethanolic extracts containing mid-polarity compounds were evaluated utilizing generalist fish and crabs as model predators. The body-wall tissues of the echinoderms examined ranged 10-fold from 0.9–9.4 mm in thickness, and four and a half-fold in level of mineralization (17.8–82.7% ash content). Holothuroids had the thickest body-wall tissues and contained the lowest levels of mineralization in their body-walls. Crinoids and ophiuroids had high levels of mineralization in their arms. Asteroid body-wall tissues varied the most in thickness and ash content (0.9–3.9 mm in thickness and 29.2–55.5% in ash content). Body-wall tissues of 19 species of echinoderms were tested for their feeding deterrent properties against the marine fishes Lagodon rhomboides (Linnaeus) and Cyprinodon variegatus (Lacepede), as well as the decapod crustacean Libinia emarginata (Leach). Equivalent sized pieces of fresh body-wall tissue of 16 species of echinoderms caused observable feeding deterrence responses in at least two of the three model predators. There was no significant correlation between body-wall thickness or percent ash and its palatability to any of the three model predators. Agar pellets containing ethanolic body-wall extracts of 12 of 18 echinoderm species caused observable feeding deterrence responses in the fish L. rhomboides. In similar experiments with the arrow crab Stenorhyncus seticornis (Herbst), using carrageenan fish-meal blocks as food models, no differences in consumption of control fish-meal and experimental body-wall extract blocks were detected. Our findings indicate that invertebrate and vertebrate predators may respond quite differently to echinoderm body-wall extracts.

Six strains of marine bacteria were isolated from benthic algal films and subtidal rocks. Films and aqueous leachates of isolates were tested for effects on larval attachment of Bugula neritina (Linnaeus) in three experiments. Confluent films of four bacterial isolates significantly inhibited larval attachment of B. neritina. A fifth isolate inhibited B. neritina attachment in two of three experiments, while the sixth isolate had no effect on attachment of B. neritina larvae. Results of additional experiments employing Petri dishes filmed by bacteria on one half of the dish bottom suggest both soluble and surface film factors are involved in mediating larval settlement. Aqueous extracts of two bacterial strains inhibited bryozoan settlement in each of the three replicate assays and were toxic to larvae. A final series of experiments tested paired bacterial films and leachates for direct comparisons. For certain isolates, the film inhibited settlement and the water soluble-leachate had no effect on settlement. Other isolates displayed opposite results. These results suggest both surface properties and water-soluble compounds released from bacterial films are responsible for settlement inhibition.

Thirteen new (1-13) and seven known (14-20) steroid glycosides were isolated from Henricia downeyae, collected from the offshore waters of the northern Gulf of Mexico. Ethanolic extracts of these starfish caused growth inhibition in bacteria and fungi, potent antifouling activity against barnacle and bryozoan larvae, and feeding deterrent activity against a marine fish. The known compounds are typical glycosides found in several species of the family Echinasteridae, i.e., Echinaster sp. and Henricia laeviuscola. One of the new compounds belongs to this group, whereas the remaining 12 new compounds represent a novel series of steroid glycosides which have aglycons with structural similarities to the "asterosaponins". They possess a delta 9(11) 3 beta, 6 alpha-dihydroxysteroidal aglycon with 23-oxo or 22,23-epoxy functionalities and often a 20-hydroxyl group in the side chain. The sulfate is located at C-6 and the saccharide moiety at C-3, in contrast with the asterosaponins which have the sulfate at C-3 and the oligosaccaride moiety at C-6. All the new compounds contain a glucuronic acid unit, which is uncommon among steroid glycosides from echinoderms. The structures of the new compounds, isolated in amounts ranging from 3.4 to 0.9 mg, were determined by interpretation of their spectral data and by comparison with spectral data of known compounds.

The ethanolic body-wall extracts of 16 species of echinoderms from 16 genera were screened for their ability to affect the attachment of the marine bacteria Deleya marina (Baumann) and Alteromonas luteo-violacea (Gauthier). Body-wall extracts were tested at concentrations which mimic mean natural tissue concentration, 3.0 mg/ml seawater, and four half-log dilutions of this initial concentration. The extracts of three echinoderm species caused significant inhibition of bacterial attachment, while extracts of eight species caused significant enhancement of attachment. The body-wall extract of the asteroid Goniaster tesselatus (Lamarck) displayed the most potent antimicrobial activity, completely inhibiting attachment of both bacterial species at a concentration of 3.0 mg/ml seawater. The ethanolic extracts of 20 echinoderm species were also tested at a similar range of concentrations for their ability to affect the settlement of cyprid larvae of the barnacle Balanus amphitrite (Darwin), and coronate larvae of the bryozoan Bugula neritina (Linné). All echinoderm extracts inhibited settlement of both barnacle and bryozoan larvae at the highest concentration tested (3.0 mg/ml seawater). Eleven of the 20 echinoderms tested (13 asteroids, 3 holothuroids, 3 ophiuroids and a crinoid) had body-wall extracts that inhibited settlement of competent barnacle and bryozoan larvae at concentrations as low as 0.12 mg/ml seawater. These extracted echinoderm compounds may function as non-toxic or toxic antifoulants, and to promote bacterial surface colonization, which could be valuable to the organisms disease resistance.

The ability of the antarctic pteropod Clione antarctica (Mollusca: Gastropoda) to synthesize sex steroids from radiolabeled steroid precursors was examined in individuals sampled near the end of their observed reproductive season from McMurdo Sound, Antarctica. The precursors, H-3-progesterone and 3H-androstenedione, were absorbed and subsequently metabolized to produce several progestin and androgen metabolites, including 5 alpha-pregnane-3,20-dione, 3 beta-hydroxy-5 alpha-pregnan-20-one, 3 beta-hydroxy-4-pregnen-20-one, 5 alpha-androstane-3,17-dione, 3 beta-hydroxy-5 alpha-androstan-17-one (androsterone), and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone). Synthesis or accumulation of testosterone and estradiol was not observed. At least 2 unidentified, water-soluble progestin metabolites were synthesized, as well as an unidentified esterified steroid. The metabolic accumulation of these steroidal compounds indicates that C. antarctica can produce potentially bioactive steroids. The activity of steroid metabolic enzymes in a polar species further indicates the universal capacity of invertebrates from various geographic regions to metabolize biologically relevant steroids.

Two new sulphated polyhydroxylated steroid glucuronides. downeyoside A and B (1, 2), C-24 methyl epimers possessing an ethereal ring linking C-16 to C-22 of the steroid, were isolated from the starfish Henricia downeyae collected from the Gulf of Mexico. Their structures were determined by spectral analysis and the stereochemistry of the highly oxygenated steroid side chain was derived from NMR data combined with molecular dynamics calculation. Compounds 1 and 2 were tested against non-small-cell lung human carcinoma cells and found to be cytotoxic with IC50 of 60 μg/ml and 36 μg/ml, respectively.

Ten new [1-10] and three known [11-13] polyhydroxysteroids were isolated, along with four known asterosaponins [14-17], from the starfish Luidia clathrata, collected from the offshore waters of the northern Gulf of Mexico. The EtOH extracts of this starfish showed feeding-deterrent properties against marine fish, and inhibited the settlement of larvae of barnacles and bryozoans, as well as the growth of several bacteria. The structures of the new compounds were determined by interpretation of their nmr spectral data and by comparison with the spectral data of known compounds. The assignment of the configurations of the side-chain stereogenic centers of compounds 1 and 3-10 were based on the comparison of their nmr data with those of the stereoisomeric model compounds after derivatization with the chiral auxiliary MTPA reagent. Larval settlement assays conducted on ten isolated compounds revealed they are all potent inhibitors of settlement. Two of these isolated compounds inhibited the growth of several bacteria.

Dense populations of the antarctic pteropod Clione antarctica (Smith) offer a rich source of potential nutrients and energy to planktivorous predators. Nonetheless, antarctic fish do not prey on C. antarctica. Employing flash and high-pressure liquid chromatographic techniques, a linear β-hydroxyketone, pteroenone (C14H24O2) was isolated from whole tissues of C. antarctica. When embedded in alginate food pellets at ecologically relevant concentrations, pteroenone caused significant feeding deterrence in Pagothenia borchgrevinki and Pseudotrematomas bernacchii, two antarctic fish known to feed on planktonic organisms. Concentrations of pteroenone were variable between pteropods (0.056 to 4.5 mg ml-1 tissue), but even those individuals with the lowest natural concentration contained levels five-fold greater than the lowest effective feeding-deterrent concentration (0.012 mg ml-1 alginate). Chemical analysis indicated that the primary dietary item of the carnivorous C. antarctica, the shelled pteropod Limacina helicina, does not contain pteroenone. This suggests that C. antarctica does not derive this defensive compound from its diet. This is the first example of a defensive secondary metabolite in a pelagic gastropod.

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The common bright yellow antarctic lamellarian gastropodMarseniopsis mollis was examined for the presence of defensive chemistry. Proton nuclear magnetic resonance (NMR) spectroscopy indicated that a major component of ethanolic extracts purified by reversed-phase column chromatography was homarine. Further high-performance liquid chromatography (HPLC) analysis of the mantle, foot, and viscera verified the presence of homarine in all body tissues at concentrations ranging from 6 to 24 mg/g dry tissue. A conspicuous macroinvertebrate predator of the shallow antarctic benthos, the sea starOdontaster validus, always rejected live individuals ofM. mollis, while readily feeding on pieces of fish tail muscle. Filter paper disks treated with shrimp elicited a broad range of feeding behaviors in the sea starO. validus (movement of disc to mouth, extrusion of cardiac stomach, humped feeding posture). Shrimp disks treated with homarine (0.4 and 4 mg/disk) were rejected byO. validus significantly more frequently than control disks treated with solvent carrier and shrimp or shrimp alone. The highest concentration of homarine tested not only caused feeding deterrence, but in several sea stars a flight response was noted. Homarine was not detected in the tunic of the antarctic ascidianCnemidocarpa verrucosa, a presumed primary prey ofM. mollis. Nonetheless, crude extracts of the epizooites that foul the tunic (primarily the bryozoans and hydroids) contain homarine, suggestingM. mollis may ingest and derive its chemistry from these organisms. This appears to be only the third example of chemical defense in a member of the Order Mesogastropoda. As the vestigial internalized shell ofM. mollis is considered a primitive condition, the findings of this study lend support to the hypothesis that chemical defense evolved prior to shell loss in shell-less gastropods.

Extracts of the dorid nudibranchTritoniella belli and stoloniferan coralClavularia frankliniana were chromatographed and analyzed by(1)H NMR and thin-layer chromatography. Three glycerol ethers were detected inT. belli, primarily 1-O-hexadecyl glycerol (chimyl alcohol). Chimyl alcohol was also detected after gradient flash chromatography and reverse-phase HPLC purification in the tissues ofC. frankliniana. The common omnivorous predatory Antarctic sea starOdontaster validus, a likely predator of benthic invertebrates, showed feeding deterrence to small cubes ofT. belli mantle tissue placed on the tube feet along the ambulacral feeding groove, while always extruding the cardiac stomach when presented with cubes of shrimp tissue of similar size. Filter-paper disks soaked in an aqueous shrimp solution and then dried were found to elicit a broad range of feeding behaviors inO. validus, including movement of the shrimp disk to the mouth, extrusion of the cardiac stomach, and the assumption of a humped feeding posture. Chimyl alcohol-treated shrimp disks caused significant feeding deterrence in sea stars when compared with control disks (solvent plus shrimp treated disks alone).T. belli andC. frankliniana appear to employ a defensive compound that has been found in a variety of temperate and tropical mollusks, where it has been demonstrated to deter fish predators. We provide evidence for further deterrent capabilities of chimyl alcohol and of its trophic relationship in the polar ecosystem of McMurdo Sound, Antarctica.