In situ ATR-FTIR characterization of primary cement interfaces of the barnacle Balanus amphitrite.

A method is presented for characterizing primary cement interfaces of barnacles using in situ attenuated total reflection-Fourier transform infrared spectroscopy. Primary cement of the barnacle, Balanus amphitrite (Amphibalanus amphitrite), was characterized without any disruption to the original cement interface, after settling and growing barnacles directly on double sided polished germanium wafers. High-quality IR spectra were acquired of live barnacle cement interfaces, providing a spectroscopic fingerprint of cured primary cement in vivo with the barnacle adhered to the substratum. Additional spectra were also acquired of intact cement interfaces for which the upper portion of the barnacle had been removed leaving only the base plate and cement layer attached to the substratum. This allowed further characterization of primary cement interfaces that were dried or placed in D(2)O. The resulting spectra were consistent with the cement being proteinaceous, and allowed analysis of the protein secondary structure and water content in the cement layer. The estimated secondary structure composition was primarily beta-sheet, with additional alpha-helix, turn and unordered components. The cement of live barnacles, freshly removed from seawater, was estimated to have a water content of 20-50% by weight. These results provide new insights into the chemical properties of the undisturbed barnacle adhesive interface.

Barnacle reattachment: a tool for studying barnacle adhesion.

Standard approaches for measuring adhesion strength of fouling organisms use barnacles, tubeworms or oysters settled and grown in the field or laboratory, to a measurable size. These approaches suffer from the vagaries of larval supply, settlement behavior, predation, disturbance and environmental stress. Procedures for reattaching barnacles to experimental surfaces are reported. When procedures are followed, adhesion strength measurements on silicone substrata after 2 weeks are comparable to those obtained using standard methods. Hydrophilic surfaces require reattachment for 2-4 weeks. The adhesion strength of barnacles in reattachment assays was positively correlated to results obtained from field testing a series of experimental polysiloxane fouling-release coatings (r = 0.89). The reattachment method allows for precise barnacle orientation, enabling the use of small surfaces and the potential for automation. The method enables down-selection of coatings from combinatorial approaches to manageable levels for definitive field testing. Reattachment can be used with coatings that combine antifouling and fouling-release technologies.

Alteration of photoresponses involved in diel vertical migration of a crab larva by fish mucus and degradation products of mucopolysaccharides.

Photoresponses involved in the descent phase of nocturnal diel vertical migration (DVM) of larvae of the crab Rhithropanopeus harrisii were measured in a laboratory system that mimicked the underwater angular light distribution. The test hypothesis was that kairomones from fish that activate zooplankton photoresponses involved in DVM are derived from polysaccharides from the external mucus of fishes. Studies considered fish mucus from the mummichog (Fundulus heteroclitus) and disaccharides (originating from chondroitin sulfate A and heparin polysaccharides) that are likely constituents of fish mucus. R. harrisii larvae descend at sunrise with an isolume and remain near the isolume during the day. Since depth maintenance near the isolume depends upon a negative phototaxis, the lowest light intensity (threshold) that induces this response was used to quantify the effects of the test chemicals. It was predicted that exposure to fish kairomones would lower the photoresponse threshold, thereby resulting in larvae remaining deeper in the water column where light for visual predation was reduced. The photoresponse threshold declined as the concentration of fish mucus increased. Disaccharides originating from chondroitin sulfate A and heparin also decreased the photoresponse threshold as compared to responses in aged, filtered seawater. Collectively, the results support the hypothesis and indicate that disaccharide degradation products of predator mucus containing sulfated and acetylated amines can serve as kairomones.

Natural product antifoulants: One perspective on the challenges related to coatings development.

Fouling of surfaces by abiotic and biotic substances has molecular, microbial, and macro-organismal levels of organization. Fouling involves molecular bonding and biological adhesives. Existing commercial solutions to fouling are antifouling or foul-release. Antifouling uses broad-spectrum biocides which kill foulers by virtue of oxidation or toxic metal ions. Foul-release coatings are dimethyl silicone polymers that foul, but clean easily. The best foul-release coatings also contain additives that kill organisms. Environmentally unacceptable consequences of toxic antifouling coatings, especially those based on organotins, have prompted interest in antifoulants found in living organisms. Laboratories worldwide now use bioassays with target fouling organisms to direct purification and identification of antifoulant compounds. Natural antifoulants are common and include toxins, anesthetics, surface-active agents, attachment and/or metamorphosis inhibitors and repellents. Development of commercial coatings using natural products is blocked by cost, the time horizon to meet government regulations and performance standards based upon coatings with unacceptable environmental impacts. If blocks are removed, the potential for environmentally acceptable solutions that combine natural products with organic biocides is high.

Antisettlement and Narcotic Action of Analogues of Diterpene Marine Natural Product Antifoulants from Octocorals.

: Prevoius studies have determined that the octocorals Renilla reniformis and Leptogorgia virgulata contain diterpenes that are potent inhibitors of barnacle settlement. These antifoulants-the renillafoulins and pukalide-are, however, comparatively complex and thus are not amenable to commercial exploitation. The present study examined 19 analogues, based on the functional groups of lactone and furan rings in the parent molecules, for antisettlement activity and toxicity. The latter parameters are presented as EC(50) values for inhibition of cypris settlement and naupliar swimming, respectively. Assays of a subset of the analogues indicated that they were active in solution rather than when bound to a surface and that at relatively high concentrations they had a narcotic action. The mechanism whereby some of the analogues were able to inhibit settlement at nontoxic concentrations has yet to be explained but suggests that there is merit in the present approach to antifoulant development.

Alteration of [14C]-testosterone metabolism after chronic exposure of Daphnia magna to tributyltin.

Tributyltin (TBT) is a marine biocide that has been shown to alter the activity of cytochrome P450 monooxygenases and elicit toxicity indicative of androgenization in some species. The present study was conducted to determine whether TBT altered P450-, reductase-, and transferase-mediated testosterone metabolic processes in Daphnia magna at sublethal exposure concentrations. Two generations of daphnids were continuously exposed for 21 days to nominal TBT concentrations ranging from 0.31 to 2.5 microg/L TBT. The highest TBT concentration (2.5 microg/L) was lethal to 60% of the exposed organisms. Lower TBT concentrations elicited no adverse effects on molting or reproduction of the daphnids. No differences were observed in the response of the first- and second-generation daphnids to the toxicity of TBT. The ability of daphnids to metabolize [14C]-testosterone in vivo was assessed following exposure of each generation to TBT. Production of hydroxylated, reduced/dehydrogenated, and glucose-conjugated metabolites of testosterone were all elevated following exposure of both generations to 1.25 microg/L TBT. These findings indicate that, under these conditions, TBT elicits no discernible effects on molting and reproduction of daphnids at sublethal concentrations, and testosterone metabolism is enhanced at concentrations approaching those that are lethal to organisms. Alterations of steroid metabolism by xenobiotics can be used as a more sensitive indicator of sublethal exposure in daphnids than reproductive endpoints.

Localization and characterization of neuropeptide Y/peptide YY receptors in the brain of the smooth dogfish (Mustelis canis).

Multiple receptor subtypes specific for the neuropeptide Y (NPY)/peptide Y (PYY) family of peptides exist in mammals, but little is known about the distribution of this receptor family in other vertebrates. Saturable binding sites for 125I-labeled porcine PYY were localized in frozen sections of the brain of the smooth dogfish (Mustelis canis) by radioligand binding and autoradiography. Saturable 125I-porcine PYY binding sites were distributed widely in the cerebral hemispheres, optic lobes, hypothalamus, cerebellum and hindbrain. Binding was saturable, specific for PYY and related peptides, and of high affinity (Kd = 2.53 nM). The specificity of the binding site was analyzed by performing competitive inhibition experiments with nonradioactive PYY, NPY, and [Leu31, Pro34]-NPY and NPY13-36, synthetic peptide analogs specific for the mammalian Y1 and Y2 receptor subtypes, respectively. Saturable 125I-porcine PYY binding sites in all regions of the dogfish brain closely resembled the mammalian Y1 NPY receptor subtype in specificity for these substances. There was no evidence for expression of multiple receptor subtypes. We conclude that a single receptor specific for the NPY/PYY family of peptides is widely expressed in the smooth dogfish brain and that this receptor closely resembles the mammalian Y1 receptor subtype, suggesting that the Y1 receptor is the ancestral receptor in this family.

Evidence for the involvement of cyclic AMP in the pheromonal modulation of barnacle settlement

The involvement of cyclic AMP in the settlement of the cypris larva of Balanus amphitrite amphitrite Darwin has been examined through the use of compounds that affect intracellular cyclic AMP levels. The activation of adenylate cyclase with forskolin, and the inhibition of phosphodiesterase with 3-isobutyl-1-methylxanthine, caffeine and theophylline, significantly increased the settlement of cyprids. Although the analogue dibutyryl cyclic AMP appeared to increase settlement, the effect was not significant. No marked increase in settlement resulted from the incubation of cyprids with dibutyryl cyclic GMP, 8-(4-chlorophenylthio) (CPT) cyclic AMP or papaverine (a phosphodiesterase inhibitor). Miconazole nitrate, an adenylate cyclase inhibitor, prevented settlement, but this effect appeared to be physico-chemical rather than pharmacological. Radioimmunoassay did not clearly show whether cyclic AMP levels changed following exposure of cyprids to a pulse of crude barnacle extract. However, exposure to forskolin significantly increased the cyclic AMP titre of cyprids. We conclude that compounds that alter intracellular cyclic AMP levels alter normal patterns of cyprid settlement. Whether this is because of an alteration in signal transduction is unclear.

Behavioral responses ofLittoraria irrorata (SAY) to water-borne odors.

Behavioral responses of the gastropod molluscLittoraria (=Littorina)irrorata indicate that it can discriminate among environmental odors. Snails were assayed for responses to 11 odors from plants and animals potentially representing food, shelter, location in the environment, and predators. Crushed conspecifics were included as an alarm odor. Except for odor of crushed conspecifics, all odor sources were water-borne from living intact organisms. Behavioral responses were categorized as no response, positive response, or negative response. For some analyses, negative responses were subdivided into withdrawing and turning responses. Snails responded positively to several plant odors. They did not respond to odors of intact conspecifics, fiddler crabs, or grass shrimp. They responded negatively to odors of a plant found at the upper limit of their minimal habitat, predatory blue crabs, crushed conspecifics, predatory gastropods, and ribbed mussels. Odors of blue crabs on different diets affect the type of negative response the snails display.

Superpotent synthetic tripeptide mimics of the mud-crab pumping pheromone.

Soluble pheromones released by the mud crab, Rhithropanopeus harrisii (Gould) during egg hatching cause the female crab to contract her abdomen rapidly (the pumping response). This stereotypical behavior can be induced in the laboratory by exposing egg-bearing females to solutions containing certain amino acids or peptides. Twelve amino acids exhibited response thresholds of 10(-3)-10(-10) M, the most potent (norleucine, methionine and tryptophan) having a hydrophobic sidechain. Four synthetic tripeptides of the form Gly-X-Arg, where the hydrophobic residue X was isoleucine, norleucine, methionine or phenylalanine, were superpotent agonists over a limited concentration range. Gly-Ile-Arg was a significant agonist at 10(-17) and 10(-16) M, both Gly-Nle-Arg and Gly-Met-Arg at 10(-20) and 10(-19) M, and Gly-Phe-Arg at 10(-21), 10(-20) and 10(-19) M. At the subattomolar concentration of 10(-20) M the superpotent pheromone mimics Gly-Met-Arg and Gly-Phe-Arg produced not only a statistically significant increase in the relative number of pumping mud crabs but also a substantial increase in the pumping rate ratio. In contrast, at 10(-13) M the tripeptide Gly-Met(O)-Arg with an internal residue of methionine sulfoxide blocked the abdominal pumping response of egg-bearing mud crabs.

Bacterial-barnacle interaction: Potential of using juncellins and antibiotics to alter structure of bacterial communities.

In preparation for studies using natural products to probe interactions between bacterial consortia and settlement stage barnacles, we isolated 16 strains of bacteria associated with barnacles and examined: (1) effects of films of bacterial isolates on barnacle settlement, and (2) bacteriostatic effects of juncellins and standard antibiotics. Bacteria were isolated from the biofilm associated withBalanus amphitrite. On the basis of morphological and biochemical characteristics, bacteria were classified into five major groups:Aeromonas, Alcaligenes, Flavobacterium, Pseudomonas, andVibrio. Barnacle settlement was inhibited by allVibrio films and 64% of the other isolates. No film stimulated barnacle settlement. Juncellins were approximately as potent as standard antibiotics for all bacterial species tested.Vibrio spp. were most resistant to juncellins.

Inhibition of Settlement by Larvae of Balanus amphitrite and Ciona intestinalis by a Surface-Colonizing Marine Bacterium.

In an attempt to isolate bacteria with inhibitory effects against settlement by larvae of sessile invertebrates, 40 marine bacterial isolates were screened for effects against laboratory-reared barnacle larvae (Balanus amphitrite) and ascidian larvae (Ciona intestinalis). Five isolates displayed non-pH-dependent inhibitory effects against the larvae. The initial characterization of a toxic component released from an isolate, designated D2 (CCUG 26757), and its effect on laboratory-reared barnacle and ascidian larvae were studied. D2 is a facultative, anaerobic, gram-negative bacterium isolated from the surface of C. intestinalis from waters off the Swedish west coast at a depth of 10 m. Results suggest that the toxic component is released by D2 during the stationary phase. Aged biofilms were more toxic to the larvae than unaged films. The biologically active compound was in the supernatant of D2 and was heat stable and <500 Da in molecular mass. No evidence of protein or peptide moieties was found. On the basis of two phase and chromatography separations, the component is polar and neutral and contains or binds to carbohydrate moieties. Metaperiodate treatment increased toxicity; undiluted supernatant from a 24-h growth culture of D2 killed barnacle and ascidian larvae within a few hours of exposure, whereas after metaperiodate treatment, the larvae were killed in approximately 30 min.

Inhibition of larval barnacle attachment to bacterial films: An investigation of physical properties.

The effects of films of two strains of a marine bacterium, Deleya marina (ATCC 25374 and 27129) on the attachment response of cypris larvae of the balanomorph barnacle, Balanus amphitrite, were examined in the laboratory. Tests showed that the cell-surface hydrophobicities of the two bacteria in suspension were different. In contrast, films derived from these cells were both highly wettable (i.e., displayed high surface free energy). Assays (22 hours) compared permanent attachment of larval barnacles to films derived from exponential and stationary phase cells for both bacteria. These films either had no effect or inhibited attachment of both 0-day- and 4-day-old cypris larvae when compared with unfilmed controls. Our data indicate that inhibition of larval barnacle attachment by films of the two bacteria is the result of factors other than surface free energy. Production of chemical barnacle settlement inhibitors by the bacteria is hypothesized.

Chemical mediation of behavior in hermit crabs: Alarm and aggregation cues.

Field studies with three common local species of hermit crabs,Clibanarius vittatus, Pagurus longicarpus, andPagurus pollicaris, showed that these crabs responded behaviorally to chemicals originating from crushed conspecifics. Hermit crabs are attracted specifically and in a manner similar to previously reported crab responses to odors from dead gastropods. Responses byC. vittatus to both kinds of odor are of three types: (1) aggregation/shell investigation responses (previously reported for odors from dead gastropods), characterized by increased locomotor activity, investigation of shells in the vicinity, and switching into empty shells; (2) alarm responses, in which crabs flee the area; and (3) withdrawal responses, in which crabs pull into their shells and do not come out. Studies withC. vittatus showed that the stimulatory chemicals originate from hemolymph, are less than 500 D, adsorb to octadecyl silica, and are recovered by elution with 20% methanol. Responses ofC. vittatus are dependent upon crab size, type of shell occupied, and shell fit. Chemicals originating from dead conspecifics provide a forum for shell acquisition by crabs in relatively small shells and alarm by crabs in relatively large shells.

Control of chemically stimulated feeding behavior in sand fiddler crabsUca pugilator: Evidence for hemolymph feeding inhibitory factor.

Uca pugilator, the sand fiddler crab, is a deposit-feeder. It feeds on exposed sand flats as the tide recedes. Feeding movements are evoked by stimulation of chemoreceptors on the dactyls. Previous studies have demonstrated that a proteinaceous factor associated with the sinus gland region inhibits chemically-stimulated feeding behavior. Here, that work is extended to show the existence of a similar inhibitory factor present in the hemolymph of fed crabs in the laboratory and in the hemolymph of crabs returning from feeding in the field. The factor is not detected in fasted laboratory crabs or in field crabs walking from burrow areas to feeding sites. Injection of glucose results in inhibition of feeding activity in intact crabs. Injection of glucose, but not galactose, stimulates feeding in eyestalk-ablated crabs. We suggest that neural responsiveness is stimulated by glucose, and that elevated glucose in intact crabs results in release of feeding inhibitory factor from the sinus gland. Release of feeding inhibitory factor into the blood enables crabs to return to burrow areas without stopping when they encounter food-laden sediments.

Peptide attraction of hermit crabsClibanarius vittatus Bosc: Roles of enzymes and substrates.

Hermit crabs are obligate users of gastropod shells. Shell availability is often the limiting factor for crab population size. Crabs have an extensive behavioral repertoire for obtaining shells. Here we extend our studies of the chemical ability of crabs to locate the shells of dead and dying gastropods from a distance. We show that peptide cues generated by the action of specific proteases on specific substrates attract crabs. The specificity of the crab response is dependent upon the type of substrate as well as the suit of enzymes attacking the substrate. Single specific enzymes are not as effective as mixtures of enzymes in generating cues from pure (and totally foreign) substrates such as ovalbumin. However, the activation of trypsinogen by enterokinase yields only a single hexapeptide and results in potent crab attraction. We conclude that the specific sequence of the peptide determines attraction. Thus, the key to crab attraction is the presence of a particular sequence in a substrate and the ability of enzymes or mixtures of enzymes to release that sequence.

Chemical Mediation of Larval Release Behaviors in the Crab Neopanope sayi.

Control of egg hatching was investigated in ovigerous females of the crab Neopanope sayi. Larval release is a brief event, generally lasting less than 15 min, during which females perform stereotypic behaviors involving vigorous abdomen pumping. Substances released by hatching eggs (pumping factors) of N. sayi, Rhithropanopeus harrisii, and Uca pugilator, but not Sesarma cinereum, evoked these stereotypic behaviors (pumping response) in ovigerous N. sayi. Spontaneous pumping and responsiveness to pumping factors varied with the age of the embryos. These results indicate that the eggs release pheromones around the time of hatching, which supports the general model for egg-hatching control described for R. harrisii (Forward and Lohmann, 1983). The chemistry of N. sayi pumping factors was investigated, and the pumping response was used as a bioassay in this study. Pumping factors adsorbed to Amberlite XAD-7 resin and could be eluted from it with methanol. Size fractionation by cascade pressure dialysis showed that the active molecules were <1000 daltons. Acid hydrolysis followed by reverse-phase HPLC amino acid analysis showed that the biologically active fraction contained peptides. Cysteine, glycine, methionine, and isoleucine were the four most common amino acids in these peptides. The responsiveness of N. sayi to hatch water from R. harrisii, the general similarity of adsorptive characteristics of hatch waters from the two species toward XAD-7 resin, and the amino acid compositional analysis suggest that the pumping factors from both species are similar. This supports the hypothesis that N. sayi pumping factors are also small peptides, as was suggested for those of R. harrisii (Rittschof et al., 1985, 1989).

Adhesion and motility of gliding bacteria on substrata with different surface free energies.

The adhesion and motility of several aquatic and terrestrial gliding bacteria on slides differing in their critical surface energies have been examined. In general, adhesion was tenacious on low-critical surface energy (hydrophobic) surfaces and tenuous on hydrophilic surfaces. Gliding was inhibited on very hydrophobic substrata and skittish on very hydrophilic surfaces.