A relaxin-like gonad-stimulating peptide identified from the starfish Astropecten scoparius.

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B-chain (21 aa), C-peptide (47 aa), and A-chain (24 aa). There are three putative processing sites (Lys-Arg) between the B-chain and C-peptide, between the C-peptide and A-chain, and within the C-peptide. This structural organization revealed that the mature AscRGP is composed of A- and B-chains with two interchain disulfide bonds and one intrachain disulfide bond. The C-terminal residues of the B-chain are Gln-Gly-Arg, which is a potential substrate for formation of an amidated C-terminal Gln residue. Non-amidated (AscRGP-GR) and amidated (AscRGP-NH2 ) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP-GR and AscRGP-NH2 induced oocyte maturation and ovulation in similar dose-dependent manners. This is the first report on a C-terminally amidated functional RGP. Collectively, these results suggest that AscRGP-GR and AscRGP-NH2 act as a natural gonadotropic hormone in A. scoparius.

Effect of chimeric relaxin-like gonad-stimulating peptides on oocyte maturation and ovulation in the starfish Asterias rubens and Aphelasterias japonica.

A relaxin-like gonad-stimulating peptide (RGP), comprising two peptide chains (A- and B-chains) linked by two interchain bonds and one intrachain disulfide bond, acts as a gonadotropin in starfish. RGP orthologs have been identified in several starfish species, including Patiria pectinifera (PpeRGP), Asterias rubens (AruRGP) and Aphelasterias japonica (AjaRGP). To analyze species-specificity, this study examined the effects on oocyte maturation and ovulation in ovaries of A. rubens and A. japonica of nine RGP derivatives comprising different combinations of A- and B-chains from the three species. All nine RGP derivatives induced spawning in A. rubens and A. japonica ovaries. However, AruRGP, AjaRGP and their chimeric derivatives were more potent than peptides containing the A- or B-chain of PpeRGP. Three-dimensional models of the structures of the RGP derivatives revealed that residues in the B-chains, such as AspB6, MetB10 and PheB13 in PpeRGP and GluB7, MetB11, and TyrB14 in AruRGP and AjaRGP, respectively, are likely to be involved in receptor binding. Conversely, it is likely that ArgA18 in the A-chain of AruRGP and AjaRGP impairs binding of these peptides to the PpeRGP receptor in P. pectinifera. In conclusion, this study provides new insights into the structural basis of RGP bioactivity and RGP receptor activation in starfish.

A Crown-of-Thorns Seastar recombinant relaxin-like gonad-stimulating peptide triggers oocyte maturation and ovulation.

The Acanthaster planci species-complex [Crown-of-Thorns Seastar (COTS)] are highly fecund echinoderms that exhibit population outbreaks on coral reef ecosystems worldwide, including the Australian Great Barrier Reef. A better understanding of the COTS molecular biology is critical towards efforts in controlling outbreaks and assisting reef recovery. In seastars, the heterodimeric relaxin-like gonad stimulating peptide (RGP) is responsible for triggering a neuroendocrine cascade that regulates resumption of oocyte meiosis prior to spawning. Our comparative RNA-seq analysis indicates a general increase in RGP gene expression in the female radial nerve cord during the reproductive season. Also, the sensory tentacles demonstrate a significantly higher expression level than radial nerve cord. A recombinant COTS RGP, generated in a yeast expression system, is highly effective in inducing oocyte germinal vesicle breakdown (GVBD), followed by ovulation from ovarian fragments. The findings of this study provide a foundation for more in-depth molecular analysis of the reproductive neuroendocrine physiology of the COTS and the RGP.

The impact of ocean acidification on the gonads of three key Antarctic benthic macroinvertebrates.

CO2 atmospheric pressure is increasing since industrial revolution, leading to a lowering of the ocean surface water pH, a phenomenon known as ocean acidification, with several reported effects on individual species and cascading effects on marine ecosystems. Despite the great amount of literature on ocean acidification effects on calcifying organisms, the response of their reproductive system still remains poorly known. In the present study, we investigated the histopathological effects of low pH on the gonads of three key macroinvertebrates of the Terra Nova Bay (Ross Sea) littoral area: the sea urchin Sterechinus neumayeri, the sea star Odontaster validus and the scallop Adamussium colbecki. After 1 month of exposure at control (8.12) and reduced (7.8 and 7.6) pH levels, we dissected the gonads and performed histological analyses to detect potential differences among treatments. Results showed significant effects on reproductive conditions of A. colbecki and S. neumayeri, while O. validus did not show any kind of alteration. Present results reinforce the need to focus on ocean acidification effects on soft tissues, particularly the gonads, whose damage may exert large effects on the individual fitness, with cascading effects on the population dynamic of the species.

Novel pentapeptide, PALAL, derived from a bony fish elicits contraction of the muscle in starfish Patiria pectinifera.

A bioactive peptide mimicking peptide-signaling molecules has been isolated from the skin extract of fish Channa argus which caused contraction of the apical muscle of a starfish Patiria pectinifera, a deuterostomian invertebrate. The primary structure of the isolated pentapeptide comprises amino acid sequence of H-Pro-Ala-Leu-Ala-Leu-OH (PALAL) with a molecular mass of 483.7 Da. Pharmacological activity of PALAL, dosage ranging from 10-9 to 10-5 M, revealed concentration-dependent contraction of the apical muscles of P. pectinifera and Asterias amurensis. However, PALAL was not active on the intestinal smooth muscle of the goldfish Carassius auratus and has presumably other physiological roles in fish skin. Investigation of structure-activity relationship using truncated and substituted analogs of PALAL demonstrated that H-Ala-Leu-Ala-Leu-OH was necessary and should be sufficient to constrict apical muscle of P. pectinifera. Furthermore, the second alanine residue was required to display the activity, and the fifth leucine residue was responsible for its potency. Comparison with PALAL's primary structure with those of other known bioactive peptides from fish and starfish revealed that PALAL does not have any significant homology. Consequently, PALAL is a bioactive peptide that elicits a muscle contraction in starfish, and the isolation of PALAL may lead to develop other bioactive peptides sharing its similar sequence and/or activity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition.

Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K-Akt pathway through the Gßγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B-Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B-Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gßγ-dependent pathway enhances PI3K-Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gßγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries.

Lethal and sub-lethal effects of elevated CO2 concentrations on marine benthic invertebrates and fish.

Concern about leakage of carbon dioxide (CO2) from deep-sea storage in geological reservoirs is increasing because of its possible adverse effects on marine organisms locally or at nearby coastal areas both in sediment and water column. In the present study, we examined how elevated CO2 affects various intertidal epibenthic (benthic copepod), intertidal endobenthic (Manila clam and Venus clam), sub-tidal benthic (brittle starfish), and free-living (marine medaka) organisms in areas expected to be impacted by leakage. Acute lethal and sub-lethal effects were detected in the adult stage of all test organisms exposed to varying concentrations of CO2, due to the associated decline in pH (8.3 to 5.2) during 96-h exposure. However, intertidal organisms (such as benthic copepods and clams) showed remarkable resistance to elevated CO2, with the Venus clam being the most tolerant (LpH50 = 5.45). Sub-tidal species (such as brittle starfish [LpH50 = 6.16] and marine medaka [LpH50 = 5.91]) were more sensitive to elevated CO2 compared to intertidal species, possibly because they have fewer defensive capabilities. Of note, the exposure duration might regulate the degree of acute sub-lethal effects, as evidenced by the Venus clam, which showed a time-dependent effect to elevated CO2. Finally, copper was chosen as a model toxic element to find out the synergistic or antagonistic effects between ocean acidification and metal pollution. Combination of CO2 and Cu exposure enhances the adverse effects to organisms, generally supporting a synergistic effect scenario. Overall, the significant variation in the degree to which CO2 adversely affected organisms (viz., working range and strength) was clearly observed, supporting the general concept of species-dependent effects of elevated CO2.

Lime Juice and Vinegar Injections as a Cheap and Natural Alternative to Control COTS Outbreaks.

Outbreaks of the corallivorous crown-of-thorns seastar Acanthaster planci (COTS) represent one of the greatest disturbances to coral reef ecosystems in the Indo-Pacific, affecting not only coral reefs but also the coastal communities which rely on their resources. While injection approaches are increasingly used in an attempt to control COTS densities, most of them display severe drawbacks including logistical challenges, high residual environmental impacts or low cost-effectiveness. We tested a new alternative control method based upon acidic injections of cheap, 100% natural products. We investigated the lethal doses, intra- and inter-specific disease transmission and immune responses of COTS when injected with fresh lime juice (extracted from local Citrus arantifolia) and white spirit vinegar. High COTS mortality was achieved with small volumes: 10-20 ml per seastar induced death in 89%/97% of injected specimens after an average 34.3 h/29.8 h for lime juice and vinegar respectively. Highest efficiency was reached for both solutions with double shots of (2 × 10 ml) in two different areas on the body: 100% mortality occurred within 12-24 h, which is similar or faster compared with other current injection methods. Multiple immune measures suggested that death was very likely caused by pH stress from the acidic solutions rather than a bacterial infection. Contagion to either conspecifics or a variety of other reef species was not observed, even at COTS densities 15 times higher than the highest naturally reported. 10 to 20 l lime juice/vinegar could kill up to a thousand COTS at a cost of less than 0.05 USD per specimen; no permits or special handling procedures are required. We conclude that injections of lime juice and vinegar offer great advantages when compared to current best practises and constitute a cheap and natural option for all reefs affected by COTS.

Saxitoxin and the Ochre Sea Star: Molecule of Keystone Significance and a Classic Keystone Species.

Saxitoxins (STXs) are paralytic alkaloids produced by marine dinoflagellates in response to biotic and abiotic stressors yielding harmful algal blooms. Because STX impacts coastal, near-shore communities to a greater extent than would be predicted by its relative abundance, it has been referred to as a "molecule of keystone significance" in reference to Robert Paine's Keystone Species Concept. Pisaster ochraceus, the predator upon which Paine's concept was founded, inhabits waters regularly plagued by harmful algal blooms, but the effects of STX on Pisaster have not yet been investigated. Here, we used laboratory and field experiments to examine the potential consequences of exposure to STX on sea stars' feeding, attachment to the substrate, and success in fertilization. Pisaster exhibited similar feeding behaviors when offered non-toxic prey, STX-containing prey, or a combination of the two. Although feeding behavior is unaffected, consumption of STX poses a physiological tradeoff. Sea stars in the laboratory and field had significantly lower thresholds of the force needed to detach them from their substrates after either being exposed to, or consuming, STX. High pressure (or high performance) liquid chromatography analysis indicated an accumulation of STX (and structural analogues) in sea stars' viscera, likely due to trophic transfer from toxic prey. Incidence of fertilization tended to decrease when gametes were exposed to high, yet ecologically relevant, STX concentrations of STX. These findings suggest that the molecule of keystone significance, STX, produced during harmful algal blooms extends its impacts to rocky intertidal communities by way of the keystone predator P. ochraceus.

Larval starvation to satiation: influence of nutrient regime on the success of Acanthaster planci.

High density populations of the crown-of-thorns seastar, Acanthaster planci, are a major contributor to the decline of coral reefs, however the causes behind periodic outbreaks of this species are not understood. The enhanced nutrients hypothesis posits that pulses of enhanced larval food in eutrophic waters facilitate metamorphic success with a flow-on effect for population growth. The larval resilience hypothesis suggests that A. planci larvae naturally thrive in tropical oligotrophic waters. Both hypotheses remain to be tested empirically. We raised A. planci larvae in a range of food regimes from starvation (no food) to satiation (excess food). Algal cell concentration and chlorophyll levels were used to reflect phytoplankton conditions in nature for oligotrophic waters (0-100 cells ml(-1); 0-0.01 µg chl a L(-1)), natural background levels of nutrients on the Great Barrier Reef (GBR) (1,000-10,000 cells ml(-1); 0.1-1.0 µg chl a L(-1)), and enhanced eutrophic conditions following runoff events (100,000 cells ml(-1); 10 µg chl a L(-1)). We determine how these food levels affected larval growth and survival, and the metamorphic link between larval experience and juvenile quality (size) in experiments where food ration per larvae was carefully controlled. Phytoplankton levels of 1 µg chl a L(-1), close to background levels for some reefs on the GBR and following flood events, were optimal for larval success. Development was less successful above and below this food treatment. Enhanced larval performance at 1 µg chl a L(-1) provides empirical support for the enhanced nutrients hypothesis, but up to a limit, and emphasizes the need for appropriate mitigation strategies to reduce eutrophication and the consequent risk of A. planci outbreaks.

Antibody inhibition of protein activity in starfish oocytes.

Antibodies are widely utilized in cell and molecule biology for immunoblots, immunostaining, immunoprecipitation, immunoaffinity purification, and immunoassay. Some antibodies can be used for in vivo inhibition experiments. These antibodies bind to their target molecules and neutralize their functions, providing functional information in the study of their biological role. Here, we describe our methods for obtaining inhibitory antibodies against desired proteins. We then describe in the starfish oocyte system how to inhibit a target protein, even in the nucleus, by injection of antibody into the cytoplasm, and how to evaluate antibody inhibition of cell cycle regulators in small numbers of oocytes.

Impacts of ocean acidification on early life-history stages and settlement of the coral-eating sea star Acanthaster planci.

Coral reefs are marine biodiversity hotspots, but their existence is threatened by global change and local pressures such as land-runoff and overfishing. Population explosions of coral-eating crown of thorns sea stars (COTS) are a major contributor to recent decline in coral cover on the Great Barrier Reef. Here, we investigate how projected near-future ocean acidification (OA) conditions can affect early life history stages of COTS, by investigating important milestones including sperm motility, fertilisation rates, and larval development and settlement. OA (increased pCO2 to 900-1200 µatm pCO2) significantly reduced sperm motility and, to a lesser extent, velocity, which strongly reduced fertilization rates at environmentally relevant sperm concentrations. Normal development of 10 d old larvae was significantly lower under elevated pCO2 but larval size was not significantly different between treatments. Settlement of COTS larvae was significantly reduced on crustose coralline algae (known settlement inducers of COTS) that had been exposed to OA conditions for 85 d prior to settlement assays. Effect size analyses illustrated that reduced settlement may be the largest bottleneck for overall juvenile production. Results indicate that reductions in fertilisation and settlement success alone would reduce COTS population replenishment by over 50%. However, it is unlikely that this effect is sufficient to provide respite for corals from other negative anthropogenic impacts and direct stress from OA and warming on corals.

Lethal doses of oxbile, peptones and thiosulfate-citrate-bile-sucrose agar (TCBS) for Acanthaster planci; exploring alternative population control options.

Effective control of outbreaks of Acanthaster planci represents the most immediate and practical intervention to reverse sustained declines in coral cover on reefs in the Indo-Pacific. This study explored the minimum doses of oxbile, oxgall, and thiosulfate-citrate-bile-sucrose agar (TCBS) that result in reliable and comprehensive mortality when injected into adult A. planci. The minimum doses required to induce 100% mortality among starfish (n=10) were 4 g l(-1) of oxbile, 8 g l(-1) of oxgall and 22 g l(-1) of TCBS. Moreover, there was no evidence of unintended side effects for other coral reef organisms (e.g., scleractinian corals, echinoderms and fishes) when using oxbile, oxgall, or TCBS at minimum doses. The effectiveness of peptones in killing crown-of-thorns starfish was also tested, but inconsistency in the results revealed that these proteins are unreliable.

Petroacetylene, a new polyacetylene from the marine sponge Petrosiasolida that inhibits blastulation of starfish embryos.

A new C30 linear polyacetylene compound designated petroacetylene (1) has been isolated from the marine sponge Petrosia solida Hoshino 1981, collected off the coast of Amami-Oshima, Kagoshima Prefecture, Japan. The structure was elucidated on the basis of spectroscopic data and chemical means. Petroacetylene (1) inhibited blastulation of starfish embryos at a concentration of 3.1 µg mL(- 1) or greater.

Bromotheoynic acid, a brominated acetylenic acid from the marine sponge Theonella swinhoei.

A new brominated C(17) acetylenic acid (1) designated as bromotheoynic acid has been isolated from the marine sponge Theonella swinhoei, collected off the coast of Tanegashima, Kagoshima Prefecture, Japan. The structure was determined on the basis of the analysis of its extensive 2D NMR spectroscopic data as well as HRMS. Bromotheoynic acid (1) inhibited maturation of starfish oocytes and cell division of fertilised starfish eggs. Bromotheoynic acid (1) also inhibited proliferation of human leukaemia U937 and HL60 cells, human lung cancer A549 and H1299 cells, and human embryonic kidney 293 (HEK293) cells.

Interspecific transmission and recovery of TCBS-induced disease between Acanthaster planci and Linckia guildingi.

The susceptibility of the coral-feeding crown-of-thorns starfish Acanthaster planci to disease may provide an avenue with which to effectively control population outbreaks that have caused severe and widespread coral loss in the Indo-Pacific. Injecting thiosulfate-citrate-bile-sucrose (TCBS) agar into A. planci tissues induced a disease characterized by dermal lesions, loss of skin turgor, collapsed spines, and accumulation of mucus on spine tips. Moreover, the symptoms (and presumably the agent) of this disease would spread rapidly intraspecifically, but interspecific transmission (to other species of echinoderms) is yet to be examined. Vibrio rotiferianus, which was previously reported as a pathogen isolated from lesions of experimentally infected A. planci, was also recovered from Linckia guildingi lesions after several days of direct contact with diseased A. planci, demonstrating disease transmission. However, all L. guildingi fully recovered after 31 ± 16 d. Further studies are in progress to understand the ecology of Vibrio infection in A. planci and the potential transmission risk to corals, fishes, and other echinoderms to evaluate whether injections of TCBS could be a viable tool for controlling A. planci outbreaks.

Chemo-ecological studies on hexactinellid sponges from the Southern Ocean.

Hexactinellids (glass sponges) are an understudied class with syncytial organization and poor procariotic associations, thought to lack defensive secondary metabolites. Poriferans, though, are outstanding sources of bioactive compounds; nonetheless, a growing suspicion suggests that many of these chemicals could be symbiont-derived. In Polar latitudes, sponges are readily invaded by diatoms, which could provide natural products. Hexactinellids are typical of deep waters; but in Antarctica, they dominate the upper shelf providing shelter and food supply to many opportunistic mesograzers and macroinvertebrates, which exert strong ecological pressures on them. Aiming to examine the incidence of defensive activities of hexactinellids against consumption, feeding experiments were conducted using their lipophilic fractions. Antarctic hexactinellid and demosponge extracts were tested against the asteroid Odontaster validus and the amphipod Cheirimedon femoratus as putative sympatric, omnivorous consumers. Hexactinellids yielded greater unpalatable activities towards the amphipod, while no apparent allocation of lipophilic defenses was noted. After chemical analyses on the lipophilic fractions from these Antarctic glass sponges, quite similar profiles were revealed, and no peculiar secondary metabolites, comparable to those characterizing other poriferans, were found. Instead, the lipidic compounds 5α(H)-cholestan-3-one and two glycoceramides were isolated for their particular outspread presence in our samples. The isolated compounds were further assessed in asteroid feeding assays, and their occurrence was evaluated for chemotaxonomical purposes in all the Antarctic samples as well as in glass sponges from other latitudes by NMR and MS. Characteristic sphingolipids are proposed as chemical markers in Hexactinellida, with possible contributions to the classification of this unsettled class.

Retrospective qualitative analysis of ecological networks under environmental perturbation: a copper-polluted intertidal community as a case study.

The coast of Chañaral Bay in northern Chile has been affected by copper mine wastes for decades. This sustained perturbation has disrupted the intertidal community in several ways, but the mechanisms behind the observed shifts in local biodiversity remain poorly understood. Our main goal was to identify the species (lumped into trophic groups) belonging to the Chañaral intertidal community that, being directly affected by copper pollution, contributed primarily to the generation of the observed changes in community structure. These groups of species were called initiators. We applied a qualitative modelling approach based only on the sign and direction of effects among species, and present a formula for predicting changes in equilibrium abundances considering stress on multiple variables simultaneously. We then applied this technique retrospectively to identify the most likely set of initiators. Our analyses allowed identification of a unique set of four initiators in the studied intertidal system (a group of algae, sessile invertebrates, a group of herbivores and starfish), which were hypothesized to be the primary drivers of the observed changes in community structure. In addition, a hypothesis was derived about how the perturbation affected these initiators. The hypothesis is that pollution affected negatively the population growth rate of both algae and sessile invertebrates and suppressed the interaction between herbivores and starfish. Our analytic approach, focused on identifying initiators, constitutes an advance towards understanding the mechanisms underlying human-driven ecosystem disruption and permits identifying species that may serve as a focal point for community management and restoration.

Two-stage dependence for 1-methyladenine induced reinitiation of meiotic maturation in starfish oocytes.

The maturation hormone 1-methyladenine (1-MA) causes meiotic resumption of prophase arrested immature starfish oocytes. Continuous exposure to > or = 0.5 microM 1-MA causes germinal vesicle breakdown (GVBD) in approximately 20 min, but oocytes pretreated for > 30 min with a subthreshold dose of 1-MA undergo GVBD much faster (approximately 10 min) when they are exposed to 1 microM 1-MA. Furthermore, a very low subthreshold 1-MA suffices to start the maturation process: oocytes exposed to 0.005 microM 1-MA for up to 10 min followed by 1 microM 1-MA is equivalent to continuous exposure to 1 microM 1-MA. These dose and timing relationships indicate that there is a two-stage dependence on 1-MA. A possible explanation for this dependence is that there are two processes involved: an initial process that is triggered by a low dose of 1-MA, and a second process that cannot start until the first process is completed and is stimulated by a higher dose of 1-MA. These subthreshold 1-MA effects on GVBD timing are not directly coupled to changes in calcium physiology that also occur during maturation. Subthreshold 1-MA was found to cause a transient accumulation of Cdc2/cyclin B into the nucleus. The two-stage dependence indicates that there are unsuspected features in this well-studied pathway leading to GVBD. In the animal, this hormone dependence may help to synchronize maturation throughout all parts of the ovary.