Reproduction In Molluscs Research Articles

Micro-CT screening of old shell collections helps to understand the distribution of viviparity in the highly diversified clausiliid clade of land snails

Current zoological research may benefit in many ways from the study of old collections of shells. These collections may provide materials for the verification of broad zoogeographical and ecological hypotheses on the reproduction of molluscs, as they include records from many areas where sampling is currently impossible or very difficult due to political circumstances. In the present paper we present data on viviparous and embryo-retention reproductive modes in clausiliid land snails (subfamily Phaedusinae) acquired from specimens collected since the nineteenth century in the Pontic, Hyrcanian, and East and Southeast Asian regions. X-ray imaging (micro-CT) enabled relatively quick screening of more than 1,000 individuals classified within 141 taxa, among which we discovered 205 shells containing embryos or eggs. Gravid individuals were found to belong to 55 species, representing, for some of these species, the first indication of brooding reproductive strategy.

Neuropeptides encoded within a neural transcriptome of the giant triton snail Charonia tritonis, a Crown-of-Thorns Starfish predator

Neuropeptides represent a diverse class of signaling molecules originating from neural tissues. These chemical modulators orchestrate complex physiological events including those associated with growth and reproduction. De novo transcriptome sequencing of a cerebral ganglion library of the endangered giant triton snail (Charonia tritonis) was undertaken in an effort to identify key neuropeptides that control or influence its physiology. The giant triton snail is considered a primary predator of the corallivore Acanthaster planci (Crown-of-Thorns Starfish) that is responsible for a significant loss in coral cover on reefs in the Indo-Pacific. The transcriptome library was assembled into contigs, and then bioinformatic analysis was used to identify a repertoire of 38 giant triton snail neuropeptide precursor genes, and various isoforms, that encode conserved molluscan neuropeptides. C. tritonis neuropeptides show overall precursor organisation consistent with those of other molluscs. These include those neuropeptides associated with mollusc reproduction such as the APGWamide, buccalin, conopressin, gonadotropin-releasing hormone (GnRH), NKY and egg-laying hormone. These data provide a foundation for further studies targeted towards the functional characterisation of neuropeptides to further understand aspects of the biology of the giant triton snail, such as elucidating its reproductive neuroendocrine pathway to allow the development of knowledge based captive breeding programs.

Spermatozeugmata structure and dissociation of the Australian flat oyster Ostera angasi: Implications for reproductive strategy.

Variation in reproductive strategy is one of the key factors contributing to recruitment success of molluscs in different habitats. Spermcasting is a unique mode in mollusc reproduction where males produce spermatozeugmata, a radially arrayed sperm cluster wrapped by gelatinous membrane. In this study, spermatozeugmata structure and their dissociation in the Australian flat oyster Ostrea angasi were investigated to elucidate the reproductive strategy in spermcasting molluscs. The histological observation indicated that spermatogonia gradually aggregated in the gonad follicle at the early gonad development stages and developed into spermatozeugmata and became tightly packed at the advanced stages. Even though mature male and female gametes could be found in a hermaphroditic individual, the animal may prevent self-fertilization by shedding different sex gametes at different time. The O. angasi sperm are similar in size and shape to broadcasting oysters, but have one additional mitochondrion. Variations in maintaining spermatozeugmata integrity and sperm motility between individuals depended on the level of masculinity or femineity. The durations of spermatozeugmata dissociation and sperm viability were longer in males than in hermaphrodites. The unique structure and capability for spermatozeugmata to maintain the functional integrity after spawning have adaptive significance for fertilization and gamete dispersal in this species.

Characterisation of two conopressin precursor isoforms in the land snail, Theba pisana

Increased understanding of the molecular components involved in mollusc reproduction may assist in understanding the evolutionary adaptations used by animals, including hermaphrodites, to produce offspring. The neuropeptide conopressin, a member of the vasopressin/oxytocin-like peptide family, can modulate various reproductive activities in invertebrates. In this study, we used the hermaphroditic land snail, Theba pisana, to investigate the presence and tissue-specific distribution of a conopressin gene. Our transcriptomic analysis of T. pisana CNS sheath tissue has revealed two conopressin gene transcripts (Tpi-conopressin-1 and Tpi-conopressin-2), each encoding for precursors containing an identical conopressin nonapeptide and a variable neurophysin. T. pisana conopressins share high identity with other land snails and slugs, as well as other mollusc and vertebrate vasopressin/oxytocin, supported by phylogenetic analysis. Conserved residues in the T. pisana neurophysin are important for peptide binding, and we present molecular dynamic models demonstrating the most likely stable structure of the Tpi-conopressin-1 peptide when associated with neurophysin. RT-PCR shows that Tpi-conopressin-1 is additionally expressed in reproductive tissues, including the dart sac, where abundant spatial expression throughout the sac region is found; this implies a role in ‘love’ dart synthesis or dart injection during mating. The presence of a conopressin receptor in the CNS sheath indicates CNS neural excitation. In summary, this study represents a detailed molecular analysis of conopressin in a land snail.

Characteristics of 17β-hydroxysteroid dehydrogenase 8 and its potential role in gonad of Zhikong scallop Chlamys farreri

17β-Hydroxysteroid dehydrogenases (17β-HSDs) are important enzymes catalyzing steroids biosynthesis and metabolism in vertebrates. Although studies indicate steroids play a potential role in reproduction of molluscs, little is known about the presence and function of 17β-HSDs in molluscs. In the present study, a full-length cDNA encoding 17β-HSD type 8 (17β-HSD8) was identified in the Zhikong scallop Chlamys farreri, which is 1104bp in length with an open reading frame of 759bp encoding a protein of 252 amino acids. Phylogenetic analysis revealed that the C. farreri 17β-HSD8 (Cf-17β-HSD8) belongs to the short chain dehydrogenase/reductase family (SDR) and shares high homology with other 17β-HSD8 homologues. Catalytic activity assay in vitro demonstrated that the refolded Cf-17β-HSD8 expressed in Escherichia coli could effectively convert estradiol-17β (E2) to estrone (E1), and weakly catalyze the conversion of testosterone (T) to androstenedione (A) in the presence of NAD+. The Cf-17β-HSD8 mRNA was ubiquitously expressed in all tissues analyzed, including gonads. The expression levels of Cf-17β-HSD8 mRNA and protein increased with gametogenesis in both ovary and testis, and were significantly higher in testis than in ovary at growing stage and mature stage. Moreover, results of in situ hybridization and immunohistochemistry revealed that the mRNA and protein of Cf-17β-HSD8 were expressed in follicle cells and gametes at all stages except spermatozoa. Our findings suggest that Cf-17β-HSD8 may play an important role in regulating gametogenesis through modulating E2 levels in gonad of C. farreri.

Proteomic Analysis of the Reproductive Organs of the Hermaphroditic Gastropod Lymnaea stagnalis Exposed to Different Endocrine Disrupting Chemicals

Many studies have reported perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of endocrine disrupting chemicals (EDCs). However, the mechanisms of action of these molecules on molluscs are still poorly understood. Investigation of the modifications of protein expression in organisms exposed to chemicals using proteomic methods can provide a broader and more comprehensive understanding of adverse impacts of pollution on organisms than conventional biochemical biomarkers (e.g., heat-shock proteins, metallothioneins, GST, EROD). In this study we have investigated the impacts of four chemicals, which exhibit different endocrine disrupting properties in vertebrates, on the proteome of the hermaphroditic freshwater pulmonate gastropod Lymnaea stagnalis after 21 days of exposure. Testosterone, tributyltin, chlordecone and cyproterone acetate were chosen as tested compounds as they can induce adverse effects on the reproduction of this snail. The 2D-DIGE method was used to identify proteins whose expression was affected by these compounds. In addition to modifying the expression of proteins involved in the structure and function of the cytoskeleton, chemicals had impacts on the expression of proteins involved in the reproduction of L. stagnalis. Exposure to 19.2 µg/L of chlordecone increased the abundance of ovipostatin, a peptide transmitted during mating through seminal fluid, which reduces oviposition in this species. The expression of yolk ferritin, the vitellogenin equivalent in L. stagnalis, was reduced after exposure to 94.2 ng Sn/L of tributyltin. The identification of yolk ferritin and the modification of its expression in snails exposed to chemicals were refined using western blot analysis. Our results showed that the tested compounds influenced the abundance of yolk ferritin in the reproductive organs. Alteration in proteins involved in reproductive pathways (e.g., ovipostatin and yolk ferritin) could constitute relevant evidence of interaction of EDCs with reproductive pathways that are under the control of the endocrine system of L. stagnalis.

Effects of test media on reproduction in Potamopyrgus antipodarum and of pre-exposure population densities on sensitivity to cadmium in a reproduction test

Molluscan species can be affected by various anthropogenic substances. Yet, these effects are disregarded in chemical risk assessment as molluscs are unrepresented in standard OECD guidelines. The project “validation of a mollusc reproduction test” (Federal Environment Agency, code 371165417) deals with the development of a test method with the mudsnail Potamopyrgus antipodarum for OECD purposes. In this context, the influence on reproduction of both, different media and varying snail density, has been observed in independent experiments. Further, the impact of density on the outcome of subsequent cadmium (Cd) toxicity in a test has been investigated to refine the existing methodology. First, adult snails were kept in different test media for 12 weeks. Second, snail density was increased for 4 weeks to induce stress. Snails from each density scenario were used for another 4 weeks in a reproduction test at an equal density with 12 μg Cd/L, respectively. Significant differences in reproduction between medium groups were noted after 4 and 8, but not 12, weeks. Further, reproduction was significantly altered by snail density in the beakers but after subsequent 4 weeks at a constant density, no differences were observed between control groups. Cd reduced reproduction and this effect increased with snail density in the pre-exposure period, demonstrating that a previous stress factor may result in increased sensitivity to chemicals and underlines the need for more standardized breeding conditions to minimize effect variations. Based on the outcome of this study, an acclimatization period of 12 weeks must be guaranteed for specimens transferred to another medium. Further, 4 weeks of acclimatization are necessary after density stress. An additional 12 weeks density experiment showed that medium volume in each replicate can be decreased by half to save on chemicals, water and space during tests.

Influence of historical exploitation and recovery of biological resources on contemporary status of Margaritifera margaritifera L. and Salmo salar L. populations in Northwestern Russia

The influence of the historical exploitiation and recovery of biological resources on populations of freshwater pearl mussel and salmon was studied in the Kozha river (Onega River Basin). The freshwater pearl mussel was characterized by a low population density (0.09 ind./m2, on the average) and number of recruits. The role of the anthropogenic factor in the population is negligible; the effect of the pearl fishing is not observed. The dramatic decrease in the number of Atlantic salmon in the Onega River Basin in the late 20th century is a major reason for the slowed down reproduction of the freshwater pearl mussel. The second factor is the reconstruction of the Onega salmon hatchery in 1984, which led to a decrease in the juvenile salmon population density below the critical level on spawning and nursery grounds. As a result, mollusc reproduction has stopped. An artificial increase in the number of recruits must be developed in order to preserve the salmon population in the Onega River Basin. It is also necessary to forbid salmon fishing, including fishing migratory paths (with the exception of trapping for hatchery needs and traditional harvesting performed by locals in historical settlements).

Characterization of mucus‐associated proteins from abalone (Haliotis) – candidates for chemical signaling

Living in groups is a widespread phenomenon in the animal kingdom. For free-spawning aquatic animals, such as the abalone (Haliotis), being in the close proximity to potential mating partners enhances reproductive success. In this study, we investigated whether chemical cues could be present in abalone mucus that enable species-specific aggregation. A comparative MS analysis of mucus obtained from trailing or fixed stationary Haliotis asinina, and from seawater surrounding aggregations, indicated that water-soluble biomolecules are present and that these can stimulate sensory activity in conspecifics. Purified extracts of trail mucus contain at least three small proteins [termed H. asinina mucus-associated proteins (Has-MAPs)-1-3], which readily diffuse into the surrounding seawater and evoke a robust cephalic tentacle response in conspecifics. Mature Has-MAP-1 is approximately 9.9 kDa in size, and has a glycine-rich N-terminal region. Has-MAP-2 is approximately 6.2 kDa in size, and has similarities to schistosomin, a protein that is known to play a role in mollusc reproduction. The mature Has-MAP-3 is approximately 12.5 kDa in size, and could only be identified within trail mucus of animals outside of the reproductive season. All three Has-MAP genes are expressed at high levels within secretory cells of the juvenile abalone posterior pedal gland, consistent with a role in scent marking. We infer from these results that abalone mucus-associated proteins are candidate chemical cues that could provide informational cues to conspecifics living in close proximity and, given their apparent stability and hydrophilicity, animals further afield.

Late Palaeozoic mollusc reproduction: cephalopod egg‐laying behavior and gastropod larval palaeobiology

Faunal analysis of an oxygen-depleted marine Lower Carboniferous succession (Late Mississippian Ruddle Shale) suggests how some cephalopod taxa laid their eggs during the Late Palaeozoic. At the Ruddle Shale collecting site in Arkansas, USA, the facies and overall fauna suggest severe oxygen depletion at the sediment/water interface. The Ammonoidea, with their small egg size, were probably laid in suspended gelatinous egg-filled masses in the water column above the bottom or by attachment of the egg masses to floating debris. The ammonitella embryos developed within the suspended or attached egg mass; hatched individuals became part of the free-swimming plankton biota. Based on shell morphology the Bactritoidea probably followed the same reproductive pattern. Coiled nautiloids (the Nautilida) and most orthoconic nautiloids (mostly the Pseudorthocerida) probably did not lay their eggs in the mid water column or as floatant attachments. This conclusion is based on the fact that, with one exception, all shells recovered of these two nautiloid orders are well past hatching. Gastropods in the Ruddle Shale are very small and cannot be visually detected in the field. However, microgastropods are abundant in washed residues. Most specimens are much smaller than 1 mm. The largest caenogastropod specimen is 1.3 mm high. These caenogastropods represent isolated larval shells and a successful metamorphosis was impossible because of oxygen depletion on the bottom. Allegations that a size of more than 1 mm is too large for pelagic larvae are refuted by examples of planktotrophic larval shells of modern gastropods (more than 1 mm high) and Triassic caenogastropods (up to 2 mm high) from the Cassian Formation (Northern Italy, South Alps). Repository information is given for the type-material of the gastropod species Nuetzelina striata Bandel, 2002 and Anozyga arkansasensis Bandel, 2002 which were both erected based on specimens from the Ruddle Shale that were illustrated by Nutzel & Mapes in 2001.

The estrogen receptor of the gastropod Nucella lapillus: Modulation following exposure to an estrogenic effluent?

The molecular targets of estrogenic endocrine disrupting chemicals have been studied in detail in vertebrates. The lack of basic endocrine knowledge impairs similar approaches for invertebrates. Evidence indicates that the signalling pathways of invertebrates may also be a target of estrogenic chemicals (ECs). In fact, the exposure to effluents containing ECs has been reported to impact mollusc reproduction. Despite the reported estrogen independence of the mollusc nuclear estrogen receptor (ER), its role in EC-induced toxicity has not been investigated in vivo. Therefore, we have cloned the ER of the gastropod Nucella lapillus and evaluated the effects of a mixture of estrogenic chemicals (sewage effluent) on its expression in the ovary. Here, we show that the exposure to a raw domestic/industrial effluent, impact ER expression with a simultaneous reproductive maturation. These results highlight the need to further investigate the role of ER on the reproductive process in prosobranch gastropods and whether this signalling pathway is prone to disruption by ECs.

Purification of a lipid-activated and Ca2+-independent protein kinase from the mantle tissue of Mytilus galloprovincialis Lmk.

A phospholipid-sensitive Ca2+-independent protein kinase (p105) was purified to homogeneity from mantle tissue of the mussel Mytilus galloprovincialis Lmk., employing consecutively DE-52 cellulose, Sephacryl S-200 and Biogel HTP chromatographies. The purified enzyme appeared as a single band on 10% SDS-PAGE, and had a molecular weight of 105 kDa. The positive Western blotting of the purified eluate for anti-human-PKCdelta and PKCepsilon suggests that the enzyme from mussel mantle may be an ancestral nPKC isoform, with the kinetic properties of the enzyme very close to those of PKCepsilon isoform of vertebrates. Western blotting of samples from different steps of purification using specific mouse anti-p105, showed two protein bands in samples from the initial steps. However, only one band was detected in the Biogel-HTP eluate, the most purified fraction. The purification steps did not affect the presence of P-serine in p105. No P-tyrosine peptides were detected in any of the purification steps. These results open a new field of work on the study of several molecular processes related to energetic metabolism and reproduction in molluscs, whose regulation is associated with the activation of protein kinases.

Localization of the Neuropeptide APGWamide in Gastropod Molluscs byin SituHybridization and Immunocytochemistry

The amidated tetrapeptide Ala-Pro-Gly-Trp-NH2(APGWamide) plays a key role in the control of male copulation behavior in the basommatophoran pulmonate freshwater snailLymnaea stagnalis.The morphological basis for a conserved role of APGWamide in the control of male reproduction in gastropod molluscs is presented. The prosobranchLittorina littorea,the opisthobranchAplysia californica,the basommatophoran pulmonateBulinus truncatus,and the stylommatophoran pulmonatesArion aterandLimax maximushave been examined for the presence of APGWamide producing neurons using immunocytochemistry andin situhybridization. In all species investigated a cluster of APGWamide expressing neurons is present in the anteromedial region of the cerebral ganglia. The asymmetrical distribution which exists inLymnaeaand which coincides with the innervation of the asymmetrically located penial complex is also found in the opisthobranchAplysia,as well as in the stylommatophoran pulmonate slugsArionandLimax,in which APGWamide immunoreactive neurons are only found in the mesocerebrum of the right cerebral ganglion. APGWamide immunoreactive varicose fibers innervate muscles of the male accessory sex organs inBulinusandAplysia,confirming the hypothesis that APGWamide may be a biochemically and functionally conserved factor in the regulation of gastropod mollusc reproduction.

Characterization of central neurons in bivalves using antibodies raised against neuropeptides involved in gastropod egg-laying behavior

Summary Immunohistochemistry was used to characterize central neurons which react to antibodies raised against neuropeptides known to control reproduction in other molluscs. Antibodies raised against αCDCP (α caudodorsal cell peptide) and CDCH (caudodorsal cell hormone), peptides which control ovulation in the pulmonate gastropod Lymnaea, labelled numerous neurons in Mytilus, Mya and Placopecten. The labelled neurons in the cerebral and visceral ganglia of Mytilus are consistent with descriptions of neurons implicated in the control of reproduction on the bases of earlier neurosecretory staining procedures. This study thus suggests that related peptides might be involved in the reproduction of gastropods and bivalve molluscs. The use of selective immunological markers for peptides which might control bivalve reproduction not only permits the tentative identification of neurosecretory cells in bivalves but also suggests a promising avenue for future research aimed at isolating and characterizing neuropeptides involved in the control of reproduction in these molluscs.

Biotechnology in marine aquaculture

Intensification of production, modernization of technique, and improvement of cultivars are needed to increase the economic efficiency of marine aquaculture industries. To help achieve such improvements, modern methods of biochemical and genetic engineering can be adapted to control biological processes which intrinsically limit the efficiency and yield of production. Such applications now have been developed for improved production of molluscs. Critical life-cycle stages of several species of abalones and other commercially valuable molluscs which thus far have proved amenable to improved control by these means include reproduction, larval settlement, metamorphosis, and acceleration of early growth. Analysis of the physiological and molecular mechanisms which control reproduction in molluscs reveals a prostaglandin-dependent regulation of spawning in abalones and certain other species. Spawning of gravid adults can be induced by the addition of prostaglandins to the surrounding seawater, or, more reliably and inexpensively, by activation of the endogenous enzymatic synthesis of prostaglandin-related spawning triggers in response to added hydrogen peroxide. Peroxide activation of the prostaglandin-dependent spawning reaction has been found widely useful for obtaining synchronous and copious release of fully competent gametes in a large number of species of abalones, oysters, scallops, mussels, clams, and other valuable molluscs; a number of these species had not been successfully spawned by other methods. Settlement and metamorphosis of many molluscan species can be brought under similar biochemical control. Most efficiently and inexpensively, the natural biochemical requirement for induction of these critical developmental processes can be met by providing competent larvae with unique amino acid constituents associated with the naturally required inducing molecules. Thus, γ-aminoburyric acid (GABA) can be used simply, safely and inexpensively to induce complete and rapid larval settlement and metamorphosis — with minimal mortality — in many commercially important abalone species. This and similar neurotransmitter-related, amino-acid-derived compounds are proving comparably effective for the reliable induction of settlement and metamorphosis in a number of other valuable molluscan species. Recent analyses of the requirements for rapid early juvenile growth in abalones reveals a mechanism of growth-control which is susceptible to exogenous hormonal acceleration. Genetic cloning and amplification of recombinant-DNA templates for the efficient production of essential growth-regulating hormones, and substances required for enhanced nutritional efficiency and resistance of cultivars, are now in progress. Genetic engineering for the direct improvement of cultivated species soon will be within reach.

Biochemical and genetic engineering for improved production of abalones and other valuable molluscs

Intensification of production, modernization of technique, and improvement of cultivars are needed to increase the economic efficiency of molluscan cultivation industries in the United States and certain other countries. To help achieve such improvements, modern methods of biochemical and genetic engineering can be adapted to control biological processes which intrinsically limit the efficiency and yield of molluscan production. Critical life-cycle stages of several species of abalones and other commercially valuable molluscs which thus far have proved amenable to improved control by these means include reproduction, larval settlement, metamorphosis, and acceleration of early growth. Analysis of the physiological and molecular mechanisms that control reproduction in molluscs reveals a prostaglandin-dependent regulation of spawning in abalones and certain other species. Spawning of gravid adults can be induced by the addition of prostaglandins to the surrounding seawater, or, more reliably and inexpensively, by activation of the endogenous enzymatic synthesis of prostaglandin-related spawning triggers in response to added hydrogen peroxide. Peroxide activation of the prostaglandin-dependent spawning reaction has been found widely useful for obtaining synchronous and copious release of fully competent gametes in a large number of species of abalones, oysters, scallops, mussels, clams, and other valuable molluses; a number of these species had not been successfully spawned by other methods. Settlement and metamorphosis of many species can be brought under similar biochemical control. The efficient induction of settlement and metamorphosis of the larvae of many Haliotis (abalone) species normally requires contact with any of several crustose red algae usually associated with natural recruiting substrates. This requirement can be met by providing competent larvae with the essential algae or with specific proteins purified from these algae, although production of these inducers is both time consuming and costly. Most conveniently and inexpensively, this natural requirement can be met by providing the larvae with certain unique amino acid constituents normally associated with the inducing proteins. Thus, γ-aminobutyric acid (GABA) can be used simply, safely, and inexpensively to induce complete and rapid larval settlement and metamorphosis — with minimal mortality — in many commercially important abalone species. This and similar neurotransmitter-related, amino acid-derived compounds are proving comparably effective for the reliable induction of settlement and metamorphosis in a number of other valuable molluscan species. Recent analyses of the requirements for rapid early juvenile growth in abalones reveals a mechanism of growth control which is susceptible to exogenous hormonal acceleration. Present experiments aimed at genetic cloning and amplification of recombinant-DNA templates for the essential growth-regulating hormones, progress in the establishment of useful molluscan gene banks, and potential uses of this technology for molluscan aquaculture are described.

Endocrinology of reproduction in molluscs

Among molluscs the optic glands of the cephalopods and the dorsal bodies of the gastropods are endocrine organs which are involved in the regulation of reproductive processes. The optic glands stimulate the differentiation of the sex cells, spermatogenesis and vitellogenesis, and the growth of the female tract. The male tract is controlled by these glands via the testis. The dorsal bodies stimulate vitellogenesis, and the growth and differentiation of the reproductive tract. In hermaphrodite gastropods the differentiation of male sex cells is dependent on the presence of a factor produced by the cerebral ganglia, whereas the female cells show autodifferentiation. Data on the role of neurosecretory cells in the reproduction of molluscs are scarce. The neurosecretory bag cells of the abdominal ganglion in Aplysia are known to stimulate ovulation. There are some indications that the optic tentacles play a part in regulating reproduction, but the mechanism is, as yet, not clear.