System Of Molluscs Research Articles

Optimising flow-cytometry methods for marine mollusc haemocyte analyses using the pearl oyster Pinctada maxima as a model

Flow-cytometry has become increasingly popular to assess the haemocytes morphology and functions of marine molluscs. Indeed, haemocytes are the first line of defence of the immune system in molluscs and are used as a proxy for oyster health. Authors publishing in the field of flow-cytometry and molluscs health seemed to utilise the same methods for all model species used, independently of their geographical location in the world (temperate, tropical, etc.). Hence, this paper dived into flow-cytometry methodology and investigated if using different plates, different thresholds, different incubation times and temperatures as well as different fluorochromes concentrations affected the results. This study revealed that the cell count did not change when using different thresholds on the FSC-H parameter of the instrument but was affected by the plate type, the temperature of incubation, and the time of incubation. Indeed, non-adherent plates yielded the highest cell count and lower cell counts were associated with a higher temperature and a longer time of incubation. Furthermore, the haemocytes functions such as the phagocytosis, the lysosomal content, the intracellular oxidative activity, and the mitochondria activity were also affected by the temperature and the time of incubation. An increase in the phagocytosis capacity, lysosomal content and mitochondria activity was observed with a higher temperature. At the exception of the phagocytosis rate, all the other parameters such as the phagocytosis capacity, the intracellular oxidative activity, and the lysosomal content increased with a longer incubation time. We also showed that it is best to optimise the amount of fluorochromes used to avoid unnecessary background or non-specific staining.

The primitive complement system in molluscs

The complement system is an important immune defense mechanism that plays essential roles in both innate and adaptive immunity of vertebrates. Since complement components are identified in deuterostome and even primitive protostome species, the origin and evolution of complement system in invertebrates have been of great interest. Recently, research on the complement system in mollusc immunity has been increasing due to their importance in worldwide aquaculture, and their phylogenetic position. Complement components including C3, C1q domain containing protein (C1qDCP), C-type lectin (CTL), ficolin-like, mannose-binding lectin (MBL)-associated serine proteases like (MASPL), and factor B have been identified, suggesting the existence of complement system in molluscs. The lectin pathway has been outlined in molluscs, which is initiated by CTL with CCP domain and MASPL protein to generate C3 cleavage fragments. The molluscan C1qDCP exhibits the capability to bind human IgG, indicating the existence of possible C1qDCP-mediated activation pathway in molluscs. The activation of C3 regulates the expressions of immune effectors (cytokines and antibacterial peptides), mediates the haemocyte phagocytosis, and inhibits the bacterial growth. Some MACPF domain containing proteins may replace the missing terminal pathway in molluscs. This article provides a review of complement system in molluscs, including its components, activation mechanisms and functions in the immune response of molluscs.

Precocious Sperm Exchange in the Simultaneously Hermaphroditic Nudibranch, Berghia stephanieae.

SynopsisSexual systems vary greatly across molluscs. This diversity includes simultaneous hermaphroditism, with both sexes functional at the same time. Most nudibranch molluscs are thought to be simultaneous hermaphrodites, but detailed studies of reproductive development and timing remain rare as most species cannot be cultured in the lab. The aeolid nudibranch, Berghia stephanieae, is one such species that can be cultured through multiple generations on the benchtop. We studied B. stephanieae reproductive timing to establish when animals first exchange sperm and how long sperm can be stored. We isolated age- and size-matched individuals at sequential timepoints to learn how early individuals can exchange sperm. Individuals isolated at 10 weeks post initial feeding (wpf; ∼13 weeks postlaying [wpl]) can produce fertilized eggs. This is 6 weeks before animals first lay egg masses, indicating that sperm exchange occurs well before individuals are capable of laying eggs. Our results indicate that male gonads become functional for animals between 6 mm (∼6 wpf, ∼9 wpl) and 9 mm (∼12 wpf, ∼15 wpl) in length. That is much smaller (and sooner) than the size (and age) of individuals at first laying (12–19 mm; ∼16 wpf, ∼19 wpl), indicating that male and female functions do not develop simultaneously. We also tracked the number of fertilized eggs in each egg mass, which remained steady for the first 10–15 egg masses, followed by a decline to near-to-no fertilization. This dataset provides insights into the precise timing of the onset of functionality of the male and female reproductive systems in B. stephanieae. These data contribute to a broader understanding of reproductive development and the potential for understanding the evolution of diverse sexual systems in molluscs.

Reconstructing the Origins of the Somatostatin and Allatostatin-C Signaling Systems Using the Accelerated Evolution of Biodiverse Cone Snail Toxins.

Somatostatin and its related peptides (SSRPs) form an important family of hormones with diverse physiological roles. The ubiquitous presence of SSRPs in vertebrates and several invertebrate deuterostomes suggests an ancient origin of the SSRP signaling system. However, the existence of SSRP genes outside of deuterostomes has not been established, and the evolutionary history of this signaling system remains poorly understood. Our recent discovery of SSRP-like toxins (consomatins) in venomous marine cone snails (Conus) suggested the presence of a related signaling system in mollusks and potentially other protostomes. Here, we identify the molluscan SSRP-like signaling gene that gave rise to the consomatin family. Following recruitment into venom, consomatin genes experienced strong positive selection and repeated gene duplications resulting in the formation of a hyperdiverse family of venom peptides. Intriguingly, the largest number of consomatins was found in worm-hunting species (>400 sequences), indicating a homologous system in annelids, another large protostome phylum. Consistent with this, comprehensive sequence mining enabled the identification of SSRP-like sequences (and their corresponding orphan receptor) in annelids and several other protostome phyla. These results established the existence of SSRP-like peptides in many major branches of bilaterians and challenge the prevailing hypothesis that deuterostome SSRPs and protostome allatostatin-C are orthologous peptide families. Finally, having a large set of predator–prey SSRP sequences available, we show that although the cone snail’s signaling SSRP-like genes are under purifying selection, the venom consomatin genes experience rapid directional selection to target receptors in a changing mix of prey.

Cloning and characterization of a novel hydrolase gene from Hong Kong oyster Crassostrea hongkongensis

Hydrolase genes play an important role in the immune defense system in mollusks. We found a new hydrolase gene in Crassostrea hongkongensis. To explore its function, we cloned and characterized the hydrolase gene (Chhd) from C. hongkongensis. The full-length cDNA of Chhd is 936 bp, which is composed of a 531 bp open reading frame (ORF), a 92 bp 5′ -untranslated region (UTR) and a 313 bp 3′ UTR, the ORF encodes a polypeptide of 176 amino acids which contains a hydrolase_2 domain. qRT-PCR results showed that Chhd was ubiquitously expressed in tissues of C. hongkongensis, and the expression was highest in the mantle. After bacterial challenge, the expression of Chhd in hemocytes was significantly up-regulated. Recombinant pEGFP-N1-Chhd was distributed in the cytoplasm of HEK293T cells. The results of prokaryotic expression showed that pET28a-Chhd protein could express in large quantities. The expression of Chhd in total protein of C. hongkongensis was detected by western blot assay. In addition, Chhd inhibited the activities of NF-κB/p21-Luc in HEK293T cells, and RNA interference of Chhd significantly reduced the apoptosis rate of C. hongkongensis hemocytes. Taken together, our finding indicated that Chhd was involved in immunity and apoptosis regulation of C. hongkongensis, and would provide basic data for understanding the immune mechanism of shellfish.

Identification of an allatostatin C signaling system in mollusc Aplysia

Neuropeptides, as pervasive intercellular signaling molecules in the CNS, modulate a variety of behavioral systems in both protostomes and deuterostomes. Allatostatins are neuropeptides in arthropods that inhibit the biosynthesis of juvenile hormones. Based on amino acid sequences, they are divided into three different types in arthropods: allatostatin A, allatostatin B, allatostatin C. Allatostatin C (AstC) was first isolated from Manduca sexta, and it has an important conserved feature of a disulfide bridge formed by two cysteine residues. Moreover, AstC appears to be the ortholog of mammalian somatostatin, and it has functions in common with somatostatin, such as modulating feeding behaviors. The AstC signaling system has been widely studied in arthropods, but minimally studied in molluscs. In this study, we seek to identify the AstC signaling system in the marine mollusc Aplysia californica. We cloned the AstC precursor from the cDNA of Aplysia. We predicted a 15-amino acid peptide with a disulfide bridge, i.e., AstC, using NeuroPred. We then cloned two putative allatostatin C-like receptors and through NCBI Conserved Domain Search we found that they belonged to the G protein-coupled receptor (GPCR) family. In addition, using an inositol monophosphate 1 (IP1) accumulation assay, we showed that Aplysia AstC could activate one of the putative receptors, i.e., the AstC-R, at the lowest EC50, and AstC without the disulfide bridge (AstC') activated AstC-R with the highest EC50. Moreover, four molluscan AstCs with variations of sequences from Aplysia AstC but with the disulfide bridge activated AstC-R at intermediate EC50. In summary, our successful identification of the Aplysia AstC precursor and its receptor (AstC-R) represents the first example in molluscs, and provides an important basis for further studies of the AstC signaling system in Aplysia and other molluscs.

Mobilizing molluscan models and genomes in biology.

Molluscs are among the most ancient, diverse, and important of all animal taxa. Even so, no individual mollusc species has emerged as a broadly applied model system in biology. We here make the case that both perceptual and methodological barriers have played a role in the relative neglect of molluscs as research organisms. We then summarize the current application and potential of molluscs and their genomes to address important questions in animal biology, and the state of the field when it comes to the availability of resources such as genome assemblies, cell lines, and other key elements necessary to mobilising the development of molluscan model systems. We conclude by contending that a cohesive research community that works together to elevate multiple molluscan systems to 'model' status will create new opportunities in addressing basic and applied biological problems, including general features of animal evolution. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Immune Control of Herpesvirus Infection in Molluscs.

Molluscan herpesviruses that are capable of infecting economically important species of abalone and oysters have caused significant losses in production due to the high mortality rate of infected animals. Current methods in preventing and controlling herpesviruses in the aquacultural industry are based around biosecurity measures which are impractical and do not contain the virus as farms source their water from oceans. Due to the lack of an adaptive immune system in molluscs, vaccine related therapies are not a viable option; therefore, a novel preventative strategy known as immune priming was recently explored. Immune priming has been shown to provide direct protection in oysters from Ostreid herpesvirus-1, as well as to their progeny through trans-generational immune priming. The mechanisms of these processes are not completely understood, however advancements in the characterisation of the oyster immune response has assisted in formulating potential hypotheses. Limited literature has explored the immune response of abalone infected with Haliotid herpesvirus as well as the potential for immune priming in these species, therefore, more research is required in this area to determine whether this is a practical solution for control of molluscan herpesviruses in an aquaculture setting.

HC-SPH: A conserved serine protease homolog of S1 superfamily in the triangle-shell pearl mussel (Hyriopsis cumingii)

Serine proteases play central roles in immune defense in invertebrates through innate immunity, and are particularly important complement system in molluscs because their susceptibility to infection due to lack of an adaptive immune ability. A gene encoding the serine protease homolog from the triangle - shell pearl mussel (Hyriopsis cumingii) was identified and designated as HC-SPH in this study. Protein sequence analysis revealed that HC-SPH consists of a typical Tryp_SPc functional domain of serine protease of S1 family lead by a signal peptide, and the molecule shares a highly conserved sequence and structural organization with other members, including a cleavage site, 3 enzymatic active sites and 3 substrate binding sites, so that it was clustered into a trypsin-like serine protease subfamily of the S1 superfamily. Semi - quantitative analysis of the amplicons separated on agarose gel by comparing to the β-actin products revealed that the digestive gland had a strong expression while the gonads were seen as weak expression sites. Infected by Aeromonas hydrophila, the gene expression was significantly up - regulated in the kidney at the 6 hours post challenge (hpc), stomach at 12 hpc and gills at 24 hpc while the expression maintained steadily unchanged in the digestive gland. However, up to 48 hpc, the expression levels in all four tissues reached significantly high, and also joined by a high level of expression in intestine that was down - regulated before 24 hpc, to build up an enhanced immune defense. The complementary up - regulation of the gene expression in these tissues suggested a temporal and spatial reinforce model for HC-SPH in immune response.

Structure and function of the digestive system in molluscs.

The phylum Mollusca is one of the largest and more diversified among metazoan phyla, comprising many thousand species living in ocean, freshwater and terrestrial ecosystems. Mollusc-feeding biology is highly diverse, including omnivorous grazers, herbivores, carnivorous scavengers and predators, and even some parasitic species. Consequently, their digestive system presents many adaptive variations. The digestive tract starting in the mouth consists of the buccal cavity, oesophagus, stomach and intestine ending in the anus. Several types of glands are associated, namely, oral and salivary glands, oesophageal glands, digestive gland and, in some cases, anal glands. The digestive gland is the largest and more important for digestion and nutrient absorption. The digestive system of each of the eight extant molluscan classes is reviewed, highlighting the most recent data available on histological, ultrastructural and functional aspects of tissues and cells involved in nutrient absorption, intracellular and extracellular digestion, with emphasis on glandular tissues.

RFamidergic neurons in the olfactory centers of the terrestrial slug Limax

BackgroundThe terrestrial slug Limax has long been used as a model for the study of olfactory information processing and odor learning. Olfactory inputs from the olfactory epithelium are processed in the tentacular ganglion and then in the procerebrum. Glutamate and acetylcholine are the major neurotransmitters used in the procerebrum. Phe-Met-Arg-Phe-NH2 (FMRFamide) has been shown to be involved in the regulation of the network activity of the procerebrum. Although there are thought to be various RFamide family peptides other than FMRFamide that are potentially recognized by anti-FMRFamide antibody in the central nervous system of mollusks, identifying the entire repertoire of RFamide peptides in Limax has yet to be achieved.MethodsIn the present study, we made a comprehensive search for RFamide peptide-encoding genes from the transcriptome data of Limax, and identified 12 genes. The expression maps of these RFamide genes were constructed by in situ hybridization in the cerebral ganglia including the procerebrum, and in the superior/inferior tentacles.ResultsTen of 12 genes were expressed in the procerebrum, and nine of 12 genes were expressed in the tentacular ganglia. Gly-Ser-Leu-Phe-Arg-Phe-NH2 (GSLFRFamide), which is encoded by two different genes, LFRFamide1 (Leu-Phe-Arg-Phe-NH2–1) and LFRFamide2 (Leu-Phe-Arg-Phe-NH2–2), decreased the oscillatory frequency of the local field potential oscillation in the procerebrum when exogenously applied in vitro. We also found by immunohistochemistry that the neurons expressing pedal peptide send efferent projections from the procerebrum back to the tentacular ganglion.ConclusionOur findings suggest the involvement of a far wider variety of RFamide family peptides in the olfactory information processing in Limax than previously thought.

Proteomic profile of Ostrea edulis haemolymph in response to bonamiosis and identification of candidate proteins as resistance markers.

European flat oyster Ostrea edulis populations have suffered extensive mortalities caused by bonamiosis. The protozoan parasite Bonamia ostreae is largely responsible for this disease in Europe, while its congener B. exitiosa has been detected more recently in various European countries. Both of these intracellular parasites are able to survive and proliferate within haemocytes, the main cellular effectors of the immune system in molluscs. Two-dimensional electrophoresis was used to compare the haemolymph protein profile between Bonamia spp.-infected and non-infected oysters within 3 different stocks, a Galician stock of oysters selected for resistance against bonamiosis, a non-selected Galician stock and a selected Irish stock. Thirty-four proteins with a presumably relevant role in the oyster-Bonamia spp. interaction were identified; they were involved in major metabolic pathways, such as energy production, respiratory chain, oxidative stress, signal transduction, transcription, translation, protein degradation and cell defence. Furthermore, the haemolymph proteomic profiles of the non-infected oysters of the 2 Galician stocks were compared. As a result, 7 proteins representative of the non-infected Galician oysters selected for resistance against bonamiosis were identified; these 7 proteins could be considered as candidate markers of resistance to bonamiosis, which should be further assessed.

Characterization and comparison of transcriptional activities of the retinoid X receptors by various organotin compounds in three prosobranch gastropods; Thais clavigera, Nucella lapillus and Babylonia japonica

Two cDNAs of RXR were isolated, for the first time, from the ivory shell, Babylonia japonica, and the transcriptional activities were tested in vitro to compare with other gastropod (Thais clavigera and Nucella lapillus) RXR isoforms. The transcriptional activities of all of these RXR isoforms were significantly induced by mammalian RXR agonist, 9-cis retinoic acid (9cRA). The transcriptional activity of T. clavigera RXR-1 was also examined by using 9cRA and 16 organotin compounds, and significant ligand-dependent transactivations were observed by 9cRA and 5 organotins (tributyltin (TBT), tetrabutyltin (TeBT), tripropyltin (TPrT), tricyclohexyltin (TcHT) and triphenyltin (TPhT)). These 5 organotins also induced significant transcriptional activities in N. lapillus and B. japonica RXR isoforms. These 4 organotins, except for TeBT, have been reported to promote the development of imposex after a month of a single injection each, using female T. clavigera. To investigate the function of gastropod RXR isoforms, the effects of mammalian specific pan-agonist, PA024, and pan-antagonist, HX531, were examined, and significant induction of transcriptional activity by PA024 was demonstrated in these gastropod RXR isoforms. The additions of HX531 significantly suppressed the transcriptional activities of these gastropod RXR isoforms by 9cRA and 5 organotins. Using the mammalian two retinoic acid response elements, the transcriptional activities by 2 agonists, 9cRA and PA024, were different among the RXR isoforms of each gastropod species. With retinoid X response element (RXRE), transcriptional activities of TcRXR-1, BjRXR-1, and NlRXRa were significantly higher than those of TcRXR-2, BjRXR-2, and NlRXRb. Transcriptional activities of TcRXR-2, BjRXR-2, and NlRXRb, however, were significantly higher than those of TcRXR-1, BjRXR-1, and NlRXRa with thyroid hormone response element, TREpal. Thus, induction of imposex in prosobranch gastropods is strongly suggested to be triggered by 9cRA and certain organotins, such as TBT and TPhT through the activation of RXRs. These gastropod RXRs might control the different gene transcription by forming homo- or heterodimer complex with their own isoforms. These findings will contribute to our understanding of the fundamentals of the endocrine system in molluscs, particularly on RXR signaling pathway.

Stability, variability, and parallelisms in the development of distant sensory systems: Olfactory and visual systems in the phylogeny and ontogeny of gastropods

The results of studies in the field of olfactory and visual systems of a number of gastropod representatives, differing in the general level of organization, phylogenetic position, habitat, and mode of life are given. The direction, general patterns, features, and parallels in the development of these sensory structures and related integrative brain regions in different phylogenetic lineages of gastropods are traced. Parallels in the development of the olfactory system of mollusks, insects, and vertebrates are shown. The important role of the habitat and mode of life of animals in the formation of specific patterns of evolutionary development of sensory organs is noted.

Infectious diseases of marine molluscs and host responses as revealed by genomic tools.

More and more infectious diseases affect marine molluscs. Some diseases have impacted commercial species including MSX and Dermo of the eastern oyster, QPX of hard clams, withering syndrome of abalone and ostreid herpesvirus 1 (OsHV-1) infections of many molluscs. Although the exact transmission mechanisms are not well understood, human activities and associated environmental changes often correlate with increased disease prevalence. For instance, hatcheries and large-scale aquaculture create high host densities, which, along with increasing ocean temperature, might have contributed to OsHV-1 epizootics in scallops and oysters. A key to understanding linkages between the environment and disease is to understand how the environment affects the host immune system. Although we might be tempted to downplay the role of immunity in invertebrates, recent advances in genomics have provided insights into host and parasite genomes and revealed surprisingly sophisticated innate immune systems in molluscs. All major innate immune pathways are found in molluscs with many immune receptors, regulators and effectors expanded. The expanded gene families provide great diversity and complexity in innate immune response, which may be key to mollusc's defence against diverse pathogens in the absence of adaptive immunity. Further advances in host and parasite genomics should improve our understanding of genetic variation in parasite virulence and host disease resistance.

Allatostatin-type A, kisspeptin and galanin GPCRs and putative ligands as candidate regulatory factors of mantle function

Allatostatin-type A (AST-A), kisspeptin (KISS) and galanin (GAL) G-protein coupled receptor (GPCR) systems share a common ancestral origin in arthropods and the vertebrates where they regulate metabolism and reproduction. The molluscs are the second most diverse phylum in the animal kingdom, they occupy an important phylogenetic position, and their genome is more similar to deuterostomes than the arthropods and nematodes and thus they are good models for studies of gene family evolution and function. This mini-review intends to extend the current knowledge about AST-A, KISS and GAL GPCR system evolution and their putative function in the mollusc mantle. Comparative evolutionary analysis of the target GPCR systems was established by identifying homologues in genomes and tissue transcriptome datasets available for molluscs and comparing them to those of other metazoan systems. Studies in arthropods have revealed the existence of the AST-A system but the loss of homologues of the KISS and GAL systems. Homologues of the insect AST-AR and vertebrate KISSR genes were found in molluscs but putative GALR genes were absent. Receptor gene number suggested that members of this family have suffered lineage specific evolution during the molluscan radiation. In molluscs, orthologues of the insect AST-A peptides were not identified but buccalin peptides that are structurally related were identified and are putative receptor agonists. The identification of AST-AR and KISSR genes in molluscs strengthens the hypotheses that in metazoans members of the AST-AR subfamily share evolutionary proximity with KISSRs. The variable number of receptors and large repertoire of buccalin peptides may be indicative of the functional diversity of the AST-AR/KISSR systems in molluscs. The identification of AST-A and KISS receptors and ligands in the mantle transcriptome indicates that in molluscs they may have acquired a novel function and may play a role in shell development or sensory detection in the mantle.

Oct-GnRH, the first protostomian gonadotropin-releasing hormone-like peptide and a critical mini-review of the presence of vertebrate sex steroids in molluscs

In protostome and deuterosome invertebrates, neurosecretory cells play major roles in the endocrine system. The optic glands of cephalopods are indicators of sexual maturation. In mature octopuses, optic glands enlarge and secrete a gonadotropic hormone. A peptide with structural features similar to that of vertebrate gonadotropin-releasing hormone (GnRH) was isolated from the octopus, Octopus vulgaris, and was named oct-GnRH. The discovery of oct-GnRH has triggered structural determinations and predictions of other mollusc GnRH-like peptides in biochemical and in silico studies. Interestingly, cephalopods studied so far are characterized by a single molecular form of oct-GnRH with a C-terminal -Pro-Gly-NH2 sequence, which is critical for gonadotropin-releasing activity in vertebrates. Other molluscan GnRH-like peptides lack the C-terminal -Pro-Gly-NH2 sequence but have -X-NH2 or -Pro-Gly although all protostome GnRH-like peptides have yet to be sequenced.In marine molluscs, relationships between GnRH-like peptides and sex steroids have been studied to verify the hypothesis that molluscs have vertebrate-type sex steroid system. However, it is currently questionable whether such sex steroids are present and whether they play endogenous roles in the reproductive system of molluscs. Because molluscs uptake and store steroids from the environment and fishes release sex steroids into the external environment, it is impossible to rule out the contamination of vertebrate sex steroids in molluscs. The function of key enzymes of steroidogenesis within molluscs remains unclear. Thus, evidence to deny the existence of the vertebrate-type sex steroid system in molluscs has been accumulated. The elucidation of substances, which regulate the maturation and maintenance of gonads and other reproductive functions in molluscs will require rigorous and progressive scientific study.

Pacific oyster (Crassostrea gigas) hemocyte are not affected by a mixture of pesticides in short-term in vitro assays

Pesticides are frequently detected in estuaries among the pollutants found in estuarine and coastal areas and may have major ecological consequences. They could endanger organism growth, reproduction, or survival. In the context of high-mortality outbreaks affecting Pacific oysters, Crassostrea gigas, in France since 2008, it appears of importance to determine the putative effects of pesticides on oyster susceptibility to infectious agents. Massive mortality outbreaks reported in this species, mainly in spring and summer, may suggest an important role played by the seasonal use of pesticides and freshwater input in estuarine areas where oyster farms are frequently located. To understand the impact of some pesticides detected in French waters, their effects on Pacific oyster hemocytes were studied through short-term in vitro experiments. Bivalve immunity is mainly supported by hemocytes eliminating pathogens by phagocytosis and producing compounds including lysosomal enzymes and antimicrobial molecules. In this study, oyster hemocytes were incubated with a mixture of 14 pesticides and metaldehyde alone, a molecule used to eliminate land mollusks. Hemocyte parameters including dead/alive cells, nonspecific esterase activities, intracytoplasmic calcium, lysosome number and activity, and phagocytosis were monitored by flow cytometry. No significant effect of pesticides tested at different concentrations was reported on oyster hemocytes maintained in vitro for short-term periods in the present study. It could be assumed that these oyster cells were resistant to pesticide exposure in tested conditions and developing in vivo assays appears as necessary to better understand the effects of pollutants on immune system in mollusks.

Distinct Expression Patterns of Glycoprotein Hormone Subunits in the Lophotrochozoan Aplysia: Implications for the Evolution of Neuroendocrine Systems in Animals

Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific β subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHβ5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHβ subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHβ5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHβ, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHβ confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHβ5 subunit the seatbelt structure is absent in AcaGPHβ. We also found that AcaGPHα and AcaGPHβ subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands.

Variations in Functional Activity of the Hormone-Sensitive Adenylate Cyclase System in Tissues of Gastropod Mollusks with Streptozotocin-Induced Diabetes

Treatment of gastropod mollusks of pond snail Lymnaea stagnalis and orb snail Coretus corneus with streptozotocin was followed by an increase in hexose content in the hemolymph and development of the diabetic state (day 1 after treatment). Functional activity of the hormone-sensitive adenylate cyclase system significantly decreased in the muscles and hepatopancreas of mollusks with diabetes. We revealed a decrease in the regulatory effects of biogenic amines and peptide hormones that were realized via stimulatory (octopamine, dopamine, serotonin, tryptamine, and relaxin) and inhibitory G proteins (somatostatin). Disturbances in the hepatopancreas were more pronounced than in the muscle. The severity of disorders in the adenylate cyclase system reached maximum 1 day after streptozotocin treatment. The sensitivity of this system to hormonal and nonhormonal agents was partially restored on days 3 and 5. Hexose content in the hemolymph was elevated after streptozotocin treatment, but returned to normal on day 3. Our results indicate that hyperglycemia is one of the key factors for dysfunction of the adenylate cyclase system in mollusks with the diabetic state.