A novel exoskeletal-derived C-type lectin facilitates phagocytosis of hemocytes in the oriental river prawn Macrobrachium nipponense.

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (MnLec3). The full-length cDNA of MnLec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. MnLec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). MnLec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of MnLec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of MnLec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant MnLec3 protein (rMnLec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli, Vibrio anguillarum and A. hydrophila). Moreover, rMnLec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What's more, MnLec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that MnLec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR.

Mantle tissue in the pearl oyster Pinctada fucata secretes immune components via vesicle transportation.

Molluscan bivalves secrete shell matrices into the extrapallial space (EPS) to guide the precipitation of rigid shells. Meanwhile, immune components are present in the EPS and shell matrices, which are pivotal in resistant to invaded pathogens, thus ensuring the shell formation process. However, the origin of these components remains unclear. In this study, we revealed numerous vesicles were secreted from the outer mantle epithelial cells by using light and electron microscopes. The secreted vesicles were isolated by gradient centrifugation and confirmed by transmission electron microscopy. Proteomics analysis showed that the secreted vesicles were composed of cytoplasmic and immune components, most of which do not have signal peptides, indicating that they were secreted by a non-classical pathway. Moreover, real-time PCR revealed that some immune components were highly expressed in the mantle tissue, compared to the hemocytes. FTIR analysis verified the presence of lipids in the shell matrices, indicating that the vesicles have integrated into the shell layers. Taken together, our results suggested that mantle epithelial cells secreted some important immune components into the EPS via secreted vesicle transportation, thus cooperating with the hemocytes to play a vital role in immunity during shell formation.

Evolution and Potential Function in Molluscs of Neuropeptide and Receptor Homologues of the Insect Allatostatins.

The allatostatins (ASTs), AST-A, AST-B and AST-C, have mainly been investigated in insects. They are a large group of small pleotropic alloregulatory neuropeptides that are unrelated in sequence and activate receptors of the rhodopsin G-protein coupled receptor family (GPCRs). The characteristics and functions of the homologue systems in the molluscs (Buccalin, MIP and AST-C-like), the second most diverse group of protostomes after the arthropods, and of high interest for evolutionary studies due to their less rearranged genomes remains to be explored. In the present study their evolution is deciphered in molluscs and putative functions assigned in bivalves through meta-analysis of transcriptomes and experiments. Homologues of the three arthropod AST-type peptide precursors were identified in molluscs and produce a larger number of mature peptides than in insects. The number of putative receptors were also distinct across mollusc species due to lineage and species-specific duplications. Our evolutionary analysis of the receptors identified for the first time in a mollusc, the cephalopod, GALR-like genes, which challenges the accepted paradigm that AST-AR/buccalin-Rs are the orthologues of vertebrate GALRs in protostomes. Tissue transcriptomes revealed the peptides, and their putative receptors have a widespread distribution in bivalves and in the bivalve Mytilus galloprovincialis, elements of the three peptide-receptor systems are highly abundant in the mantle an innate immune barrier tissue. Exposure of M. galloprovincialis to lipopolysaccharide or a marine pathogenic bacterium, Vibrio harveyi, provoked significant modifications in the expression of genes of the peptide precursor and receptors of the AST-C-like system in the mantle suggesting involvement in the immune response. Overall, our study reveals that homologues of the arthropod AST-systems in molluscs are potentially more complex due to the greater number of putative mature peptides and receptor genes. In bivalves they have a broad and varying tissue distribution and abundance, and the elements of the AST-C-like family may have a putative function in the immune response.

Identification of a long non-coding RNA (LncMSEN2) from pearl oyster and its potential roles in exoskeleton formation and LPS stimulation.

Long non-coding RNAs (lncRNAs) play regulatory roles in various biological processes, including exoskeleton formation and immune response. The exoskeleton-based mantle-shell defense system is an important defense mechanism in shellfish. In this study, we found a novel lncRNA, herein formally named, LncMSEN2, from the pearl oyster Pinctada fucuta martensii, and its sequence was validated via polymerase chain reaction (PCR). LncMSEN2 was highly expressed in mantle tissues, especially in the central region (P < 0.05), and was also expressed in the pearl sac as detected by quantitative real-time PCR. In situ hybridization experiments revealed that LncMSEN2 had a strong positive signal in the inner and outer epidermal cells of the mantle pallial and central regions. RNA interference experiments showed that interference of LncMSEN2 expression with dsRNA in mantle tissues led to an abnormal crystal structure of the nacre. In addition, LncMSEN2 expression significantly increased 6 h after lipopolysaccharide stimulation in mantle tissues (P < 0.05). These results indicated that LncMSEN2 may be a novel regulator of the mantle-shell defense system of pearl oyster.

Response of a novel selenium-dependent glutathione peroxidase from thick shell mussel Mytilus coruscus exposed to lipopolysaccharide, copper and benzo[α]pyrene.

Glutathione peroxidase (GPx) plays an important antioxidant role in cellular defense against environmental stress. In the present study, a novel selenium-dependent glutathione peroxidase termed McSeGPx firstly identified in thick shell mussel Mytilus coruscus. McSeGPx consists of 197 amino acid residues, characterized with one selenocysteine residue encoded by an opal stop codon TGA, one selenocysteine insertion sequence (SECIS) in the 3' untranslated region (UTR), two active site motifs and one signature sequence motif. McSeGPx transcripts were constitutively expressed in all examined tissues, and were significantly induced in gills and digestive glands with the stimulations of lipopolysaccharide (LPS), copper (Cu) and benzo[α]pyrene (B[α]P). Additionally, rough increases in McSeGPx activity were detected in both tissues under the challenge of LPS, Cu and B[α]P. Collectively, these results suggested that McSeGPx affiliate to selenocysteine dependent GPx (SeGPx) family and might play an important role in mediating the environmental stressors and antioxidant response in M. coruscus.

Transcriptional changes in the Japanese scallop (Mizuhopecten yessoensis) shellinfested by Polydora provide insights into the molecular mechanism of shell formation and immunomodulation.

The Japanese scallop (Mizuhopecten yessoensis) is one of the most important aquaculture species in Asian countries; however, it has suffered severe infection by Polydora in northern China in recent years, causing great economic losses. The Polydora parasitizes the shell of scallops, badly destroying the shell's structure. To investigate the molecular response mechanism of M. yessoensis to Polydora infestion, a comprehensive and niche-targeted cDNA sequence database for diseased scallops was constructed. Additionally, the transcriptional changes in the edge mantle, central mantle and hemocytes, tissues directly related to the disease, were first described in this study. The results showed that genes involved in shell formation and immunomodulation were significantly differentially expressed due to the infestation. Different transcriptional changes existed between the two mantle regions, indicating the different molecular functions likely responsible for the formation of different shell layers. The differential expression of genes for immune recognition, signal transduction and pathogen elimination presented an integrated immune response process in scallops. Moreover, neuromodulation and glycometabolism involved in the regulation process with relevant function significantly enriched. The study provides valuable information for mechanism study of shell formation and immunomodulation in scallops.

Hemocytes in the extrapallial space of Pinctada fucata are involved in immunity and biomineralization.

In bivalves, the mantle tissue secretes organic matrix and inorganic ions into the extrapallial space (EPS) to form the shells. In addition, more and more evidences indicate the participation of hemocytes in shell mineralization, but no direct evidence has been reported that verifies the presence of hemocytes in the EPS, and their exact roles in biomineralization remain uncertain. Here, we identified hemocytes from the EPS of Pinctada fucata. Numerous components involved in cellular and humoral immunity were identified by proteome analysis, together with several proteins involved in calcium metabolism. The hemocytes exerted active phagocytosis and significantly upregulated the expression of immune genes after immune stimulation. A group of granulocytes were found to contain numerous calcium-rich vesicles and crystals, which serve as a calcium pool. During shell regeneration, some genes involved in calcium metabolism are upregulated. Strikingly, most of the shell matrix proteins were absent in the hemocytes, suggesting that they might not be solely responsible for directing the growth of the shell. Taken together, our results provided comprehensive information about the function of hemocytes in immunity and shell formation.

Effects of fluorine-containing usnic acid and fungus Beauveria bassiana on the survival and immune-physiological reactions of Colorado potato beetle larvae.

BACKGROUND: The search for compounds that interact synergistically with entomopathogenic fungi is aimed at enhancing the efficacy and stability of biological products against pest insects, for example, against the Colorado potato beetle (CPB). We hypothesized that fluorine-containing derivatives of usnic acid (FUA) might be candidates for the development of multicomponent bio-insecticides. The aim of this study was to analyze the co-influence of FUA and Beauveria bassiana on the survival and immune-physiological reactions of CPB larvae. RESULTS: Synergy between FUA and B. bassiana was observed after treatment of second, third and fourth larvae instars under laboratory conditions. Furthermore, synergy was observed in field trials in continental climate conditions in southeastern Kazakhstan. In a field experiment, the median lethal time was shortened three-fold, and cumulative mortality for 15 days increased by 36% in the combined treatment compared with a fungal infection alone. FUA treatment delayed larval development, decreased the total hemocyte count, and increased both the phenoloxidase activity in integuments and the detoxification enzyme rate in hemolymph. A combined treatment with fungus and FUA led to increases in the aforementioned changes. CONCLUSION: Toxicosis caused by FUA provides a stable synergistic effect between FUA and B. bassiana. The combination can be promising for the development of highly efficient products against CPB. © 2017 Society of Chemical Industry.

The gastropod shell has been co-opted to kill parasitic nematodes.

Exoskeletons have evolved 18 times independently over 550 MYA and are essential for the success of the Gastropoda. The gastropod shell shows a vast array of different sizes, shapes and structures, and is made of conchiolin and calcium carbonate, which provides protection from predators and extreme environmental conditions. Here, I report that the gastropod shell has another function and has been co-opted as a defense system to encase and kill parasitic nematodes. Upon infection, cells on the inner layer of the shell adhere to the nematode cuticle, swarm over its body and fuse it to the inside of the shell. Shells of wild Cepaea nemoralis, C. hortensis and Cornu aspersum from around the U.K. are heavily infected with several nematode species including Caenorhabditis elegans. By examining conchology collections I show that nematodes are permanently fixed in shells for hundreds of years and that nematode encapsulation is a pleisomorphic trait, prevalent in both the achatinoid and non-achatinoid clades of the Stylommatophora (and slugs and shelled slugs), which diverged 90-130 MYA. Taken together, these results show that the shell also evolved to kill parasitic nematodes and this is the only example of an exoskeleton that has been co-opted as an immune system.

Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization.

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels.

Antimicrobial activity in the cuticle of the American lobster, Homarus americanus.

American lobster, Homarus americanus, continues to be an ecologically and socioeconomically important species despite a severe decline in catches from Southern New England and Long Island Sound (USA) and a high prevalence of epizootic shell disease in these populations. A better understanding of lobster immune defenses remains necessary. Cuticle material collected from Long Island Sound lobsters was found to be active against a broad spectrum of bacteria, including Gram-negative and -positive species. The antimicrobial activity was characterized by boiling, muffling, and size fractioning. Boiling did not significantly reduce activity, while muffling did have a significant effect, suggesting that the active component is organic and heat stable. Size fractioning with 3 and 10 kDa filters did not significantly affect activity. Fast protein liquid chromatography fractions were also tested for antimicrobial activity, and fractions exhibiting protein peaks remained active. MALDI mass spectrometry revealed peptide peaks at 1.6, 2.8, 4.6, and 5.6 kDa. The data presented suggest that one or several antimicrobial peptides contribute to antimicrobial activity present in the American lobster cuticle.

Shell colouration and parasite tolerance in two helicoid snail species.

The polymorphism of shell colouration in helicoid snails is a well-known phenomenon attributed to different factors such as predation and climatic effects. Another aspect contributing to this polymorphism could be the interplay of melanin production and phenoloxidase-related immunity. Therefore, in this study we aimed at answering the questions whether there is a differential sensitivity of different snail shell colour morphs to nematode infection, and whether this can be related to differences in phenoloxidase (PO) activity levels using the two helicoid, polymorphic snail species Cepaea hortensis and Cernuella virgata. Snails of both species were artificially infected with the parasitic nematode Phasmarhabditis hermaphrodita, and analysed for mortality and PO activity levels. We found C. virgata to be more severely affected by P. hermaphrodita infection than C. hortensis, and the dark C. virgata morphs to be more resistant to lethal effects of this infection than pale morphs. However, these differences in sensitivity to the parasite could not clearly be related to different PO activity levels.

Differential expression of genes involved in immunity and biomineralization during Brown Ring Disease development and shell repair in the Manila clam, Ruditapes philippinarum.

Severe drop in Manila clams production in French aquacultured fields since the end of the 1980's is associated to Brown Ring Disease (BRD). This disease, caused by the bacteria Vibrio tapetis, is characterized by specific symptoms on the inner face of the shell. Diseased animals develop conchiolin deposit to enrobe bacteria and form new calcified layers on the shell. Suppression subtractive hybridization was performed to identify genes differentially expressed during the early interaction of V. tapetis and Ruditapes philippinarum. Results revealed 301 unique genes differentially expressed during V. tapetis challenge. Several candidates involved in immune and biomineralization processes were selected from libraries. Transcriptional expression of selected candidates was determined in hemolymph and mantle tissues and revealed spatial and temporal variations. At 56 days after infection, when clams were in phase of shell repair, transcripts of galectin and ferritin in hemocytes showed higher expression. Ca-like and serpin transcripts were specifically expressed in mantle and could contribute to defense against BRD.