Littoporins: Novel actinoporin-like proteins in caenogastropod genus Littorina.

In the course of searching for genes controlling the immune system in caenogastropod mollusks, we characterized and phylogenetically placed five new actinoporin-like cytolysins expressed in periwinkles of the genus Littorina. These newly discovered proteins, named littoporins (LitP), contain a central cytolysin/lectin domain and exhibit a predicted protein fold that is almost identical to the three-dimensional structures of actinoporins. Two of these proteins, LitP-1 and LitP-2, were found to be upregulated in L. littorea kidney tissues and immune cells in response to natural and experimental infection with the trematode Himasthla elongata, suggesting their potential role as perforins in the systemic anti-trematode immune response. The primary sequence divergence of littoporins is hypothesized to be attributed to the taxonomic range of cell membranes they can recognize and permeabilize.

Experimental and molecular predictions of the adjuvanticity of snail mucin on hepatitis B vaccine in albino mice.

Although aluminum-containing adjuvants are widely used in human vaccination due to their excellent safety profile, they exhibit low effectiveness with many recombinant antigens. This study investigated the adjuvanticity of snail mucin with recombinant Hepatitis B Vaccine (rHBsAg). Twenty-five (25) female mice distributed unbiasedly into 5 groups were used in the study and were administered different rHBsAg/Mucin formulation at 7 days intervals. Blood samples were collected a day following the administration for analysis. The results of liver function and body weight analysis were indications that snail mucin had no adverse effect on the mice. The treatment group (administer mucin and rHBsAg) showed significantly (P<0.05) higher mean titres of anti-HBsAg antibodies when compared with the negative controls and the positive control administered with two doses of rHBsAg after the boost doses (day 28). Furthermore, a comparable immune response to positive control administered with three doses rHBaAG was recorded. In silico prediction, studies of the protein-protein interaction of a homology modelled snail mucus protein and HBsAg gave an indication of enhanced HBV antigen-antibody interaction. Therefore, this study has shown that snail mucin possesses some adjuvant properties and enhances immune response towards rHBsAg vaccine. However, there is a need for further molecular dynamics studies to understand its mechanism of action.

Both quiescent and proliferating cells circulate in the blood of the invasive apple snail Pomacea canaliculata.

Gastropod hematopoiesis occurs at specialized tissues in some species, but the evidence also suggests that hemocyte generation is maybe widespread in the connective tissues or the blood system in others. In Ampullariidae (Caenogastropoda), both the kidney and the lung contain putative hematopoietic cells, which react to immune challenges. In the current study, we wanted to explore if hematopoiesis occurs in the blood of Pomacea canaliculata. Thus, we obtained circulating hemocytes from donor animals and tested their ability to proliferate in the blood of conspecific recipients. We tracked cell proliferation by labeling the donors' hemocytes with the fluorescent cell proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE). Transferred CFSE-labeled hemocytes survived and proliferated into the recipients' circulation for at least 17 days. We also determined the cell cycle status of circulating hemocytes by using the propidium iodide (PI) and acridine orange (AO) staining methods. Flow cytometry analyses showed that most PI-stained hemocytes were in the G1 phase (~96%), while a lower proportion of cells were through the G2/S-M transition (~4%). When we instead used AO-staining, we further distinguished a subpopulation of cells (~5%) of low size, complexity-granularity, and RNA content. We regarded this subpopulation as quiescent cells. In separate experimental sets, we complemented these findings by assessing in circulating hemocytes two evolutionary conserved features of quiescent, undifferentiated cells. First, we used JC-1 staining to determine the mitochondrial membrane potential (Ψm) of circulating hemocytes, which is expected to be low in quiescent cells. Most hemocytes (~87%) showed high aggregation of JC-1, which indicates a high Ψm. Besides that, a small hemocyte subpopulation (~11%) showed low aggregation of the dye, thus indicating a low Ψm. It is known that the transition from a quiescent to a proliferating state associates with an increase of the Ψm. The specificity of these changes was here controlled by membrane depolarization with the Ψm disruptor CCCP. Second, we stained hemocytes with Hoechst33342 dye to determine the efflux activity of ABC transporters, which participate in the multixenobiotic resistance system characteristic of undifferentiated cells. Most hemocytes (>99%) showed a low dye-efflux activity, but a small proportion of cells (0.06-0.12%) showed a high dye-efflux activity, which was significantly inhibited by 100 and 500 µM verapamil, and thus is indicative of an undifferentiated subpopulation of circulating hemocytes. Taken together, our results suggest that, among circulating hemocytes, there are cells with the ability to proliferate or to stay in a quiescent state and behave as progenitor cells later, either in the circulation or the hematopoietic tissues/organs.

Combined transcriptomics and proteomics to identify differential proteins involved in the immune response to the parasite schistosoma japonicum in snail hosts pre-infected with exorchis sp.

Oncomelania hupensis is the obligate intermediate host of Schistosoma japonicum, and it also serves as the first intermediate host for Exorchis sp., which uses Parasilurus asoyus as its definitive host rather than humans. In previous studies, Tang et al. found that all S. japonicum larvae can be blocked and killed in O. hupensis pre-infected with Exorchis sp. eggs. However, the molecular and cellular mechanisms involved in this process remain unclear. Therefore, in the present study, a combined transcriptomic and proteomic analysis was performed to identify the differential proteins involved in the immune response to the parasite S. japonicum in the O. hupensis snail host pre-infected with Exorchis sp. trematodes. The results showed that a total of 46,162 unigenes were obtained with 23,535 (50.98%) unigenes annotated in relevant databases, and 3811 proteins from O. hupensis were identified. In addition, iTRAQ-based quantitative proteomic analysis demonstrated that among three groups (OhSj-1_vs_OhN-1, OhE-1_vs_OhN-1 and OhES-1_vs_OhN-1), there were 146 common differential proteins including 44 up-regulated proteins and 90 down-regulated proteins, and 195 differential proteins exclusive to only one experimental group, including 91 up-regulated proteins and 104 down-regulated proteins, which were defined as the Common group and the Only group, respectively. KEGG analysis showed that 15 and 11 differential proteins were annotated in "Infectious diseases" in the Common group and the Only group, respectively, indicating that these proteins may be involved in the snail host immune response to parasite infection. These data will be helpful for better understanding the host-parasite interaction, and could pave the way towards exploring the mechanisms involved in the biological control on S. japonicum in O. hupensis. They also provide valuable information about developing new anti-schistosomiasis strategies.

RNA-seq: The early response of the snail Physella acuta to the digenetic trematode Echinostoma paraensei.

To analyze the response of the snail Physella acuta to Echinostoma paraensei, a compatible digenetic trematode, Illumina RNA-seq data were collected from snails with early infection (5 snails at 2 days post-exposure [DPE]) and established infection (4 snails, 8 DPE), and 7 control (unexposed) snails. A reference transcriptome (325,563 transcripts, including 98% of eukaryotic universal single-copy orthologs; BUSCO) and a draft P. acuta genome (employing available genomic Illumina reads; 799,945 scaffolds, includes 88% BUSCO genes) were assembled to guide RNA-seq analyses. Parasite exposure of P. acuta led to 10,195 differentially expressed (DE) genes at 2 DPE and 8,876 DE genes at 8 DPE with only 18% of up-regulated and 22% of down-regulated sequences shared between these time points. Gene ontology (GO) analysis yielded functional annotation of only 1.2% of DE genes but did not indicate major changes in biological activities of P. acuta between 2 and 8 DPE. Increased insights were achieved by analysis of expression profiles of 460 immune-relevant DE transcripts, identified by BLAST and InterProScan. Physella acuta has expanded gene families that encode immune-relevant domains, including CD109/TEP, GTPase IMAP, Limulus agglutination factor (dermatopontin), FReD (≥82 sequences with fibrinogen-related domains), and transcripts that combine C-type lectin (C-LECT) and C1q domains, novel among metazoa. Notably, P. acuta expressed sequences from these immune gene families at all time points, but the assemblages of unique transcripts from particular immune gene families differed between 2 and 8 DPE. The shift in profiles of DE immune genes, from early exposure to parasite establishment, suggests that compatible P. acuta initially respond to infection but switch to express immune genes that likely are less effective against E. paraensei but counter other types of (opportunistic) pathogens and parasites. We propose that the latter expression profile is part of an extended phenotype of E. paraensei, imposed upon P. acuta through parasite manipulation of the host, following successful parasite establishment in the snail after 2 DPE.

Novel Role for Animal Innate Immune Molecules: Enterotoxic Activity of a Snail Egg MACPF-Toxin.

Gastropod Molluscs rely exclusively on the innate immune system to protect from pathogens, defending their embryos through maternally transferred effectors. In this regard, Pomacea snail eggs, in addition to immune defenses, have evolved the perivitellin-2 or PV2 combining two immune proteins into a neurotoxin: a lectin and a pore-forming protein from the Membrane Attack Complex/Perforin (MACPF) family. This binary structure resembles AB-toxins, a group of toxins otherwise restricted to bacteria and plants. Many of these are enterotoxins, leading us to explore this activity in PV2. Enterotoxins found in bacteria and plants act mainly as pore-forming toxins and toxic lectins, respectively. In animals, although both pore-forming proteins and lectins are ubiquitous, no enterotoxins have been reported. Considering that Pomacea snail eggs ingestion induce morpho-physiological changes in the intestinal mucosa of rodents and is cytotoxic to intestinal cells in culture, we seek for the factor causing these effects and identified PmPV2 from Pomacea maculata eggs. We characterized the enterotoxic activity of PmPV2 through in vitro and in vivo assays. We determined that it withstands the gastrointestinal environment and resisted a wide pH range and enzymatic proteolysis. After binding to Caco-2 cells it promoted changes in surface morphology and an increase in membrane roughness. It was also cytotoxic to both epithelial and immune cells from the digestive system of mammals. It induced enterocyte death by a lytic mechanism and disrupted enterocyte monolayers in a dose-dependent manner. Further, after oral administration to mice PmPV2 attached to enterocytes and induced large dose-dependent morphological changes on their small intestine mucosa, reducing the absorptive surface. Additionally, PmPV2 was detected in the Peyer's patches where it activated lymphoid follicles and triggered apoptosis. We also provide evidence that the toxin can traverse the intestinal barrier and induce oral adaptive immunity with evidence of circulating antibody response. As a whole, these results indicate that PmPV2 is a true enterotoxin, a role that has never been reported to lectins or perforin in animals. This extends by convergent evolution the presence of plant- and bacteria-like enterotoxins to animals, thus expanding the diversity of functions of MACPF proteins in nature.

Recent advances on the immunobiology of Bithynia spp. hosts of Opisthorchis viverrini.

This article reviews the past and present scientific reports regarding Bithynia spp. focusing on the biology, ecology and life cycle of Bithynia snails and their responses to Opisthorchis viverrini infection. Moreover, new data regarding comparative molecular genomics and proteomic approaches have recently revealed novel molecular components involved in the immune defence responses from Bithynia spp., providing additional perspectives for future studies. Studies on the specific interaction between Bithynia snails and their trematodes will contribute to further understanding the snail-parasite relationship with regards to epidemiology and control of Opisthorchiasis and broaden the scope on comparative immunology of gastropod snails.

The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: Hemocytes in the spotlight.

The Fasciola hepatica/Pseudosuccinea columella interaction in Cuba involves a unique pattern of phenotypes; while most snails are susceptible, some field populations are naturally resistant to infection and parasites are encapsulated by snail hemocytes. Thus, we investigated the hemocytes of resistant (R) and susceptible (S) P. columella, in particular morphology, abundance, proliferation and in vitro encapsulation activity following exposure to F. hepatica. Compared to susceptible P. columella, hemocytes from exposed resistant snails showed increased levels of spreading and aggregation (large adherent cells), proliferation of circulating blast-like cells and encapsulation activity of the hemocytes, along with a higher expression of the cytokine granulin. By contrast, there was evidence of a putative F. hepatica-driven inhibition of host immunity, only in susceptible snails. Additionally, (pre-)incubation of naïve hemocytes from P. columella (R and S) with different monosaccharides was associated with lower encapsulation activity of F. hepatica larvae. This suggests the involvement in this host-parasite interaction of lectins and lectins receptors (particularly related to mannose and fucose sensing) in association with hemocyte activation and/or binding to F. hepatica.

Immune response of a caenogastropod host: A case study of Littorina littorea and its digenean parasites.

The caenogastropod mollusk Littorina littorea is a promising experimental model for comparative studies on host/parasite immune conflict. Several different digenean parasites use L. littorea as the first intermediate host, overcoming snail immune reactions by a wide range of tactics that are radically different among different digenean species and at different developmental parasite stages. The immune system of L. littorea is rather effective against digenean Himasthla elongata invasion, and even successfully established parasite induces a chronic host immune reaction, present at a low but stable level, that may be involved in the selection of derived parasitic clones in long lived self-sustaining infrapopulations (SSI) of rediae. An anti-digenean response in L. littorea is not systemic (non-generalized) yet tissue specific, mostly reliant on cellular rather than humoral reactions. The repertoire of immune pattern-recognizing receptors in the common periwinkle comprises diverse secreted and membrane-attached lectin molecules, as the main drivers of snail immune discrimination of digenean parasites. Comparative studies suggest that the characteristic vulnerability to digenean parasitism of L. littorea, and gastropods in general, is in part due the overall organization of immunity relative to other classes of molluscs, e.g. the immune strategy of bivalves seems to rely on less specific cellular reactions and a more generalized systemic humoral immunity. This difference may arise from the molecular features of the selective retention of their taxon-specific complement-like molecular complexes, which diverged in common ancestors of Bivalvia and Gastropoda.

Natural resistance to Fasciola hepatica (Trematoda) in Pseudosuccinea columella snails: A review from literature and insights from comparative "omic" analyses.

The snail Pseudosuccinea columella is one of the main vectors of the medically-important trematode Fasciola hepatica. In Cuba, the existence of natural P. columella populations that are either susceptible or resistant to F. hepatica infection offers a unique snail-parasite for study of parasite-host compatibility and immune function in gastropods. Here, we review all previous literature on this system and present new "omic" data that provide a molecular baseline of both P. columella phenotypes from naïve snails. Comparison of whole snail transcriptomes (RNAseq) and the proteomes of the albumen gland (2D-electrophoresis, MS) revealed that resistant and susceptible strains differed mainly in an enrichment of particular biological processes/functions and a greater abundance of proteins/transcripts associated with immune defense/stress response in resistant snails. These results indicate a differential allocation of molecular resources to self-maintenance and survival in resistant P. columella that may cause enhanced responsiveness to stressors (i.e. F. hepatica infection or tolerance to variations in environmental pH/total water hardness), possibly as trade-off against reproduction and the ecological cost of resistance previously suggested in resistant populations of P. columella.

Effects of azinphos-methyl on enzymatic activity and cellular immune response in the hemolymph of the freshwater snail Chilina gibbosa.

The use of a battery of biomarkers, especially those more closely related to species integrity, is desired for more complete ecotoxicological assessments of the effects of pesticide contamination on aquatic organisms. The phosphorodithioate azinphos-methyl has been intensively used in agriculture worldwide and have been found in the habitat of Chilina gibbosa, a freshwater snail endemic to South America. This snail has been proposed as a good model organism for ecotoxicity bioassays on the basis of studies focused mainly on enzymatic responses in whole tissue homogenates. Our aim was to evaluate the effect of an acute 48 h exposure to an environmental concentration of azinphos-methyl on C. gibbosa hemolymph enzymatic activity and cellular immune response. Our results show that cholinesterase activity was strongly inhibited (94%) in hemolymph of exposed snails. Carboxylesterase activity measured with p-nitrophenyl butyrate and glutathione S-transferase activity were augmented 47% and 89% respectively after exposure. No differences were found for hemolymph carboxylesterase activity measured with p-nitrophenyl acetate. These results differ from those reported for whole tissue homogenates and reveal that tissue-specific responses of enzymatic biomarkers exist in this species. Regarding immune cell response, hemocytes were identified for the first time for C. gibbosa. Their viability and phagocytic activity decreased after azinphos-methyl exposure although total number of circulating cells did not differ between treatments. We conclude that concentrations of azinphos-methyl that can be found in the environment can compromise both hemolymph cholinesterase activity and the immune system of C. gibbosa. Furthermore, we propose that carboxylesterase and glutathione S-transferase activities measured in hemolymph and hemocyte viability and phagocytic activity could be incorporated as sensitive biomarkers to evaluate the effects of pesticide exposure on this and related species.

Comparative immunological study of the snail Physella acuta (Hygrophila, Pulmonata) reveals shared and unique aspects of gastropod immunobiology.

The freshwater snail Physella acuta was selected to expand the perspective of comparative snail immunology. Analysis of Physella acuta, belonging to the Physidae, taxonomic sister family to Planorbidae, affords family-level comparison of immune features characterized from Biomphalaria glabrata, the model snail often used to interpret general gastropod immunity. To capture constitutive and induced immune sequences, transcriptomes of an individual Physella acuta snail, 12 h post injection with bacteria (Gram -/+) and one sham-exposed snail were recorded with 454 pyrosequencing. Assembly yielded a combined reference transcriptome containing 24,288 transcripts. Additionally, genomic Illumina reads were obtained (∼15-fold coverage). Recovery of transcripts for two macin-like antimicrobial peptides (AMPs), 12 aplysianins, four LBP/BPIs and three physalysins indicated that Physella acuta shares a similar organization of antimicrobial defenses with Biomphalaria glabrata, contrasting a modest AMP arsenal with a diverse set of antimicrobial proteins. The lack of predicted transmembrane domains in all seven Physella acuta PGRP transcripts supports the notion that gastropods do not employ cell-bound PGRP receptors, different from ecdysozoan invertebrates yet similar to mammals (vertebrate deuterostomes). The well-documented sequence diversification by Biomphalaria glabrata FREPs (immune lectins comprising immunoglobulin superfamily domains and fibrinogen domains), resulting from somatic mutations of a large FREP gene family is hypothesized to be unique to Planorbidae; Physella acuta revealed just two bonafide FREP genes and these were not diversified. Furthermore, the flatworm parasite Echinostoma paraensei, confirmed here to infect both snail species, did not evoke from Physella acuta the abundant expression of FREP proteins at 2, 4 and 8 days post exposure that was previously observed from Biomphalaria glabrata. The Physella acuta reference transcriptome also revealed 24 unique transcripts encoding proteins consisting of a single fibrinogen-related domain (FReDs), with a short N-terminal sequence encoding either a signal peptide, transmembrane domain or no predicted features. The Physella acuta FReDs are candidate immune genes based on implication of similar sequences in immunity of bivalve molluscs. Overall, comparative analysis of snails of sister families elucidated the potential for taxon-specific immune features and investigation of strategically selected species will provide a more comprehensive view of gastropod immunity.

Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites.

The anthropozoonotic metastrongyloid nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis, as well as Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior are currently considered as emerging gastropod-borne parasites and have gained growing scientific attention in the last years. However, the knowledge on invertebrate immune responses and on how metastrongyloid larvae are attacked by gastropod immune cells is still limited. This work aims to describe an in vitro system to investigate haemocyte-derived innate immune responses of terrestrial gastropods induced by vital axenic metastrongyloid larvae. We also provide protocols on slug/snail management and breeding under standardized climate conditions (circadian cycle, temperature and humidity) for the generation of parasite-free F0 stages which are essential for immune-related investigations. Adult slug species (Arion lusitanicus, Limax maximus) and giant snails (Achatina fulica) were maintained in fully automated climate chambers until mating and production of fertilized eggs. Newly hatched F0 juvenile specimens were kept under parasite-free conditions before experimental use. An improved protocol for gastropod haemolymph collection and haemocyte isolation was established. Giemsa-stained haemolymph preparations showed adequate haemocyte isolation in all three gastropod species. Additionally, a protocol for the production of axenic first and third stage larvae (L1, L3) was established. Haemocyte functionality was tested in haemocyte-nematode-co-cultures. Scanning electron microscopy (SEM) and light microscopy analyses revealed that gastropod-derived haemocytes formed clusters as well as DNA-rich extracellular aggregates catching larvae and decreasing their motility. These data confirm the usefulness of the presented methods to study haemocyte-mediated gastropod immune responses to better understand the complex biology of gastropod-borne diseases.

Identification of a phenoloxidase- and melanin-dependent defence mechanism in Achatina fulica infected with Angiostrongylus vasorum.

BACKGROUND: Angiostrongylus vasorum has different freshwater aquatic and terrestrial gastropod molluscs as an intermediate host, e.g. Arion spp. The mollusc Achatina fulica is a danger to public health, given the large diversity of nematodes utilizing it as an intermediate host, such as the parasites of the genus Angiostrongylus, of importance in human and veterinary medicine. Achatina fulica has been shown to have an excellent capacity for maintaining outbreaks and natural infections with A. cantonensis in Asia. Within the mollusc, the nematode parasites activate haemocytes and/or haemolymph factors and in some invertebrates, phenoloxidase (PO), that induces the release of toxic elements and eliminates the parasites. Despite the importance of A. fulica in the life-cycle of nematodes, little is known regarding the defence mechanisms involving PO in molluscs infected with nematodes. Here, the presence of PO and nitric oxide (NO) in the haemolymph and haemocytes of A. fulica infected with first-stage (L1) larvae of Angiostrongylus vasorum was evaluated, together with the presence of melanin in the cephalopod mollusc tissue. RESULTS: An increase in PO at one day post infection (dpi), in comparison with the control using the substrates L-tyrosine (F(4,90) = 6.73, P = 0.00006), L-DOPA (F(4,90) = 22.67, P = 0.02) and p-phenylenediamine (PPD) (F(4,90) = 27.58, P = 0.0019), was observed. PO increase coincided with the presence of melanin in the cephalopodal tissue. At 8 dpi, PO activity, compared to L-DOPA (F(4,90) = 22.67, P = 0.00002) and PPD (F(4,90) = 27.58, P = 0.079) decreased, while melanin increased. At 13 dpi, PO decreased with PPD (F(4,90) = 27.58, P = 0.000015) and also the amount of melanin observed in histology. At 30 dpi, PO increased along with the substrates L-DOPA and PPD, while melanin decreased. NO levels increased until 8 dpi, and decreased after 13 dpi. CONCLUSIONS: To our knowledge, this is the first study that illustrates PO activity in a helminth-infected A. fulica and provides the first observation of an L-tyrosine dependent PO activity in molluscs infected with A. vasorum. This work suggests that PO pathway may help to control A. vasorum infection in A. fulica.

Identification of Toll-like receptor family members in Oncomelania hupensis and their role in defense against Schistosoma japonicum.

The amphibious snail, Oncomelania hupensis, primarily distributed in the Far East, is the only intermediate host of Schistosoma japonicum, which causes the most virulent form of schistosomiasis. Obligatory parasitism of snails is the main vehicle for human and livestock infection and depends primarily on parasite infectivity, snail defense capacity and specificity, and parasite-snail compatibility. Therefore, the schistosome-snail interaction is biomedically significant, particularly the molecular mechanisms involved in the innate immune response against S. japonicum. Several immune effectors and signaling pathways have been successfully identified in mollusks, especially in Biomphalaria glabrata, the intermediate snail host of S. mansoni; however, limited information is available for O. hupensis. Here, we identified 16 Toll-like receptors (TLRs) in O. hupensis. These O. hupensis TLRs (OhTLRs) are highly expressed in haemocytes, the primary immune cell of mollusks. Most of the OhTLRs were more highly expressed in female gonads than in other tissues, which may suggest maternal immune transfer in O. hupensis. After S. japonicum challenge, the expression levels of all of the OhTLRs were significantly up-regulated at 6 h post-challenge; many of the OhTLR expression levels were inhibited at later time points in haemocytes, while they were inhibited and fluctuated to varying degrees in other tissues. Additionally, we further determined the tissue-specific expression and dynamic response against S. japonicum of one of the TLR signaling adaptors, myeloid differentiation factor 88 (MyD88), from O. hupensis. Three OhMyD88 genes were highly expressed in haemocytes, and were up-regulated in haemocytes and inhibited in the head-foot muscle at the early time-point after S. japonicum challenge; however, these had slower changes and longer durations compared to OhTLRs. These results provide evidence suggesting that immune effectors are involved in innate immune responses of O. hupensis against S. japonicum and may play a role in the activation of different haemocytes, and not limited for the early response to S. japonicum invasion. Further investigation into the varied expression of OhTLRs in other tissues after S. japonicum challenge will improve our understanding of TLR function in innate immunity of O. hupensis.

Extracellular trap-like fiber release may not be a prominent defence response in snails: evidence from three species of freshwater gastropod molluscs.

The discovery that mammalian neutrophils generate extracellular chromatin fibers that entrap/kill bacteria supported a new paradigm for innate immunity in animals. Similar findings in other models across diverse taxa have led to the hypothesis that the phenomenon is ancient and evolutionary conserved. Here, using a variety of synthetic (e.g. peptidoglycan) and biological (e.g. trematode larvae) components to investigate extracellular trap-like (ET-like) fiber production in vitro by haemocytes of Lymnaea stagnalis, Radix lagotis and Planorbarius corneus snails, ET-like fibers were rarely observed. We suggest, therefore, that ET-like fibers play a marginal role in defence of these snail species and thus the fiber production may not be a critical process underpinning immunity in all invertebrate species.

Transcriptome mining of immune-related genes in the muricid snail Concholepas concholepas.

The population of the Chilean endemic marine gastropod Concholepas concholepas locally called "loco" has dramatically decreased in the past 50 years as a result of intense activity of local fisheries and high environmental variability observed along the Chilean coast, including episodes of hypoxia, changes in sea surface temperature, ocean acidification and diseases. In this study, we set out to explore the molecular basis of C. concholepas to cope with biotic stressors such as exposure to the pathogenic bacterium Vibrio anguillarum. Here, 454pyrosequencing was conducted and 61 transcripts related to the immune response in this muricid species were identified. Among these, the expression of six genes (CcNFκß, CcIκß, CcLITAF, CcTLR, CcCas8 and CcCath) involved in the regulation of inflammatory, apoptotic and immune processes upon stimuli, were evaluated during the first 33 h post challenge (hpc). The results showed that CcTLR, CcCas8 and CcCath have an initial response at 4 hpc, evidencing an up-regulation from 4 to 24 hpc. Notably, the response of CcNFKB occurred 2 h later with a statistically significant up-regulation at 6 hpc and 10 hpc. Furthermore, the challenge with V. anguillarum induced a statistically significant down-regulation of CcIKB between 2 and 10 hpc as well as a down-regulation of CcLITAF between 2 and 4 hpc followed in both cases by an up-regulation between 24 and 33 hpc. This work describes the first transcriptomic effort to characterize the immune response of C. concholepas and constitutes a valuable transcriptomic resource for future efforts to develop sustainable aquaculture and conservations tools for this endemic marine snail species.

Potential for adaptation to climate change: family-level variation in fitness-related traits and their responses to heat waves in a snail population.

BACKGROUND: On-going global climate change poses a serious threat for natural populations unless they are able to evolutionarily adapt to changing environmental conditions (e.g. increasing average temperatures, occurrence of extreme weather events). A prerequisite for evolutionary change is within-population heritable genetic variation in traits subject to selection. In relation to climate change, mainly phenological traits as well as heat and desiccation resistance have been examined for such variation. Therefore, it is important to investigate adaptive potential under climate change conditions across a broader range of traits. This is especially true for life-history traits and defences against natural enemies (e.g. parasites) since they influence organisms' fitness both directly and through species interactions. We examined the adaptive potential of fitness-related traits and their responses to heat waves in a population of a freshwater snail, Lymnaea stagnalis. We estimated family-level variation and covariation in life history (size, reproduction) and constitutive immune defence traits [haemocyte concentration, phenoloxidase (PO)-like activity, antibacterial activity of haemolymph] in snails experimentally exposed to typical (15 °C) and heat wave (25 °C) temperatures. We also assessed variation in the reaction norms of these traits between the treatments. RESULTS: We found that at the heat wave temperature, snails were larger and reproduced more, while their immune defence was reduced. Snails showed high family-level variation in all examined traits within both temperature treatments. The only negative genetic correlation (between reproduction and antibacterial activity) appeared at the high temperature. However, we found no family-level variation in the responses of most examined traits to the experimental heat wave (i.e. largely parallel reaction norms between the treatments). Only the reduction of PO-like activity when exposed to the high temperature showed family-level variation, suggesting that the cost of heat waves may be lower for some families and could evolve under selection. CONCLUSION: Our results suggest that there is genetic potential for adaptation within both thermal environments and that trait evolution may not be strongly affected by trade-offs between them. However, rare differences in thermal reaction norms across families indicate limited evolutionary potential in the responses of snails to changing temperatures during extreme weather events.

Identification and functional characterization of Oncomelania hupensis macrophage migration inhibitory factor involved in the snail host innate immune response to the parasite Schistosoma japonicum.

Schistosomiasis, caused by parasitic trematodes of the genus Schistosoma, remains a devastating public health problem, with over 200 million people infected and 779 million people at risk worldwide, especially in developing countries. The freshwater amphibious snail Oncomelania hupensis is the obligate intermediate host of Schistosoma japonicum. This unique and long-standing host-parasite interaction highlights the biomedical importance of the molecular and cellular mechanisms involved in the snail immune defense response against schistosome infection. In recent years, a number of immune-related effectors and conserved signalling pathways have been identified in molluscs, especially in Biomphalaria glabrata, which is an intermediate host for Schistosoma mansoni, but few have been reported in O. hupensis. Here we have successfully identified and functionally characterized a homologue of mammalian macrophage migration inhibitory factor (MIF) from O. hupensis (OhMIF). MIF, a pleiotropic regulator of innate immunity, is a constitutively expressed mediator in the host's antimicrobial defense system and stress response that promotes the pro-inflammatory functions of immune cells. In the present study, we detected the distribution of OhMIF in various snail tissues, especially in immune cell types (hemocytes) and found that OhMIF displays significantly increased expression in snails following challenge with S. japonicum. Knockdown of OhMIF was conducted successfully in O. hupensis and significantly reduced the percentage of phagocytic cell populations in circulating hemocytes. Furthermore, OhMIF is not only implicated in the activation and differentiation of hemocytes, but also essential to promote the migration and recruitment of hemocytes towards the infected sites. These results provide the first known functional evidence in exploring the molecular mechanisms involved in the O. hupensis innate immune defense response to the parasite S. japonicum and help to better understand the complex host-parasite interaction.