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.

Hyperdiverse gene cluster in snail host conveys resistance to human schistosome parasites.

Schistosomiasis, a neglected global pandemic, may be curtailed by blocking transmission of the parasite via its intermediate hosts, aquatic snails. Elucidating the genetic basis of snail-schistosome interaction is a key to this strategy. Here we map a natural parasite-resistance polymorphism from a Caribbean population of the snail Biomphalaria glabrata. In independent experimental evolution lines, RAD genotyping shows that the same genomic region responds to selection for resistance to the parasite Schistosoma mansoni. A dominant allele in this region conveys an 8-fold decrease in the odds of infection. Fine-mapping and RNA-Seq characterization reveal a <1Mb region, the Guadeloupe Resistance Complex (GRC), with 15 coding genes. Seven genes are single-pass transmembrane proteins with putative immunological roles, most of which show strikingly high nonsynonymous divergence (5-10%) among alleles. High linkage disequilibrium among three intermediate-frequency (>25%) haplotypes across the GRC, a significantly non-neutral pattern, suggests that balancing selection maintains diversity at the GRC. Thus, the GRC resembles immune gene complexes seen in other taxa and is likely involved in parasite recognition. The GRC is a potential target for controlling transmission of schistosomiasis, including via genetic manipulation of snails.

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.

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.

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.

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.

Susceptibility of the Giant African snail (Achatina fulica) exposed to the gastropod parasitic nematode Phasmarhabditis hermaphrodita.

The Giant African snail (Achatina fulica) is a major pest in tropical countries. Current control methods involve the use of slug pellets (metaldehyde) but they are ineffective, therefore new methods of control are needed. We investigated whether A. fulica is susceptible to the gastropod parasitic nematode Phasmarhabditis hermaphrodita, which has been developed as a biological control agent for slugs and snails in northern Europe. We exposed A. fulica to P. hermaphrodita applied at 30 and 150nematodes per cm(2) for 70days and also assessed feeding inhibition and changes in snail weight. We show that unlike the susceptible slug species Deroceras panormitanum, which is killed less than 30days of exposure to P. hermaphrodita, A. fulica is remarkably resistant to the nematode at both doses. Also P. hermaphrodita does not reduce feeding in A. fulica nor did it have any effect on weight gain over 70days. Upon dissection of infected A. fulica we found that hundreds of P. hermaphrodita had been encapsulated, trapped and killed in the snail's shell. We found that A. fulica is able to begin encapsulating P. hermaphrodita after just 3days of exposure and the numbers of nematodes encapsulated increased over time. Taken together, we have shown that A. fulica is highly resistant to P. hermaphrodita, which could be due to an immune response dependent on the snail shell to encapsulate and kill invading parasitic nematodes.

Identification and characterization of a goose-type lysozyme from sewage snail Physa acuta.

Freshwater snail Physa acuta has been considered as an important invasive species and medical mollusc. Field investigation has shown that this snail could survive better than other snails in polluted water bodies. To understand the immune mechanisms of P. acuta, suppression subtractive hybridization hepatopancreas cDNA library has been constructed with bacterial challenge. In this study, a full-length cDNA of a novel goose-type lysozyme (PALysG) has been identified from P. acuta by EST and RACE technique. The conservative structure domains share high homology with other molluscan g-type lysozymes including the SLT domain, the substrate binding sites, the catalytic residues, three alpha-helices structures and six molluscan specific cysteines. Meanwhile, PALysG is the first record of goose-type lysozyme in Gastropoda. Real-time PCR indicated that PALysG mRNA had been expressed significantly at high levels in hepatopancreas for 8-48 h. PALysG recombinant protein displayed the lytic activity of g-type lysozyme with other organisms against Micrococcus lysodikicus.

Molecular cloning of α-2-macroglobulin from hemocytes of common periwinkle Littorina littorea.

We report the sequence of the proteinase inhibitor with a wide inhibition spectrum, α-2-macroglobulin (α2M), belonging to the thioester superfamily of proteins. This is the first α2M sequence from coenogastropod prosobranch snails. The full-length cDNA was cloned by RACE method, spans 7897 bp and contains an open reading frame of 5460 bp. The ORF encodes a protein of 1819 amino acids. The deduced mature protein contains 1795 amino acids with a molecular weight of 200 kDa and isoelectric point of 5.00. Littorina littorea α2M bears 4 conserved α2M domains and one internal thioester. Phylogenetic analysis showed that the sequence forms well supported cluster with Mollusca species and other representatives of Lophotrochozoa.

Identification of a novel molluscan short-type peptidoglycan recognition protein in disk abalone (Haliotis discus discus) involved in host antibacterial defense.

Peptidoglycan recognition proteins (PGRPs) are a widely studied group of pattern recognition receptors found in invertebrate as well as vertebrate lineages, and are involved in bacterial pathogen sensing. However, in addition to this principal role, they can also function in multiple host defense processes, including cell phagocytosis and hydrolysis of peptidoglycans (PGNs). In this study, a novel invertebrate short-type PGRP was identified in disk abalone (Haliotis discus discus) designated as AbPGRP. The complete coding sequence of AbPGRP was 534 bp, encoding a 178-amino acid protein with a predicted molecular mass of 20 kDa. The AbPGRP gene had a bipartite arrangement consisting of two exons separated by a single intron. Homology analysis revealed that AbPGRP shares conserved features, including amino acid residues critical for substrate and ion binding as well as for its amidase activity, with homologs of other species. Phylogenetic analysis of AbPGRP revealed that it likely evolved from a common ancestor of invertebrates, having significant homology with other molluscan PGRPs. Recombinant AbPGRP exhibited detectable, dose-dependent PGN-hydrolyzing activity with the presence of Zn(2+), and strong antibacterial activity against Vibrio tapetis, consistent with the functional properties previously reported for PGRPs in other mollusks. Moreover, AbPGRP transcription was induced upon treatment of healthy abalones with bacterial peptidoglycan and lipopolysaccharide, although the expression profiles differed with treatment, suggesting a capacity for discriminating between bacterial pathogens through molecular pattern recognition. Collectively, the findings of this study indicate that AbPGRP is a true homolog of invertebrate PGRPs and likely plays an indispensable role in host immunity.

Molecular cloning, characterization and expression analysis of three heat shock responsive genes from Haliotis diversicolor.

In this study, molecular characterization and expression of three heat shock responsive genes were analyzed as indicators to understand the mechanism of heat shock response of small abalone Haliotis diversicolor under stresses. The full length cDNA of heat shock transcriptional factor 1 (HdHSF1), heat shock factor binding protein 1(HSBP1), and heat shock protein 90 (HdHSP90) are 1548 bp, 809 bp, and 2592 bp respectively, encoding a protein of 515 aa, 75 aa, and 728 aa respectively. Real time quantitative PCR analysis revealed that these three genes are constitutively expressed in 7 selected tissues. The expression level of HdHSF1 in gills was higher than that in other tissues (p < 0.05). The highest expression level of HdHSBP1 was detected in hemocytes. The highest expression level of HdHSP90 was in the digestive tract and colleterial gland. The HdHSF1 expression level in the gills was up-regulated significantly (p < 0.05) after thermal stress and hypoxia exposure respectively. On the contrary, HdHSBP1 was down-regulated both in gills and hemocytes after thermal stress and the same as in gills after hypoxia stress. HdHSP90 expression level was also up-regulated in gills and hemocytes after both thermal and hypoxia stresses. These results indicated that these three heat shock responsive genes play important roles in response to thermal and hypoxia stress.

Immune response of apoptosis-related cysteine peptidases from the red abalone Haliotis rufescens (HrCas8 and HrCas3): molecular characterization and transcription expression.

Caspases play an important role in the different stages of programmed cell death, or apoptosis, which has been related to the immune response in multicellular organisms. The present study characterized an initiator caspase (HrCas8) and an effector caspase (HrCas3) from the red abalone Haliotis rufescens using the RACE method and qPCR analysis. HrCas8 showed a complete sequence of 2529 base pairs (bp) with an open-reading frame (ORF) of 1911 bp, a 5'UTR of 201 bp, and a 3'UTR of 417 bp. The estimated molecular mass for the 636 amino acids from HrCas8 was 71.5 kDa with an isoelectric point of 6.2. The HrCas8 sequence had two death-effector domains (DEDs) and the subunits p20 and p10, in addition to an active site characteristic of cysteine proteins. Meanwhile, the effector caspase HrCas3 showed a complete sequence of 1404 bp, a 5'UTR of 82 bp, and a 3'UTR of 574 bp. The ORF of this caspase had 747 bp that coded for 248 residues. Moreover, the predicted molecular mass of HrCas3 was 29.4 kDa; the theoretical isoelectric point was 5.70, and the sequence evidenced a conserved caspase recruitment domain (CARD). The distribution of the caspases in distinct tissues revealed that HrCas8 was principally expressed in the hemolymph, while HrCas3 had a higher expression in the gills. A basal level of expression was found for both caspases in muscle tissue. The immune response of caspases in H. rufescens was evaluated through an injection of Vibrio anguillarum. The results showed an increase in the transcription of HrCas8 post-challenge, as well as an activation of HrCas3, which together suggest the initiation of apoptosis as a response to bacterial infection in H. rufescens.

Molecular cloning and expression of IRAK-4, IL-17 and I-κB genes in Haliotis rufescens challenged with Vibrio anguillarum.

The candidate genes interleukin-1 receptor associated kinase 4 (IRAK-4), Interleukin 17 (IL-17) and Inhibitor of NF-κB (I-κB) were cloned and evaluated in Californian abalone (Haliotis rufescens) hemocytes in response to Vibrio anguillarum. Molecular characterization evidenced that HrI-κB has a full cDNA sequence of 3027 bp with an encoding region of 401 amino acids (aa), HrIRAK-4 comprised 1969 bp that encoded for 516 aa, and Hr-IL17 had a full sequence of 806 bp encoding for 165 aa. qPCR analysis showed the higher constitutive expression level of Hr-IL17 in hemocytes; meanwhile Hr-IκB and Hr-IRAK4 gene expression levels were higher in gills and mantle. The assessment of gene expression in hemocytes after infection with V. anguillarum evidences the immune responses of Hr-IκB, Hr-IRAK4, and Hr-IL17 and their relationships through the NF-κB signaling pathway.

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.

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata.

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches. Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.

Phagocytosis and respiratory burst activity of haemocytes from the ivory snail, Babylonia areolata.

Haemocytes from the ivory snail, Babylonia areolata phagocytized Saccharomyces cerevisiae and Vibrio parahaemolyticus after 30 min. Haemocytes phagocytized V. parahaemolyticus at a greater rate than they phagocytized S. cerevisiae. The phagocytic rate (PP) of V. parahaemolyticus by granulocytes to was a little higher than that of S. cerevisiae. The phagocytic index (PI) of V. parahaemolyticus by granulocytes was significantly higher than that of S. cerevisiae. The same was true of hyalinocytes. The PP of granulocytes was significantly higher than that of hyalinocytes for each pathogen. No difference in PI was observed in granulocytes and hyalinocytes. Two defense mechanisms of B. areolata were quantified using flow cytometry. Haemocyte phagocytosis was quantified using fluorescent microbeads and respiratory burst activity was measured using H2O2 increases detected by 2', 7'-dichlorofluorescein diacetate. Both phagocytosis and respiratory burst activity of the haemocytes increased over time. After 90 min the phagocytic rate no longer increased. In the case of respiratory burst, the greatest increase in fluorescence occurred between 30 and 120 min, no further increase was seen after 120 min. These results showed unequivocally that a native (unstimulated) haemocyte oxidative burst was active in B. areolata. The aim of this study was to further the knowledge of immunology in gastropods.

A comparative analyses of morphological variations, phagocytosis and generation of cytotoxic agents in flow cytometrically isolated hemocytes of Indian molluscs.

A comparative analyses of hemocytes of molluscs, Pila globosa (Gastropoda: Prosobranchia), Bellamya bengalensis (Gastropoda: Prosobranchia) and Lamellidens marginalis (Bivalvia: Eulamellibranchiata) were carried out for morphotype and subpopulation identification, analyses of phagocytosis and generation of cytotoxic agents. Flow cytometry and microscopic analyses of hemocytes revealed the existence of agranulocytes (blast like cells, round hyalinocytes and spindle hyalinocytes), semigranulocytes (semigranular asterocytes and round semigranulocytes) and granulocytes (round granulocytes, spindle granulocytes and granular asterocytes) as three morphotypes. In P. globosa, granulocytes and semigranulocytes and in B. bengalensis granulocytes and agranulocytes are the chief phagocytes and major producers of superoxide anion and nitric oxide. In L. marginalis, granulocytes were identified as principal phagocytes with prominent activity of superoxide anion and nitric oxide. Highest activity of phenoloxidase was recorded in the agranulocytes of P. globosa with moderate activities among other morphotypes of all three species. Differential result may be due to species specific response, non-identical habitat preference and related adaptation of the species to their different ecological niches.

Molecular characterization and expression analysis of IκB from Haliotis discus discus.

Innate immune system relies on the recognition of pathogen associated molecular patterns present in the microbes by the pattern recognition receptors leading to the activation of signaling cascade and subsequent synthesis of cytokines. NF-κB is a major stimulus activated transcription factor, which regulates the expression of a diverse array of genes. IκB is an inhibitor of NF-κB, retaining NF-κB in an inactive state in the cytoplasm. In this study, we have reported the characterization of first abalone IκB (HdIκB). The cDNA possessed an ORF of 1200 bp coding for a protein of 400 amino acids with molecular mass of 45 kDa and isoelectric point of 4.7. HdIκB protein possessed a conserved phosphorylation site (58)DSGIFS(63) in the N-terminal region, six ankyrin repeats, and a PEST sequence in the C-terminal region. A casein kinase II phosphorylation site could also be observed in the PEST sequence. Constitutive expression of HdIκB revealed its physiological significance since NF-κB is known to be activated by various stimuli. Elevated expression of HdIκB transcripts could be observed in abalones challenged with various mitogens and live microbes. This novel characterization of abalone IκB would further be a positive approach in the affirmation of evolutionary conservation and significance of this protein as a repressor/inhibitor of a pleiotropic transcription factor like NF-κB.

Molecular insights of the first gastropod TLR counterpart from disk abalone (Haliotis discus discus), revealing its transcriptional modulation under pathogenic stress.

Toll-like receptors (TLRs) are well-characterized pattern recognition receptors of innate immunity, known to induce immune responses against the pathogens by interacting with evolutionarily conserved pathogen-associated molecular patterns (PAMPs). In this study, a novel TLR homolog from disk abalone (Haliotis discus discus) was identified and characterized at molecular level. The open reading frame (ORF) of AbTLR is 3804 bp in length and encodes a 1268 amino acid peptide with a calculated molecular mass of 143.5 kDa. The deduced protein shows typical TLR domain architecture, with leucine rich repeats (LRR) and the toll-interleukin receptor (TIR) domain. Phylogenetic analysis revealed a close evolutionary relationship for AbTLR to its invertebrate counterparts, with close clustering to the molluscan homologs. Quantitative real-time PCR detected ubiquitous transcription of AbTLR in healthy tissues, but with highest levels in hemocytes. Differential transcriptional modulation of AbTLR was observed in abalone hemocytes and gills upon immune challenge, whereby Vibrio parahaemolyticus and purified lipopolysaccharide (LPS) enhanced the transcript level prominently. In addition, the viral hemorrhagic septicemia virus induced AbTLR transcription in hemocytes and gills, representing the first evidence of viral-induced immune response in mollusks to date. Collectively, our findings support a putative role for AbTLR in abalone antiviral and antibacterial defense.