Unveiling novel RNA viruses in trematodes parasitizing common periwinkle: Implications for host-parasite interactions.

This study characterized novel RNA viruses, parasites of parasites, or hyperparasites identified during transcriptomic analyses of two trematode species, Cryptocotyle lingua and Himasthla elongata, infecting a sea snail, Littorina littorea. According to the viral genome structures and phylogenetic analysis, Cryptolin alternavirus (ClRNAV1), Cryptolin calicivirus (ClRNAV2) and Himastelon rhabdovirus 1 (HeRNAV1) were respectively classified within the families Alternaviridae, Caliciviridae and Rhabdoviridae. They replicate species-specifically in two studied phases of trematode live cycle: intramolluscan parthenogenetic rediae and free-swimming cercariae. ClRNAV1 showed significantly higher expression in C. lingua cercariae relative to rediae. HeRNAV1's similarity to rabies viruses raises questions about its potential effects on the nervous system of H. elongata. This 'trematode rabies' could enable the use of genetically modified viruses for developing new methods to control the spread and intensity of diseases caused by trematodes.

Transcriptomic profiling of immune-associated molecules in the coelomocytes of lugworm Arenicola marina (Linnaeus, 1758).

Organization and functioning of immune system remain unevenly studied in different taxa of lophotrochozoan animals. We analyzed transcriptomic data on coelomocytes of the lugworm Arenicola marina (Linnaeus, 1758; Annelida, Polychaeta) to gain insights into the molecular mechanisms involved in polychaete immunity. Coelomocytes are specialized motile cells populating coelomic fluid of annelids, responsible for cellular defense reactions and providing humoral immune factors. The transcriptome was enriched with immune-related transcripts by challenging the cells in vitro with lipopolysaccharides of Escherichia coli and Zymosan from Saccharomyces cerevisiae. Our analysis revealed a multifaceted and complex internal defense system of the lugworm. A. marina possesses orthologs of proto-complement-like factors: six thioester-containing proteins, a complement-like receptor, and a MASP-related serine protease (MReM2). A. marina coelomocytes employ pattern-recognition receptors to detect pathogens and regulate immune responses. Among them, there are 18 Toll-like receptors and various putative lectin-like proteins with evolutionary conserved and taxa-specific domains. C-type lectins and a novel family of Gal-binding and CUB domains containing receptors were the most abundant in the transcriptome. The array of pore-forming proteins in the coelomocytes was surprisingly reduced compared to that of other invertebrate species. We characterized a set of conserved proteins metabolizing reactive oxygen species and nitric oxide and expanded the arsenal of potential antimicrobial peptides. Phenoloxidase activity in immune cells of lugworm is mediated only by laccase enzyme. The described repertoire of immune-associated molecules provides valuable candidates for further functional and comparative research on the immunity of annelids.

Identification of peptidoglycan recognition proteins in hemocytes and kidney of common periwinkle Littorinalittorea.

Peptidoglycan Recognition Proteins (PGRPs) are a diverse group of proteins involved in innate immunity. In particular, PGRPs have been shown to participate in immune pattern recognition in various mollusks. However, they have not been described in Caenogastropoda, a large molluscan group comprising sea, freshwater and land snails. In this study, four short PGRPs with molecular weights ranging from 21 to 34 kDa and their isoforms were identified and structurally characterized in the kidney and hemocytic transcriptomes of a caenogastropod mollusk Littorina littorea. All of them (LlPGRP1-4) are secretory, possess a signal peptide and a characteristic N-terminal N-acetylmuramoyl-l-alanine amidase (Ami) domain with conserved Zn2+ binding- and amidase catalytic sites. The shortest proteins, LlPGRP1 and LlPGRP2, have no additional conserved motifs on the N-terminus. In longer and most abundantly expressed LlPGRP3 and LlPGRP4 the Ami-domain is combined with an N-terminal SH3-domain and a cysteine-rich motif, respectively. Expression analysis showed that LlPGRPs of the common periwinkle were uninvolved in the immune response to infection with trematode Himasthla elongata though they might act in antibacterial defense.

Toll-like signaling pathway in the transcriptome of Littorina littorea.

The transcriptome of the caenogastropod mollusk Littorina littorea was scanned for the presence of sequences encoding Toll-like receptors (TLRs) and corresponding proteins involved in downstream TLR signaling pathway. In the transcriptomic snapshots of hemocytes and kidney tissues, 45 complete TLRs encoded by 35 genes were identified. Out of the 59 non-TLR molecules involved in a canonical TLR signaling pathway, 35 genes were classified as homologous and could be placed within the TLR-mediated MyD88-and MAPK-dependent circuitries. No reference vertebrate adapters TIRAP, TRIF and TRAM were identified in the transcriptome. The results of RNA-seq experiments with an immune challenge (rediae of the digenean Himasthla elongata) indicate that four TLRs (LlTLR1, 3, 5 and 8) and a set of upregulated genes involved in signal transduction (LlMyd88, LlTNFα, LlCASP8, LlFADD, LlNFKBIA (IkBα), LlIRAK1, LlSTAT1, LlMAPK14 (P38), LlMAP2K1 (MEK1/2), LlIRF3 and LlIRF5) may participate in the anti-digenean immune response of L. littorea.

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.

Derivatives of the lectin complement pathway in Lophotrochozoa.

A plethora of non-overlapping immune molecular mechanisms in metazoans is the most puzzling issue in comparative immunobiology. No valid evolutionary retrospective on these mechanisms has been developed. In this study, we aimed to reveal the origin and evolution of the immune complement-like system in Lophotrochozoa. For this, we analyzed publicly available transcriptomes of prebilaterian and lophotrochozoan species, mapping lineage-specific molecular events on the phylogenetic tree. We found that there were no orthologs of mannose-binding lectin (MBL) and ficolins (FCN) in Lophotrochozoa but C1q-like proteins (C1qL), bearing both a collagen domain and a globular C1q domain, were omnipresent in them. This suggests that among all complement-like activators the C1qL-specific domain architecture was an evolutionarily first. Two novel protostomian MASP-Related Molecules, MReM1 and MReM2, might hypothetically compensate for the loss of a prebilaterian MASP-orthologous gene and act in complex with C1qL and C1qDC as a "proto-activator" of an ancient "proto-complement". We proposed a new model of the complement evolution predicting that numerous lineage-specific complement-like systems should have evolved from a stem "antique" molecular complex. First evolved in the common ancestor of coelomic animals, the "antique" humoral complex consisted of a TEP molecule, the common ancestor of TEP-associated proteases (C2/Bf/Сf/Lf), the common ancestor of MASP-like proteases (MASP/C1r/C1s, MReM1/MReM2) and multimeric recognition proteins (C1q-, MBL- and FCN-homologs). Further evolutionary specialization and expansion of the complex was independent and lineage-specific, examples being the mammalian complement system and the Apogastropoda complement-like complex. The latter includes an impressive array of multimeric recognition proteins, the variable immunoglobulin and lectin domain containing molecules (VIgL), homologous to C1q, MBL, FCN and other lectins. Four novel polymorphic subfamilies of VIgLs were found to be expressed in Apogastropoda: C1q-related proteins (QREP), zona pellucida-related proteins (ZREP), Scavenger Receptor Cys-Rich-related proteins (SREP) and HPA-lectin related proteins (HREP). The transcriptional response of fibrinogen-related proteins of VIgL family (LlFREP), LlQREP and LlSREP to infestation of common periwinkle, Littorina littorea, with digenean parasite Himasthla elongata correlates with that of LlMReM1, supporting the model suggested in this study.

Immune repertoire in the transcriptome of Littorina littorea reveals new trends in lophotrochozoan proto-complement evolution.

The evolution of complement system in invertebrates is poorly investigated. While the repertoire of complement genes in several Ecdysozoa lineages is found substantially different from that of Deuterostomia, the composition and function of the complement in the second protostome lineage, Lophotrochozoa, remains unclear. Here we report the general description of new transcriptomic data on the common periwinkle, Littorina littorea, and trace the evolutionary trajectories of the ancestral proto-complement repertoire. The repertoire is defined as immune cascade providing the minimum set of C3-associated molecules required for C3b amplification, opsonization of the targets and their phagocytosis: thioester protein (TEP) C3, serine protease C2/factor B (Bf) and complement receptors (CR). The reference transcriptome of L. littorea was built from the dual-species RNA-seq experiment with the periwinkle and its tissue digenean parasite Himasthla elongata. Five TEPs, including the ortholog of the C3, are found expressed in the in the mollusk's inflamed tissues. The homolog of the complement receptors CR1/CR2 is also expressed, however the ortholog of Bf is not. The extensive phylogenetic analysis showed that the C3 ortholog and the complement receptors are retained in all key lophotrochozoan taxa: Mollusca, Annelida and Brachiopoda. However, the Bf ortholog was lost at least three times independently in different lineages: i) Cephalopoda, ii) a common ancestor of all Gastropoda and iii) one of the Annelida lineage, Clitellata. Both C3 and Bf molecules were retained in bivalve species, brachiopods and annelid worms from the Polychaeta lineage. Hypothetically, the function of the lost Bf in these animals can be compensated by Factor L (Lf) - the serine protease first found in L. littorea and homologous to both, the Bf and the arthropod factor C (Cf). The contrast differences in proto-complement repertoire between the sister mollusk' taxa, Bivalvia and Gastropoda (the conserved and modified sets, respectively), can underlie differences in their susceptibility to digenean infection.

Membrane Attack Complex/Perforin domain-containing proteins in a dual-species transcriptome of caenogastropoda Littorina littorea and its trematode parasite Himasthla elongata.

The MACPF domain-containing proteins potentially able to build a transmembrane pore structure are found in the transcriptome of a common periwinkle kidney loaded with trematode rediae. Two homologs of mammalian Macrophage expressed gene 1 (Mpeg1), LlMpeg1-1 and LIMpeg1-2, share similar domain structure with the only difference such as LIMpeg1-2 transcript lacks of a C-terminal transmembrane helix. Expression of membrane-anchored protein LlMpeg1-1 is similar in kidneys of naturally infected with trematode Himasthla elongata and uninfected snails. The expression of the second soluble LIMpeg1-2 protein is 4-fold upregulated under infection. The third MACPF protein found in Littorina littorea kidney is homologous with Perivitellin - 2 67 kDa subunit named LlPV2-67 and the expression of the transcript is 3-fold upregulated in the kidney of infected snails. The last two molecules are candidate effectors that may participate in the immune response of common periwinkles to trematode infestation. A single parasite-expressed MACPF-like protein was recorded from the transcriptome of Himasthla elongata.

Lectin-like molecules in transcriptome of Littorina littorea hemocytes.

The common periwinkle Littorina littorea was introduced in the list of models for comparative immunobiology as a representative of phylogenetically important taxon Caenogastropoda. Using Illumina sequencing technology, we de novo assembled the transcriptome of Littorina littorea hemocytes from 182 million mRNA-Seq pair-end 100 bp reads into a total of 15,526 contigs clustered in 4472 unigenes. The transcriptome profile was analyzed for presence of carbohydrate-binding molecules in a variety of architectural contexts. Hemocytes' repertoire of lectin-like proteins bearing conserved carbohydrate-recognition domains (CRDs) is highly diversified, including 11 of 15 lectin families earlier described in animals, as well as the novel members of lectin family found for the first time in mollusc species. The new molluscan lineage-specific domain combinations were confirmed by cloning and sequencing, including the fuco-lectin related molecules (FLReMs) composed of N-terminal region with no sequence homology to any known protein, a middle Fucolectin Tachylectin-4 Pentaxrin (FTP) domain, and a C-terminal epidermal growth factor (EGF) repeat region. The repertoire of lectin-like molecules is discussed in terms of their potential participation in the receptor phase of immune response. In total, immune-associated functions may be attributed to 70 transcripts belonging to 6 lectin families. These lectin-like genes show low overlap between species of invertebrates, suggesting relatively rapid evolution of immune-associated genes in the group. The repertoire provides valuable candidates for further characterization of the gene functions in mollusc immunity.

Himasthla elongata: Implantation of rediae to the specific iteroparous long-living host, Littorina littorea, results in the immune rejection.

All semelparous short-lived gastropods studied so far for the experimental transplantation of trematode parthenitae, from one specific host to another, showed more or less successful acceptance of implanted parasites. We implanted echinostomatid rediae, Himasthla elongata, to the specific iteroparous long-living host, coenogastropod Littorina littorea. Using simple and low-invasive implantation techniques we have tested 680 snails injected with 75 redia microhemipopulations (MHP) harvested from naturally infected snails. Neither young nor mature rediae were able to survive in the recipient periwinkles in the course of 30 days post-implantation. A strong immune response from the host was already evident within the first week after implantation: initial inactivation of motile rediae with toxic humoral immune factors, following encapsulation of the implants and increased hemocyte counts. In contrast, rediae from the same MHPs showed perfect survival rates in primary in vitro axenic cultures. The failure of the transplantation experiments is explained in terms of the compatibility matching phenotype model. In the studied host-parasite combination all periwinkles are potentially susceptible and all rediae MHPs are potentially infective, however the probability of the compatible phenotypes matching is virtually low. Low investment in L. littorea annual reproduction would result in increased investment in self maintenance and immune mechanisms, causing the general resistance to the trematode infestation. Presumably, this resistance is relatively higher in long-lived iteroparous gastropods compare to semelparous short-lived mollusks such as pulmonates.

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.

The enigma of the haemogram "left-shift" in periwinkles infected with trematodes.

To make further progress in the understanding of digenean immune evasive tactics in their snail host, we compared the haemopoietic parameters and haemocyte functional potencies in the prosobranch Littorina littorea, which were either healthy or infected with echinostome trematode Himasthla elongata. The haemocyte concentration in the circulation of infected individuals was significantly higher than in uninfected ones, 3300 microl(-1) and 1882 microl(-1), respectively. Intense haemopoiesis in haemolymph of infected snails was evidenced by 4-fold higher BrdU incorporation into nuclei of haemocytes as well as elevated level of cyclin D expression in these cells. Evident skewing of the haemocyte population toward a higher frequency of immature, undifferentiated haemocytes in infected L. littorea was found. Haemocytes in infected snails had a much lower functional potency in production of reactive oxygen intermediates (ROI) and cell-mediated cytotoxicity. Correlation analysis shows that both cytotoxic and ROI generation values were significantly and negatively correlated with proportion of juvenile cells in circulation. Experimental injection of H. elongata excretory/secretory products modulated haemopoiesis toward increasing a juvenile cells proportion by 7th day post-injection. This can be considered a haemogram "left-shift" by analogy to that seen in the human neutrophil compartment, when more immature bandforms are found in blood during acute inflammation or bone marrow disorders. We hypothesize that echinostomatide trematodes may interfere in the normal neuroendocrine management of haemopoiesis in the host and cause a haemopoietic signal to initiate multiplication to near neoplastic levels.

Modulation of mitogen-activated protein kinases (MAPK) activity in response to different immune stimuli in haemocytes of the common periwinkle Littorina littorea.

The modulation of mitogen-activated protein kinase (MAPK) activity in haemocytes of the common periwinkle (Littorina littorea) in response to immune challenges by lipopolysaccharide from Echerichia coli (LPS), mannan from baker's yeast Saccharomyces cerevisiae and secretory-excretory products (SEP) of trematodes Himasthla elongata (Echinostomatidae) or after the treatment with phorbol ester (PMA) has been studied by Western blotting using affinity purified rabbit polyclonal antibodies. Exposure of the cells in suspension to PMA, LPS and mannan triggered an activation of p38 and ERK2. The JNK-mediated cascade was modulated differently by the elicitors examined. PMA treatment caused a transient activation of the JNK54 isoform, LPS exposure resulted in a decrease in activity of JNK46, and mannan had no effect on JNK phosphorylation status. Incubation of periwinkle haemocytes in culture medium containing trematode SEP did not affect the activity of any MAPK.

Rediae of echinostomatid and heterophyid trematodes suppress phagocytosis of haemocytes in Littorina littorea (Gastropoda: Prosobranchia).

A modulation of the phagocytic activity of hemocytes from the common periwinkle Littorina littorea by secretory-excretory products (SEP) released by trematode rediae during axenic in vitro cultivation was studied. The SEP released by the parasites Himasthla elongata (Echinostomatidae) and Cryptocotyle lingua (Heterophyidae) were found to inhibit the phagocytosis of zymozan particles by periwinkle hemocytes. The specificity of SEP effects was assessed: SEP of Himasthla militaris and Cryptocotyle concavum, two trematodes belonging to the same genera but infecting another closely related prosobranch snail Hydrobia ulvae, were also shown to be able to suppress L. littorea hemocytes phagocytic activity. However, no decrease in phagocytosis rate was observed when SEP of H. elongata and C. lingua were applied to monolayers of hemocytes from the bivalve mollusc Mytilus edulis. SEP from H. elongata was fractionated; only those fractions containing proteins of molecular weight more than 50 kDa were shown to possess inhibitory activity. Different H. elongata SEP concentrations were tested in for their ability to suppress phagocytosis by L. littorea hemocytes. Even very low SEP concentrations were shown to retain their ability to decrease phagocytosis rate, the inhibitory effect being dose-dependent. Hemocytes derived from snails naturally infected with H. elongata were also found to have lower phagocytic ability as compared to healthy individuals.

In vitro culture of the avian echinostome Himasthla elongata: from redia to marita.

Axenic primary cultures of Himasthla elongata rediae harvested from hepatopancreas of naturally infected marine prosobranch snail Littorina littorea were maintained in Leibovitz's L-15 medium (osmolarity of approximately 780 mOsm, pH 7.8, temperature 14 degrees C under normal atmospheric conditions). Cultured rediae were active, motile and demonstrated high synthetic activity in metabolic labelling experiment. Long-term cultivation experiment showed 50% survival level of the rediae for up to 70 days and significant differences between mortality in redia groups derived from different host individuals. Half of the rediae in the most robust group survived for up to 163 days, when the experiment was terminated. Development and emergence of in vivo preformed cercariae and daughter rediae was observed. Cercariae in the culture also encysted, transformed into metacercariae and some of them in one to two weeks after the transformation spontaneously excysted into juvenile maritae. The employed culture system is characterized by a very low level of proteolytic activity. This system is suggested as a method permitting to obtain rediae secretory-excretory products free of host-derived contaminants.