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Schistosomes are human-infective blood flukes characterised by their unique sex biology with genetically determined male and female individuals. The worms must physically couple heterosexually to enable the full maturation of their gonads, particularly of the female, and must remain paired to sustain maturation and egg production. Recent studies have highlighted that soluble molecules could provide a previously overlooked means of inter-sex communication, including over distance, which promotes development/maturation. We argue that developing a deeper understanding of the tentative remote molecular interactions between sexes is essential given: (i) the remarkable sexual biology of schistosomes, and (ii) the requirement to develop novel control strategies for human schistosomiasis. Persistent physical interaction between male and female schistosome adult worms has long been shown to be crucial for their development and sexual maturation, particularly for the female. Although not fully understood, worm pairing promotes local molecular communication between sexes, driving gonad and vitellaria differentiation. In this opinion article we (i) summarise evidence concerning molecular interactions underlying the physical pairing, and (ii) propose a new paradigm whereby remote male–female molecular communication may play an overlooked role in parasite sexual maturation. In this context we discuss recent research that supports both physical and remote male–female interactions driving differentiation of the gonads/vitellaria. This remote communication between sexes may be mediated by excretory–secretory products (ESPs). Integrated hypotheses are presented to stimulate research in this important and emerging field. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Regulation of the Aedes albopictus cecropin A1 promoter was studied to provide insight into the transcriptional control of this antimicrobial peptide (AMP) gene in mosquitoes. Gene expression levels of cecropin A1 increased in A. albopictus C6/36 cells in response to heat-killed Escherichia coli. Reporter gene assays incorporating −757 to +32 of the A. albopictus cecropin A1 promoter revealed that E. coli could induce expression in these cells with more pronounced expression than that seen with lipopolysaccharide (LPS). Analysis of deletion constructs demonstrated that the 5′ boundary of the regulatory region for the activation of this AMP was located between −173 and −64. Western blotting with anti-phospho-specific antibodies demonstrated that p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) were activated by LPS, whereas only p38 MAPK was activated by E. coli. Moreover, pharmacological experiments revealed that pre-incubation of cells with the p38 MAPK inhibitor SB203580 resulted in a striking activation of the cecropin A1 promoter following immune challenge, demonstrating that p38 MAPK negatively regulates cecropin A1 promoter activity. Finally the region required for the negative regulation by p38 MAPK was identified as being between −173 and −64. This report is the first to show involvement of the p38 MAPK pathway in the negative regulation of AMP production in a mosquito. [Copyright &y& Elsevier]

Abstract: The crustacean family Gnathiidae Leach, 1814 (Peracarida: Isopoda) comprises 12 genera known mostly from marine environments. Juvenile gnathiid isopods are fish ectoparasites, feeding on blood and tissue fluids in order to complete their life cycles. Gnathiid juvenile development generally includes three moults, the last involving metamorphosis to non-feeding, adult stages. The blood meal ingested by juveniles provides resources for adult survival, reproduction and embryological development. Reproductive biology in the brackish water gnathiid, Paragnathia formica (Hesse, 1864), is unusual amongst crustaceans, since brooding females have paired internal uterine sacs, rather than an external brood pouch. Known embryological development for P. formica includes three post gastrulation stages. In the current study, brooding and embryological development in this gnathiid were reexamined using histological and fluorescence methods, and by scanning electron microscopy. Novel observations were made of the blastodisc and germ cell migration within developing eggs, release of Stage 2 embryos by rupture of embryonic membranes, the in utero moult of Stage 2 to Stage 3 embryos, and the asynchronous development of the brood within the paired uterine sacs. These findings highlight the remarkable nature of brooding in P. formica and expand the paucity of knowledge of embryological development in gnathiids in general. [Copyright &y& Elsevier]

More than a decade ago, it was postulated that components derived from trematode parasites block receptors on the defence cells of their snail intermediate hosts, thus preventing host-cell activation and parasite elimination. This phenomenon has still not been investigated extensively. However, recent work concerning the molecular regulation of the molluscan defence response provides a new framework for studies that focus on an extension of this original concept – subversion of host cell signalling by trematode parasites. The hypothesis is that, to facilitate survival and replication in their intermediate hosts, trematode parasites downregulate host defence responses by interfering with key signal-transduction pathways in snail defence cells. [Copyright &y& Elsevier]

Our knowledge of cell signalling pathways in the molluscan immune system and their response to immunological challenge is currently poor. The present study focused on the Protein Kinase C (PKC) pathway in the immune cells (haemocytes) of Lymnaea stagnalis and its response following exposure to bacterial lipopolysaccharide (LPS). Western blotting of haemocyte proteins with either anti-PKC (pan) or anti-phospho-PKC (Ser 660) antibodies revealed the presence of two PKC-like immuno-reactive proteins of approximately 76 and 85 kDa. Challenge of haemocytes with LPS transiently increased the phosphorylation of the 85 kDa isoform, with a 2.2-fold increase in phosphorylation levels at 5 min and a return to basal levels after 20 min. This LPS-mediated response was blocked following treatment of haemocytes with GF109203X. PKC activities measured in anti-phospho-PKC immunocomplexes following haemocyte treatment with LPS and GF109203X correlated well with the observed PKC phosphorylation levels. These data show for the first time that the activity of the PKC pathway in molluscan immune cells is modulated by LPS, as it is in mammals, and suggest that cell signalling in the innate immune response may have been conserved through evolution. [Copyright &y& Elsevier]

Lymnaea stagnalis is a common freshwater gastropod. Importantly, the snail serves as the intermediate host for more than one hundred species of digenetic trematodes, including the avian schistosome Trichobilharzia szidati , a causative agent of cercarial dermatitis in humans. Infection of L. stagnalis by T. szidati initiates a dynamic confrontation between the host and the parasite that culminates in immunocompatibility ensuring survival and development of larvae. Unfortunately, the molecular mechanisms determining this immunocompatibility remain poorly characterised. By employing a variety of immune elicitors, including chemical compounds, PAMPs and bacteria, research in the last two decades has elucidated some of the molecular processes that regulate the snail internal defence response such as haemocyte signalling pathways. These discoveries provide a framework for future studies of molecular interactions between T. szidati and L. stagnalis to help elucidate factors and mechanisms enabling transmission of schistosome parasites. Moreover, support from recently available next generation sequence data and CRISPR-enabled functional genomics should further enable L. stagnalis as an important model for comparative immunology and contribute to a more comprehensive understanding of immune functions in gastropod molluscs. • Lymnaea stagnalis is immunocompatible with Trichobilharzia szidati. • Trichobilharzia szidati alters Lymnaea stagnalis defence responses. • Secretions/surface components of Trichobilharzia szidati facilitate immunomodulation. • Molecular basis of Lymnaea stagnalis - Trichobilharzia szidati compatibility is unknown. [ABSTRACT FROM AUTHOR]

• Adult schistosome worms do not produce many viable eggs after being placed in culture. • Female worms do not produce eggs in the absence of males. • Lack of the host-derived cytokine transforming growth factor-beta (TGF-β) may be a reason for poor female worm fecundity. • TGF-β can complex with the host blood-borne protease inhibitor alpha 2-macroglobulin (A2M). • TGF-β complexed with host A2M may be the source of the cytokine needed by schistosomes to enable egg production. When adult schistosome worm pairs are transferred from experimental hosts to in vitro culture they cease producing viable eggs within a few days. Female worms in unisexual infections fail to mature, and when mature adult females are separated from male partners they regress sexually. Worms cultured from the larval stage are also permanently reproductively defective. The cytokine transforming growth factor beta derived from the mammalian host is considered important in stimulating schistosome female worm maturation and maintenance of fecundity. The means by which schistosomes acquire TGF-β have not been elucidated, but direct uptake in vivo seems unlikely as the concentration of free, biologically active cytokine in host blood is very low. Here we review the complexities of schistosome development and male–female interactions, and we speculate about two possibilities on how worms obtain the TGF-β they are assumed to need: (i) worms may have mechanisms to free active cytokine from the latency-inducing complex of proteins in which it is associated, and/or (ii) they may obtain the cytokine from alpha 2-macroglobulin, a blood-borne protease inhibitor to which TGF-β can bind. These ideas are experimentally testable. [ABSTRACT FROM AUTHOR]

Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A regulates multiple processes in eukaryotes by phosphorylating diverse cellular substrates, including metabolic and signalling enzymes, ion channels and transcription factors. Here we provide insight into protein kinase A signalling in cercariae and 24 h in vitro cultured somules of the blood parasite, Schistosoma mansoni , which causes human intestinal schistosomiasis. Functional mapping of activated protein kinase A using anti-phospho protein kinase A antibodies and confocal laser scanning microscopy revealed activated protein kinase A in the central and peripheral nervous system, oral-tip sensory papillae, oesophagus and excretory system of intact cercariae. Cultured 24 h somules, which biologically represent the skin-resident stage of the parasite, exhibited similar activation patterns in oesophageal and nerve tissues but also displayed striking activation at the tegument and activation in a region resembling the germinal ‘stem’ cell cluster. The adenylyl cyclase activator, forskolin, stimulated somule protein kinase A activation and produced a hyperkinesia phenotype. The biogenic amines, serotonin and dopamine known to be present in skin also induced protein kinase A activation in somules, whereas neuropeptide Y or [Leu 31 ,Pro 34 ]-neuropeptide Y attenuated protein kinase A activation. However, neuropeptide Y did not block the forskolin-induced somule hyperkinesia. Bioinformatic investigation of potential protein associations revealed 193 medium confidence and 59 high confidence protein kinase A interacting partners in S. mansoni , many of which possess putative protein kinase A phosphorylation sites. These data provide valuable insight into the intricacies of protein kinase A signalling in S. mansoni and a framework for further physiological investigations into the roles of protein kinase A in schistosomes, particularly in the context of interactions between the parasite and the host. [ABSTRACT FROM AUTHOR]

Abstract: Notocotylus attenuatus (Digenea: Notocotylidae) is a monostome fluke parasitizing the intestinal caeca of waterfowl that uses an injection apparatus to infect its intermediate snail host. Morphology of the invading larva (a sporocyst), and the intramolluscan larval development of this fluke have not been characterized extensively. In this study, experimental infections of Lymnaea stagnalis using N. attenuatus eggs resulted in the development of sporocysts containing one germ ball or mother redia between 12 and 21days post exposure (p.e.) within the hepatopancreas. Independent mother rediae and developing daughter rediae were present between day 25 and day 42 p.e. Cercariae, within the body of rediae, were detected 42days p.e. The development of daughter rediae and cercariae started posteriorly in the body of parent redia and these larvae migrated anteriorly during development towards the birth pore. A cercaria was also observed emerging from the birth pore and released cercariae maturated further within the snail hepatopancreas prior to leaving the snail. The intramolluscan development was completed 45days p.e. when the first fully formed cercariae were shed into the outer environment. These data detail the fascinating post-embryonic development of N. attenuatus and highlight the intricate nature of larval transitions within its snail host. [Copyright &y& Elsevier]

Abstract: High levels of polymorphism in DNA sequences of tetraspanin-23 (TSP-23) were revealed within and between nine different species of Schistosoma from Africa including Schistosoma mansoni, Schistosoma rodhaini, Schistosoma margrebowiei, Schistosoma mattheei, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma guineensis, Schistosoma curassoni and Schistosoma bovis. The greatest levels of diversity coincided with evidence of positive selection (dN /dS >1) within regions that code for extracellular loops of TSP-23 believed to interact with the host immune system. Kolaskar and Tongaonkar antigenicity predictions of protein sequences were compared across species and high levels of variation in antigenicity were also identified with each species which possessed their own unique antigenic profile. Phylogenetic analysis of TSP-23 proteins suggested evidence of convergent evolution in antigenic lineages as no true inter-species phylogenetic relationships were seen. This could be indicative of host-specific evolution of antigens in different species of schistosomes, a factor that should be considered carefully when developing vaccine targets. [Copyright &y& Elsevier]

Abstract: Biomphalaria glabrata is the major intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. Much remains to be discovered concerning specific molecules mediating the defence events in these intermediate hosts, triggered by invading schistosomes. An expressed sequence tag (EST) gene discovery strategy known as ORESTES has been employed to identify transcripts that might be involved in snail–schistosome interactions in order to examine gene expression patterns in infected B. glabrata. Over 3930 ESTs were sequenced from cDNA libraries made from both schistosome-exposed and unexposed snails using different tissue types, producing a database of 1843 non-redundant clones. The non-redundant set has been assessed for gene ontology and KEGG pathway assignments. This approach has revealed a number of signalling, antioxidant and immune-related gene homologues that, based on current understanding of molluscan and other comparative systems, might play an important role in the molluscan defence response towards infection. [Copyright &y& Elsevier]

[Display omitted] • HybridoMed Diff 1000 (HM) was further evaluated for Schistosoma mansoni juvenile worm culture. • Although Basch medium outperformed HM, HM supported development to the early liver stage. • HM is a good alternative to Basch and could be invaluable for drug screening assays. Schistosomules of the human parasite Schistosoma mansoni are vital for research focusing on the fundamental functional/developmental biology of schistosomes and many anti-schistosomal drug discovery programmes. Through the further evaluation and validation of a recently tested media, HybridoMed Diff 1000 (HM), for the cell-free culture of juvenile schistosomules, we show that while Basch medium was superior to HM for the survival/development of schistosomules, HM represents a viable and attractive alternative for somule culture, particularly to the early liver stage. Adoption of HM for schistosomule culture could facilitate more standardised approaches, which for drug screening should enable improved multi-centre target-hit evaluation. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining β cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of β cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by Western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in β cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared with controls. Purified SPARC inhibits growth factor-induced signaling in both INS-1 β cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 β cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and β cell growth. [ABSTRACT FROM AUTHOR]

Abstract: For schistosomes, development of the miracidium to mother sporocyst within a compatible molluscan host requires considerable physiological and morphological changes by the parasite. The molecular mechanisms controlling such development have not been explored extensively. To begin to elucidate the importance of kinase-mediated signal transduction to this process, the phosphorylation (activation) of protein kinase C (PKC) in larval stages of Schistosoma mansoni undergoing in vitro transformation was explored. Mining of the S. mansoni genomic database revealed two S. mansoni PKC proteins with high homology to human PKCβ and containing the conserved autophosphorylation (activation) site represented by serine 660 of human PKCβII. Western blotting with anti-phosphospecific antibodies directed to this site demonstrated that miracidia freshly-hatched from eggs possessed PKC (78kDa) which was phosphorylated (activated) when miracidia were exposed to phorbol ester, and dephosphorylated (inhibited) following exposure to the PKC inhibitor GF109203X. Miracidia treated with the phospholipase C (PLC) inhibitor U73122 also displayed decreased PKC phosphorylation. S. mansoni PKC was phosphorylated during the initial 24h development of miracidia into mother sporocysts; after 31h and 48h development, phosphorylation was reduced by 72% and 86%, respectively. Confocal microscopy of miracidia revealed phosphorylated PKC associated with the neural mass, excretory vesicle, tegument, ciliated plates, terebratorium and germinal cells; in larvae undergoing transformation for 31h, phosphorylated PKC was only occasionally detected, being present in regions likely corresponding to the ridge cyton. Inhibition of PKC in miracidia by GF109230X resulted in accelerated transformation, particularly to the postmiracidium stage; ciliated plates were also shed from developing larvae more rapidly. These results highlight the dynamic nature of PKC signalling during S. mansoni postembryonic development and support a role for active PKC in restricting transformation of S. mansoni miracidia into mother sporocysts. [Copyright &y& Elsevier]

Summary: Biomphalaria glabrata is an intermediate snail host for the human blood fluke Schistosoma mansoni. To survive in B. glabrata, S. mansoni must suppress the snail''s haemocyte-mediated defence response; the molecular mechanisms by which this is achieved remain largely unknown. We report here that S. mansoni excretory–secretory products (ESPs) attenuate phosphorylation of extracellular signal-regulated kinase (ERK) in haemocytes from a B. glabrata strain susceptible to S. mansoni. Whole S. mansoni sporocysts also impair ERK signalling in these cells. In striking contrast, ERK signalling in haemocytes from a B. glabrata strain refractory to schistosome infection is unaffected by ESPs or sporocysts. Effects of ESPs on ERK are similar in the presence or absence of snail plasma, thus ESPs seem to affect haemocytes directly. These findings reveal novel schistosome interference mechanisms; as ERK regulates various haemocyte defence reactions, we propose that disruption of ERK signalling in haemocytes facilitates S. mansoni survival within susceptible B. glabrata. [Copyright &y& Elsevier]

Abstract: Molluscs are intermediate hosts for helminth parasites such as Schistosoma spp. that possess an immunogenic surface coat of high carbohydrate content, with fucose as the predominant saccharide. More than a decade ago, it was postulated that such components could block receptors on snail haemocytes thus preventing recognition of intra-molluscan schistosome stages. Although more recent studies have shown that carbohydrates can suppress processes such as phagocytosis by haemocytes, interference of the haemocyte cell signalling pathways that regulate immunity by saccharides has not yet been investigated. We have recently reported the presence of extracellular-signal regulated kinase and protein kinase C in Lymnaea stagnalis haemocytes. Here we show that extracellular-signal regulated kinase and protein kinase C activities are down-regulated when haemocytes are exposed to albumin-linked fucose and galactose in the absence of haemolymph. Moreover, we demonstrate that phagocytosis is reduced under these conditions. Interestingly, in the presence of haemolymph, only protein kinase C activity is down-regulated and only galactose suppresses phagocytosis, implying a role for serum factors in the preservation of haemocyte function following exposure. We therefore propose that the establishment of a compatible relationship between a schistosome and its snail host is at least in part due to down-regulation of cell signalling events in haemocytes. [Copyright &y& Elsevier]