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Robust RNA purification and analysis methods are required to support the development of RNA vaccines and therapeutics as well as RNA interference-based crop protection solutions. Asymmetrical flow field -flow fractionation (AF4) is a gentle native purification method that applies liquid flows to separate sample components based on their hydrodynamic sizes. We recently showed that AF4 can be utilized to separate RNA molecules that are shorter than 110 nucleotides (nt), but the performance of AF4 in the analysis and purification of longer RNA molecules has not been previously evaluated. Here, we studied the perfor-mance of AF4 in separation of single-stranded (ss) and double-stranded (ds) RNA molecules in the size range of 75-6400 nt. In addition, we evaluated the power of AF4 coupling to different detectors, allow-ing separation to be combined with data collection on yield as well as molecular weight ( MW ) and size distribution. We show that AF4 method is applicable in RNA purification, quality control, and analytics, and results in good recoveries of ssRNA and dsRNA molecules. In addition, our results demonstrate the utility of AF4 multidetection platforms to study biophysical properties of long RNA molecules.(c) 2022 The Author(s). Published by Elsevier B.V.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

SummaryMACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) is a pleiotropic protein with chemotactic, pro-inflammatory, and growth-promoting activities first discovered in mammals. In parasites, MIF homologs are involved in immune evasion and pathogenesis. Here, we present the first comprehensive analysis of a MIF protein from the devastating plant pathogen Magnaporthe oryzae (Mo). The fungal genome encodes a single MIF protein (MoMIF1) that, unlike the human homolog, harbors multiple low-complexity regions (LCRs) and is unique to Ascomycota. Following infection, MoMIF1 is expressed in the biotrophic phase of the fungus, and is strongly down-regulated during subsequent necrotrophic growth in leaves and roots. We show that MoMIF1 is secreted during plant infection, affects the production of the mycotoxin tenuazonic acid and inhibits plant cell death. Our results show that MoMIF1 is a novel key regulator of fungal virulence that maintains the balance between biotrophy and necrotrophy during the different phases of fungal infection.

The journal of biological chemistry : JBC 296, 100611 (2021). doi:10.1016/j.jbc.2021.100611, Published by American Society for Biochemistry and Molecular Biology, Bethesda, MD.

Macrophage Migration Inhibitory Factors (MIF) are pivotal cytokines/chemokines for vertebrate immune systems. MIFs are typically soluble single-domain proteins that are conserved across plant, fungal, protist, and metazoan kingdoms but their functions have not been determined in most phylogenetic groups. Here we describe an atypical multidomain MIF protein. The marine dinoflagellate Lingulodinium polyedra produces a transmembrane protein with an extra-cytoplasmic MIF domain, which localizes to cell wall-associated membranes and vesicular bodies. This protein is also present in the membranes of extracellular vesicles accumulating at the secretory pores of the cells. Upon exposure to biotic stress, L. polyedra exhibits reduced expression of the MIF gene and reduced abundance of the surface-associated protein. These findings indicate that the transmembrane MIF may contribute to intercellular communication and/or interactions between free-living organisms in multispecies planktonic communities and raise the question of possible analogies in MIF functions between cells of metazoan organisms and protist communities.

International audience; RNA interference (RNAi) is a biological process in which small RNA (sRNA) molecules sequence-specifically silence gene expression at the transcriptional or post-transcriptional level, either by directing inhibitory chromatin modifications or by decreasing the stability or translation potential of the targeted mRNA. The trigger for gene silencing is double-stranded RNA (dsRNA) generated from an endogenous genomic locus or a foreign source, such as a transgene or virus. The process of gene silencing can be exploited in agriculture to control plant diseases and pests. Of the pests that impact crop yield (including nematodes, arthropods, rodents, snails, slugs and birds), insects constitute the largest and most diverse group. Here, we review the "pros" and "cons" of using RNAi technology mediated by dsRNA-expressing transgenic plants (host-induced gene silencing, HIGS) or direct application of chemically synthesized dsRNA to control plant-damaging insects. Rapid progress in elucidating RNAi mechanisms has led to the first commercial products on the market. Given the high potential of RNAi strategies, their use in agriculture, horticulture, and forestry will likely be extensive in the future. However, further studies are needed to improve the efficacy of RNAi-based plant protection strategies and to assess their associated safety risks.

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International audience; LBPs (lipopolysaccharide binding proteins) and BPIs (bactericidal permeability increasing proteins) are important proteins involved in defense against bacterial pathogens. We recently discovered a novel biocidal activity of a LBP/BPI from the gastropod Biomphalaria glabrata and demonstrated its role in parental immune protection of eggs, highlighting the importance of LBP/BPIs in invertebrate immunity. Here we characterize four additional LBP/BPI from B. glabrata, presenting conserved sequence architecture and exon-intron structure. Searches of invertebrate genomes revealed that existence of LBP/BPIs is not a conserved feature since they are absent from phyla such as arthropods and platyhelminths. Analyses of LBP/BPI transcripts from selected mollusk species showed recent parallel duplications in some species, including B. glabrata. In this snail species, LBP/BPI members vary in their expression tissue localization as well as their change in expression levels after immune challenges (Gram-negative bacterium; Gram-positive bacterium or yeast). These results, together with the predicted protein features provide evidences of functional specialization of LBP/BPI family members in molluscs.

Genes 10(10), E740 (2019). doi:10.3390/genes10100740 special issue: "Invertebrate Immunity", Published by MDPI, Basel

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3 pages; International audience; Pinaud et al. recently provided the first global investigation of the molecular processes underlying innate immune memory in an invertebrate species. They showed that the memory response of the snail Biomphalaria glabrata to Schistosoma mansoni infection is associated with a shift from cellular to humoral mechanisms.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis., Biomphalaria glabrata is a fresh water snail that acts as a host for trematode Schistosoma mansoni that causes intestinal infection in human. This work describes the genome and transcriptome analyses from 12 different tissues of B glabrata, and identify genes for snail behavior and evolution.

Historically, the prevailing view in the field of invertebrate immunity was that invertebrates that do not possess acquired adaptive immunity rely on innate mechanisms with low specificity and no memory. Several recent studies have shaken this paradigm and suggested that the immune defenses of invertebrates are more complex and specific than previously thought. Mounting evidence has shown that at least some invertebrates (mainly Ecdysozoa) show high levels of specificity in their immune responses to different pathogens, and that subsequent reexposure may result in enhanced protection (recently called ‘immune priming'). Here, we investigated immune priming in the Lophotrochozoan snail species Biomphalaria glabrata, following infection by the trematode pathogen Schistosoma mansoni. We confirmed that snails were protected against a secondary homologous infection whatever the host strain. We then investigated how immune priming occurs and the level of specificity of B. glabrata immune priming. In this report we confirmed that immune priming exists and we identified a genotype-dependent immune priming in the fresh-water snail B. glabrata.

La bilharziose ou schistosomiase, seconde endemie parasitaire mondiale apres le paludisme, n’existerait pas sans l’intervention cruciale de mollusques aquatiques. Les schistosomes debutent en effet leur developpement chez un mollusque ou ils effectuent une extraordinaire multiplication clonale au terme de laquelle emergent des milliers de larves nageuses, infestantes pour l’homme et d’autres animaux. La comprehension des interactions moleculaires entre mollusques et trematodes pourrait suggerer de nouveaux moyens de lutte contre ces parasitoses, mais les particularites de ces modeles biologiques ont longtemps freine la recherche dans ce domaine. Les avancees technologiques recentes permettent d’explorer l’immunite innee de ces invertebres, de tracer les premieres ebauches fonctionnelles de ces systemes immunitaires et de reveler une complexite inattendue.

As an approach to investigate the suspected involvement of cellular factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns from hemocytes collected from susceptible and resistant snails. This proteomic approach revealed that twelve hemocytic proteins exhibited significant differences in their apparent abundance. The genes corresponding to five of them were characterized by a combination of mass spectrometry and molecular cloning. They encode an aldolase, an intermediate filament protein, a cytidine deaminase, the ribosomal protein P1 and the histone H4. Furthermore, we investigated their expression in parasite-exposed or -unexposed snails. These last experiments revealed changes in transcript levels corresponding to intermediate filament and histone H4 proteins post-infection.

To date, glyceroneogenesis has only been described in mammals but we demonstrate in this paper that it could exist in the invertebrate Schistosoma mansoni, the parasitic helminth transmitted by fresh water molluscs and responsible for the major human endemic disease, schistosomiasis. Glyceroneogenesis is a phosphoenolpyruvate carboxykinase (PEPCK)-dependent process by which glycerol can be produced from precursors like glutamine and therefore represents a truncated gluconeogenic pathway. We have previously demonstrated the possible central role of glutamine in mollusc-schistosome interactions. In this work, we show that glutamine effectively promotes in vitro survival and protein synthesis in sporocysts, the intramolluscan larval stages of S. mansoni, possibly through its role as an energy source. However, the demonstration that PEPCK is massively expressed in these larval forms as compared to adult parasites, together with the observation that 3-mercaptopicolinate, a specific inhibitor of PEPCK, significantly reduces the effect of glutamine on sporocyst growth, suggest that glutamine could also be used for glucose or glycerol production. Results of [14C] glutamine incorporation confirmed that neosynthesis of glucose and mainly of glycerol occurred in sporocysts and was dependent on PEPCK activity. Therefore, our results strongly indicate that glyceroneogenesis could exist in schistosomes. Several hypotheses can be proposed concerning the importance of glycerol for the adaptation of this helminth to its host osmotic and energetic environment.

The immune effector cells (hemocytes) of the snail host Biomphalaria glabrata are known to play a key role in recognition and elimination of larval helminths such as the human blood fluke Schistosoma mansoni. To identify novel immune-relevant genes, we undertook an expressed sequence tag program. A hemocyte cDNA library was constructed using snails that were not exposed to a particular pathogen or parasite but maintained in non-axenic conditions. Putative function could be assigned to 53% of the 1613 high quality cDNAs analysed. Based on sequence similarities, we identified 31 immune-relevant genes corresponding either to cellular defence effectors, proteases and protease inhibitors, pattern recognition receptors, cell adhesion molecules or immune regulators. In order to further investigate the potential involvement of these genes in snail-trematode immunobiological interactions, we analysed their expression in unchallenged and parasite-challenged snails, using the immunosuppressive trematode Echinostoma caproni and snail strains selected for resistance or susceptibility to this parasite. Real-time PCR analysis of expression ratios at 7 time-points post-exposure revealed both (i) genes displaying constitutive expression differences between the two strains; and (ii) genes differentially modulated after parasite exposure.

Specific studies on schistosome gene functions require both access to the parasite stages, preferably the larvae, and to complete the life cycle. In the present study, we investigated whether short-term in vitro cultivation of sporocysts and surgical transplantation into snails could be combined to produce cercariae. Miracidia were maintained in vitro in the presence of Biomphalaria glabrata embryonic (Bge) cells or, alternatively, in Bge-cell-conditioned medium. The transformation of miracidia to mother sporocysts was observed in both cases. Two day-old sporocysts were transplanted into the cephalopedal sinus of recipient snails. Transplantation efficiencies varied between 16% and 43%, depending on the culture of the sporocysts in terms of the number of cercariae producing snails. Cercariae recovered from these snails were used to successfully infect hamsters, demonstrating that short term in vitro-generated sporocysts undergo normal cercariogenesis following transplantation. This combination of in vitro cultivation and transplantation may be useful for novel experimental approaches to investigate the genes involved in larval development or host-parasite molecular interactions.

Biomphalaria glabrata embryonic (Bge) cells have been shown to provide favourable environmental conditions for the development of Schistosoma mansoni sporocysts. We investigated the effect of Bge excretory-secretory products on metabolic activity and gene transcription in S. mansoni mother sporocysts. Using the differential-display technique, we identified several sporocyst transcripts regulated by exposure to Bge soluble components. Research in databases indicated that six of the eight differential products analysed were homologous to sequences already present in databases. Two transcripts appeared of interest for schistosome development since they could be associated with cell division and protein synthesis in developing sporocysts. Their up-regulation following contact with cell products was confirmed by semi-quantitative RT-PCR. The first fragment coded for a part of the chaperonin containing T-complex protein gamma subunit-like protein of S. mansoni (SmTCP 1-C). The second one represented a new S. mansoni expressed sequence tag encoding a protein homologous to various glutaminyl-tRNA synthetases (GlnRS). The full-length sequence of SmGlnRS was cloned from adult schistosomes and its primary sequence was compared to other GlnRS. The overexpression of SmTCP-1 and SmGlnRS could be correlated with the metabolic changes observed in Bge-exposed sporocysts.

Nature Communications 8: Article number: 15451 (2017); Published 16 May 2017; Updated 23 August 2017 The original version of this Article contained an error in the spelling of the author Leon Di Stefano, which was incorrectly given as Leon di Stephano. This has now been corrected in both the PDF andHTML versions of the Article.

SummaryAphids attack virtually all plant species and cause serious crop damages in agriculture [1]. Despite their dramatic impact on food production, little is known about the molecular processes that allow aphids to exploit their host plants. To date, few aphid salivary proteins have been identified that are essential for aphid feeding, and their nature and function remain largely unknown [2–4]. Here, we show that a macrophage migration inhibitory factor (MIF) is secreted in aphid saliva. In vertebrates, MIFs are important pro-inflammatory cytokines regulating immune responses [5, 6]. MIF proteins are also secreted by parasites of vertebrates, including nematodes, ticks, and protozoa, and participate in the modulation of host immune responses [7–9]. The finding that a plant parasite secretes a MIF protein prompted us to question the role of the cytokine in the plant-aphid interaction. We show here that expression of MIF genes is crucial for aphid survival, fecundity, and feeding on a host plant. The ectopic expression of aphid MIFs in leaf tissues inhibits major plant immune responses, such as the expression of defense-related genes, callose deposition, and hypersensitive cell death. Functional complementation analyses in vivo allowed demonstrating that MIF1 is the member of the MIF protein family that allows aphids to exploit their host plants. To our knowledge, this is the first report of a cytokine that is secreted by a parasite to modulate plant immune responses. Our findings suggest a so-far unsuspected conservation of infection strategies among parasites of animal and plant species.

International audience; This review summarizes the research progress made over the past decade in the field of gastropod immunity resulting from investigations of the interaction between the snail Biomphalaria glabrata and its trematode parasites. A combination of integrated approaches, including cellular, genetic and comparative molecular and proteomic approaches have revealed novel molecular components involved in mediating Biomphalaria immune responses that provide insights into the nature of host-parasite compatibility and the mechanisms involved in parasite recognition and killing. The current overview emphasizes that the interaction between B. glabrata and its trematode parasites involves a complex molecular crosstalk between numerous antigens, immune receptors, effectors and anti-effector systems that are highly diverse structurally and extremely variable in expression between and within host and parasite populations. Ultimately, integration of these molecular signals will determine the outcome of a specific interaction between a B. glabrata individual and its interacting trematodes. Understanding these complex molecular interactions and identifying key factors that may be targeted to impairment of schistosome development in the snail host is crucial to generating new alternative schistosomiasis control strategies

International audience; The nature and cost of single genes of major effect is one of the longest running controversies in biology. Resistance, whether to xenobiotics or to parasites, is often paraded as an obvious example of a single gene effect that must carry an associated fitness 'cost'. However, a review of the xenobiotic resistance literature shows that empirical evidence for this hypothesis is, in fact, scarce. We postulate that such fitness costs can only be fully interpreted in the light of the molecular mutations that might underlie them. We also derive a theoretical framework both to encompass our current understanding of xenobiotic resistance and to begin to dissect the probable cost of parasite resistance.

In vitro cultivation of parasitic helminths, including the digenetic trematodes, has long been a valuable tool in medical and veterinary parasitology, permitting and/or facilitating the development of diagnostic reagents, chemotherapeutic agents, and vaccines and providing insights into naturally complex host-parasite interactions. In vitro cultivation of the intramolluscan stages of trematodes has been particularly challenging, given the ontogenic complexities involved in the production of multiple larval generations from germinal tissues through an asexual "budding" process. Recently, however, advanced larval development has been achieved by incorporating the Biomphalaria glabrata embryonic (Bge) cell line into cocultivation systems. Most notably, the entire intramolluscan cycle (from miracidium to cercaria) has been completed for the human blood fluke Schistosoma mansoni, while significant primary sporocyst development has been attained for several other digeneans including S. japonicum and Fascioloides magna. Here we review recent advances in the cultivation of several larval trematode species and discuss the potential use of this culture system for addressing fundamental questions of host-parasite compatibility.

Les redies du trematode Echinostoma caproni (souche egyptienne) ont ete isolees des tissus de Biomphalaria glabrata entre 13 et 34 jours apres infestation, mises en culture avec la lignee cellulaire Bge (Bge cells) derivee d'embryons de B. glabrata, et maintenues jusqu'a 51 jours dans ces conditions experimentales. Les redies ingeraient activement des cellules Bge, et presentaient une meilleure survie en presence de cellules dans le milieu de culture. Les redies cultivees avec des cellules Bge dans du milieu Bge produisaient des cercaires mobiles tout au long de la periode d'observation, lorsqu'elles etaient cultivees avec des cellules Bge mais en presence de milieu 199, elles produisaient a la fois des redies et des cercaires. Certaines cercaires produites in vitro se sont enkystees en metacercaires. Le suivi des cultures a mis en evidence l'ingestion par les redies de cercaires produites in vitro, mais jamais de phases de cannibalisme entre redies appartenant ou non a la meme espece.

The Biomphalaria glabrata/Schistosoma mansoni system represented the only model available so far, for investigating snail resistance to parasites. A new host parasite model has been recently provided by selection of B. glabrata strains that are genetically resistant and susceptible to Echinostoma caproni. As a first approach in investigating resistance mechanisms in this model, we compared the hemocytic response and its effect on E. caproni development in a susceptible and a resistant strains. Histological analysis revealed that resistance does not prevent penetration or migration through the snail tissues. However, all mother sporocysts (MS) settled in the ventricle and the aorta of resistant snails were encapsulated and killed by 4 days post-exposure. MS abnormally settled in the pericardial cavity were not encapsulated and could survive for 5 days. Regardless of their level of encapsulation, all live MS observed during the first days of infection in resistant snails showed abnormal development and degeneration, raising the question of the possible role of humoral factors in the resistance to E. caproni. Finally, we observed an increased number of adherent hemocytes in the resistant as compared with the susceptible snails after parasite exposure. This different effect on circulating hemocyte subpopulations may reflect the failure of E. caproni to interfere with the hemocytic response of resistant snails.

Background The widespread use of genome sequencing provided evidences for the high degree of conservation in innate immunity signalling pathways across animal phyla. However, the functioning and evolutionary history of immune-related genes remains unknown for most invertebrate species. A striking observation coming from the analysis of the pea aphid Acyrthosiphon pisum genome is the absence of important conserved genes known to be involved in the antimicrobial responses of other insects. This reduction in antibacterial immune defences is thought to be related to their long-term association with beneficial symbiotic bacteria and to facilitate symbiont maintenance. An additional possibility to avoid elimination of mutualistic symbionts is a fine-tuning of the host immune response. To explore this hypothesis we investigated the existence and potential involvement of immune regulators in aphid agonistic and antagonistic interactions. Results In contrast to the limited antibacterial arsenal, we showed that the pea aphid Acyrthosiphon pisum expresses 5 members of Macrophage Migration Inhibitory Factors (ApMIF), known to be key regulators of the innate immune response. In silico searches for MIF members in insect genomes followed by phylogenetic reconstruction suggest that evolution of MIF genes in hemipteran species has been shaped both by differential losses and serial duplications, raising the question of the functional importance of these genes in aphid immune responses. Expression analyses of ApMIFs revealed reduced expression levels in the presence, or during the establishment of secondary symbionts. By contrast, ApMIFs expression levels significantly increased upon challenge with a parasitoid or a Gram-negative bacteria. This increased expression in the presence of a pathogen/parasitoid was reduced or missing, in the presence of facultative symbiotic bacteria. Conclusions This work provides evidence that while aphid’s antibacterial arsenal is reduced, other immune genes widely absent from insect genomes are present, diversified and differentially regulated during antagonistic or agonistic interactions. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-762) contains supplementary material, which is available to authorized users.

In vitro cultivation of Schistosoma japonicum miracidia to the mother sporocyst (MS) and then to the daughter sporocyst (DS) stage was achieved using the Biomphalaria glabrata embryonic (Bge) cell line as a coculture system. When comparing the effect of Bge cell and MS density on MS development, it was apparent that Bge cell density had a highly significant effect on both MS viability and growth. Viability and growth rate of MS cultured under high cell density conditions (350 cells/mm2) were almost 2 times greater than those of MS cultured under conditions of low cell density (60 cells/mm2). Growth under high cell density conditions corresponded to a 20 to 30 times increase in MS estimated volume within the first 9 weeks of cultivation. Emergence of fully formed motile DS was first observed after 11 weeks of cocultivation. A few DS lived for 14 weeks after emergence and attained a size of 770 +/- 100 microns in length and 48 +/- 13 microns in width. In contrast to what was observed in Bge cell/Schistosoma mansoni cocultures, Bge cells did not encapsulate S. japonicum MS. Our results show that, although the cellular interactions between Bge cells and schistosomes MS display some level of specificity, Bge cells apparently secrete soluble factors that permit excellent survival and can trigger advanced in vitro development of S. japonicum.

Vertebrate females transfer antibodies via the placenta, colostrum and milk or via the egg yolk to protect their immunologically immature offspring against pathogens. This evolutionarily important transfer of immunity is poorly documented in invertebrates and basic questions remain regarding the nature and extent of parental protection of offspring. In this study, we show that a lipopolysaccharide binding protein/bactericidal permeability increasing protein family member from the invertebrate Biomphalaria glabrata (BgLBP/BPI1) is massively loaded into the eggs of this freshwater snail. Native and recombinant proteins displayed conserved LPS-binding, antibacterial and membrane permeabilizing activities. A broad screening of various pathogens revealed a previously unknown biocidal activity of the protein against pathogenic water molds (oomycetes), which is conserved in human BPI. RNAi-dependent silencing of LBP/BPI in the parent snails resulted in a significant reduction of reproductive success and extensive death of eggs through oomycete infections. This work provides the first functional evidence that a LBP/BPI is involved in the parental immune protection of invertebrate offspring and reveals a novel and conserved biocidal activity for LBP/BPI family members., Author Summary Vertebrate immune systems not only protect adult organisms against infections but also increase survival of offspring through parental transfer of innate and adaptive immune factors via the placenta, colostrum and milk or via the egg yolk. This maternal transfer of immunity is critical for species survival as embryos and neonates are immunologically immature and unable to fight off infections at early life stages. Parental immune protection is poorly documented in invertebrates and how the estimated 1.3 million of invertebrate species protect their eggs against pathogens remains an intriguing question. Here, we show that a fresh-water snail, Biomphalaria glabrata massively loads its eggs with a lipopolysaccharide binding protein/bactericidal permeability increasing protein (LBP/BPI) displaying expected antibacterial activities. Remarkably, this snail LBP/BPI also displayed a strong biocidal activity against water molds (oomycetes). This yet unsuspected activity is conserved in human BPI. Gene expression knock-down resulted in the reduction of snail reproductive success and massive death of eggs after water mold infections. This work reveals a novel and conserved biocidal activity for LBP/BPI family members and demonstrates that the snail LBP/BPI represents a major fitness-related protein transferred from parents to their clutches and protecting them from widespread and lethal oomycete infections.

Two well-described elements of the immune response of insects include encapsulation of metazoan parasites (blood-cell-mediated) and the production of antibacterial peptides (humoral and/or cellular). However, the possible functional interrelationship between cellular encapsulation and antibacterial responses, and the extent to which the two components may be co-regulated, are poorly understood. We used a novel approach involving strains of Drosophila resistant (R) or susceptible (S) to the wasp parasitoid Leptopilina boulardi to study the expression of three genes involved in the antibacterial response: Dorsal-related immunity factor (Dif), Cecropin (CecA1) and Diptericin (Dip). Both S and R strains produced high levels of all antibacterial transcripts upon bacterial injection. However, when parasitized the R strain showed no induction whilst the S strain did. This lack of antibacterial transcript induction in the parasitized R strain not only clarifies the separation of these two types of immune response but also raises the fascinating possibility of a link in their genetic regulation.

Cyclodiene insecticide resistance is associated with replacements of a single amino acid within the putative lining of a δ-aminobutyric acid (GABA)-gated chloride ion channel gene Resistance to dieldrin (Rdl). Only two resistance-associated amino acid replacements have been identified; alanine to serine in Drosophila melanogaster, D. simulans, Aedes aegypti, and Tribolium castaneum and alanine to glycine as a second allele in D. simulans. Here we report that single stranded conformational polymorphism (SSCP) analysis of genomic DNA, amplified by the polymerase chain reaction (PCR) for exon 7 of the Rdl gene, can be used to genotype strains or individuals of all of these insects. This technique also appears simultaneously to distinguish between D. melanogaster and D. simulans, sibling species only reliably identifiable by examination of male genitalia. The relative advantages of this genotyping technique against other PCR-mediated techniques in monitoring for insecticide resistance are discussed.

The design of three PCR-based monitoring techniques for the genotyping of cyclodiene-resistant insects are described: (1) PCR followed by a diagnostic restriction enzyme digest or PCR/REN, (2) PCR amplification of specific alleles or PASA and (3) single-stranded conformational polymorphism analysis of PCR-amplified DNA or SSCP. The relative disadvantages and potential applications of each of these techniques are discussed and compared to the use of insecticide bioassays. It is concluded that, although such technique will probably never replace bioassays for routine monitoring, they can more readily address several fundamental questions relating to the evolution and spread of specific resistance alleles in insect populations

© 2012 Lockyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The outcome of infection in the host snail Biomphalaria glabrata with the digenean parasite Schistosoma mansoni is determined by the initial molecular interplay occurring between them. The mechanisms by which schistosomes evade snail immune recognition to ensure survival are not fully understood, but one possibility is that the snail internal defence system is manipulated by the schistosome enabling the parasite to establish infection. This study provides novel insights into the nature of schistosome resistance and susceptibility in B. glabrata at the transcriptomic level by simultaneously comparing gene expression in haemocytes from parasite-exposed and control groups of both schistosome-resistant and schistosome-susceptible strains, 2 h post exposure to S. mansoni miracidia, using an novel 5 K cDNA microarray. Differences in gene expression, including those for immune/stress response, signal transduction and matrix/adhesion genes were identified between the two snail strains and tests for asymmetric distributions of gene function also identified immune-related gene expression in resistant snails, but not in susceptible. Gene set enrichment analysis revealed that genes involved in mitochondrial electron transport, ubiquinone biosynthesis and electron carrier activity were consistently up-regulated in resistant snails but down-regulated in susceptible. This supports the hypothesis that schistosome-resistant snails recognize schistosomes and mount an appropriate defence response, while in schistosome-susceptible snails the parasite suppresses this defence response, early in infection. This work was carried out with funding from the Wellcome Trust (068589/Z/02/Z). CMA and BH acknowledge support from the United States National Institutes of Health (NIH), USA R01AI052363 (CMA) and COBRE 1P20RR018754 IdEA, NCRR.

Activité anti-oomycète des lipopolysaccharides (LPS)-binding proteins/bactericidal/permeability-increasing proteins

International audience; Our present understanding of the functioning and evolutionary history of invertebrate innate immunity derives mostly from studies on a few model species belonging to ecdysozoa. In particular, the characterization of signaling pathways dedicated to specific responses towards fungi and Gram-positive or Gram-negative bacteria in Drosophila melanogaster challenged our original view of a non-specific immunity in invertebrates. However, much remains to be elucidated from lophotrochozoan species. To investigate the global specificity of the immune response in the fresh-water snail Biomphalaria glabrata, we used massive Illumina sequencing of 5'-end cDNAs to compare expression profiles after challenge by Gram-positive or Gram-negative bacteria or after a yeast challenge. 5'-end cDNA sequencing of the libraries yielded over 12 millions high quality reads. To link these short reads to expressed genes, we prepared a reference transcriptomic database through automatic assembly and annotation of the 758,510 redundant sequences (ESTs, mRNAs) of B. glabrata available in public databases. Computational analysis of Illumina reads followed by multivariate analyses allowed identification of 1685 candidate transcripts differentially expressed after an immune challenge, with a two fold ratio between transcripts showing a challenge-specific expression versus a lower or non-specific differential expression. Differential expression has been validated using quantitative PCR for a subset of randomly selected candidates. Predicted functions of annotated candidates (approx. 700 unisequences) belonged to a large extend to similar functional categories or protein types. This work significantly expands upon previous gene discovery and expression studies on B. glabrata and suggests that responses to various pathogens may involve similar immune processes or signaling pathways but different genes belonging to multigenic families. These results raise the question of the importance of gene duplication and acquisition of paralog functional diversity in the evolution of specific invertebrate immune responses.

The genetic structure of populations of the Mytilus edulis (L.)-M. galloprovincialis (Lmk.) complex has been characterized by studying polymorphism by means of isoenzyme electrophoresis. Three known loci (MPI, EST-D, GPI) have been used to characterize samples taken from two hybridization sites on the French Atlantic coast. The purpose of this study was to investigate the genotypical composition of a mussel brood during two consecutive years (1990 and 1991) in relation to the genetic structure of parental populations analyzed in 1989. The 1989 parental samples showed a strong hybridization at the two sites, and there was a correspondingly high degree of evolution in the 1990 and 1991 broods towards M. edulis. This indicates that genetic structure is not stable in the syntopic zones over time, and that there can be a marked evolution of genetic components, as demonstrated by the results from Groix Island between 1990 and 1991, where brood structure did not conform to that of the parental population. Our study takes into account environmental characteristics, gene flow, and the selection of dependent genotypes responsible for the evolution of the observed biodiversity at hybridization sites where bivalve culture is a contributory factor.

Previous studies have shown that hemocytes from inbred susceptible and resistant strains of the snail, Biomphalaria glabrata , differentially react to primary sporocysts of the blood fluke, Schistosoma mansoni . It has been hypothesized that this differential reactivity may be due to differences in the expression of hemocyte surface determinants serving as parasite recognition and/or hemocyte activating factors. In order to begin addressing this hypothesis the hemodynamics and surface polypeptides of circulating hemocytes from susceptible and resistant B. glabrata strains were compared in two morphologically distinct cellular subpopulations that differed in their substrate adherence properties. When compared to a S. mansoni -resistant snail strain, two susceptible strains exhibited overall lower total circulating cell numbers and a consistently lower proportion of nonadherent hemocytes. Exposure of snails to S. mansoni miracidia induced significant increases in total circulating cell number in all strains by 2 days postexposure (PE). However, the proportion of adherent and nonadherent hemocytes remained constant in each snail strain at 2 and 4 days PE. A sensitive technique involving biotin labeling of polypeptides at the surface of living hemocytes, followed by nonreducing SDS-PAGE separation and electrotransfer to nitrocellulose, revealed a similar pattern of hemocyte surface polypeptides ranging in apparent molecular weights from 210 to 30 kDa in the three B. glabrata strains. One exception was the presence of 66-kDa polypeptide expressed at the surface of both adherent and nonadherent hemocytes in the two susceptible snail strains, which was only weakly expressed or absent from hemocytes of the resistant 13-16-R1 B. glabrata strain . In addition, exposure of snails to S. mansoni miracidia appeared to modulate (enhance) expression of the 66-kDa polypeptide in susceptible B. glabrata cell populations. Although the functional importance of this membrane polypeptide is unknown, the differential enhancement of expression in susceptible vs resistant snail hemocytes may reflect potentially important biochemical changes related to previously observed strain-specific reactivities to invading larval S. mansoni . Finally, a complex of high-molecular-weight surface polypeptides (approx 210 kDa) associated with adherent hemocytes was consistently reduced or absent in nonadherent cell preparations suggesting a possible role in substrate adhesion.

1. 1. Prosorhynchus squamatus invariably castrates its host, Mytilus edulis . 2. 2. The tissue and species specificities of the action of uninfested M. edulis —mantle extracts and P. squamatus —sporocyst extracts have been tested in vitro . 3. 3. Both extracts inhibited gonadial mitosis in M. edulis M. galloprovincialis and Pecten maximus . 4. 4. Only the parasite extract inhibited gonial mitosis in Ostrea edulis . 5. 5. Both extracts inhibited mitosis in dispersed M. edulis mantle-edge cell suspensions (somatic tissue). Neither extract affected protein synthesis by this cell preparation. 6. 6. The activities from the two sources both had an apparent M r of 2–3.5 kDa.