New insights into the Manila clam – Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges

The Manila clam (Ruditapes philippinarum) is the bivalve species with the highest global production from both fisheries and aquaculture, but its production is seriously threatened by perkinsosis, a disease caused by the protozoan parasite Perkinsus olseni. To understand the molecular mechanisms underlying R. philippinarum–P. olseni interactions, we analysed the gene expression profiles of in vitro challenged clam hemocytes and P. olseni trophozoites, using two oligo-microarray platforms, one previously validated for R. philippinarum hemocytes and a new one developed and validated in this study for P. olseni. Manila clam hemocytes were in vitro challenged with trophozoites, zoospores, and extracellular products from P. olseni in vitro cultures, while P. olseni trophozoites were in vitro challenged with Manila clam plasma along the same time-series (1 h, 8 h, and 24 h). The hemocytes showed a fast activation of the innate immune response, particularly associated with hemocyte recruitment, in the three types of challenges. Nevertheless, different immune-related pathways were activated in response to the different parasite stages, suggesting specific recognition mechanisms. Furthermore, the analyses provided useful complementary data to previous in vivo challenges, and confirmed the potential of some proposed biomarkers. The combined analysis of gene expression in host and parasite identified several processes in both the clam and P. olseni, such as redox and glucose metabolism, protease activity, apoptosis and iron metabolism, whose modulation suggests cross-talk between parasite and host. This information might be critical to determine the outcome of the infection, thus highlighting potential therapeutic targets. Altogether, the results of this study aid understanding the response and interaction between R. philippinarum and P. olseni, and will contribute to developing effective control strategies for this threatening parasitosisThis work was funded by the Ministerio de Economía y Competitividad of the Spanish Government, through the projects (AGL2011-30449-C02-01 and AGL2012-37981), the European Regional Development Funds (FEDER 2007–2013) and the Regional Government of Galicia, Xunta de Galicia, Spain, through the projects ED431C 2018/28 and ED431D 2017/21. The study was also supported by the Centro de Supercomputación de Galicia (CESGA). The first author would like to acknowledge the PhD scholarship awarded by the EXPERTS III Consortium of the European Community Mobility Programme “Erasmus Mundus Action 2, Strand 1” (EMA2). SFB was supported by a scholarship of the Consellería do Mar da Xunta de GaliciaS

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases

Evaluation of the intra and interspecific variability in the genus Perkinsus. Proteomic analysis of the parasite and its interaction with the host

Clam culture industry is rising every year worldwide. The most produced species is the Manila clam Ruditapes philippinarum. China is the main producer country followed by Italy, South Korea, USA and Spain. The introduction of the Manila clam in Europe was between 1970 and 1980. R. philippinarum is well adapted and grows faster than native species R. decussatus, for this, is the most produced species in Europe. The production of venerids in Galicia is a very important socio-economic resource. The production of Manila clam in Galicia rose in the last years and now is the most produced species too. The infection by Perkinsus olseni is one of the most serious diseases affecting clams. The parasite is highly distributed worldwide and affect to a large list of molluscs. Mortality of R. decussatus and R. philippinarum was associated with P. olseni infection in south Europe as well as with R. philippinarum mortality in several Asian countries. Two species of the genus Perkinsus, P. olseni and P. marinus, are included in the list of notifiable diseases of the World Organization for Animal Health, which indicates the interest to stop the expansion of the disease. The knowledge of P. olseni is very scarce in comparison with P. marinus which provokes massive mortalities in oysters Crassostrea virginica in USA. The economic transcendence of the perkinsosis justifies the research for a better knowledge of the disease and minimizes their effects. With this purpose, this study was developed in order to know (1) the variability of P. olseni along the Spanish coast, with the emphasis in virulence variability among populations, and (2) the modulation of the Manila clam protein expression due to P. olseni infection, with emphasis in the search of protein markers of resistance to the disease. The study of the variability was done analyzing the genetic population structure as well as the proteome of P. olseni clons derived from several regions of the Spanish coast. Furthermore, the proteome of P. olseni was compared with the proteome of two species of the genus, P. marinus and P. chesapeaki, in order to amplify the perspective of the variability of P. olseni. The analysis of the modulation of the Manila clam protein expression due to P. olseni infection was focused in haemocytes and haemolymph. The effect of the exposition to P. olseni in clams was measured at short time and a long time with the aim in the modulation of the immune response of the clam to the parasite

MgC1q, a novel C1q-domain-containing protein involved in the immune response of Mytilus galloprovincialis

9 páginas, 6 figuras, 1 tablaIn this study, we present the characterization of a newly identified gene, MgC1q, revealed in suppression subtractive hybridization and cDNA libraries from immunostimulated mussels. Based on sequence homology, molecular architecture and domain similarity, this new C1q-domain-containing gene may be classified as a member of the C1q family and, therefore, part of the C1q–TNF superfamily. The expression of MgC1q was detected all along the mussel ontogeny, being detectable within 2 h post-fertilization, with a notable increase after 1 month and continuing to increase until 3 months. Measurable transcript levels were also evident in all analyzed tissues of naïve adult mussels, and the hemocytes showed the highest expression levels. Experimental infection of adult mussels with Gram positive or Gram negative bacteria significantly modulated the MgC1q expression, and confirmed it as an immune-related gene. Intra- and inter-individual sequence analyses revealed extraordinary diversity of MgC1q at both the DNA and cDNA levels. While further research is needed to define its function, our data indicate that MgC1q is a pattern recognition molecule able to recognize pathogens during innate immune responses in Myitilus galloprovincialis. The high sequence variability suggests that somatic diversification of these nonself recognition molecules could have occurred.This work has been funded by the EU Integrated Project FOOD-CT-2005-007103 and AGL2008-05111/ACU from the Spanish Ministerio de Ciencia e Innovación. Camino Gestal wishes to acknowledge additional funding from the Spanish Ministerio de Educación y Ciencia through the “Ramón y Cajal” Contract.Peer reviewe

The Alveolate Perkinsus marinus: Biological Insights from EST Gene Discovery

<p>Abstract</p> <p>Background</p> <p><it>Perkinsus marinus</it>, a protozoan parasite of the eastern oyster <it>Crassostrea virginica</it>, has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of the United States. It is classified as a member of the Perkinsozoa, a recently established phylum considered close to the ancestor of ciliates, dinoflagellates, and apicomplexans, and a key taxon for understanding unique adaptations (<it>e.g</it>. parasitism) within the Alveolata. Despite intense parasite pressure, no disease-resistant oysters have been identified and no effective therapies have been developed to date.</p> <p>Results</p> <p>To gain insight into the biological basis of the parasite's virulence and pathogenesis mechanisms, and to identify genes encoding potential targets for intervention, we generated >31,000 5' expressed sequence tags (ESTs) derived from four trophozoite libraries generated from two <it>P. marinus </it>strains. Trimming and clustering of the sequence tags yielded 7,863 unique sequences, some of which carry a spliced leader. Similarity searches revealed that 55% of these had hits in protein sequence databases, of which 1,729 had their best hit with proteins from the chromalveolates (E-value ≤ 1e-5). Some sequences are similar to those proven to be targets for effective intervention in other protozoan parasites, and include not only proteases, antioxidant enzymes, and heat shock proteins, but also those associated with relict plastids, such as acetyl-CoA carboxylase and methyl erythrithol phosphate pathway components, and those involved in glycan assembly, protein folding/secretion, and parasite-host interactions.</p> <p>Conclusions</p> <p>Our transcriptome analysis of <it>P. marinus</it>, the first for any member of the Perkinsozoa, contributes new insight into its biology and taxonomic position. It provides a very informative, albeit preliminary, glimpse into the expression of genes encoding functionally relevant proteins as potential targets for chemotherapy, and evidence for the presence of a relict plastid. Further, although <it>P. marinus </it>sequences display significant similarity to those from both apicomplexans and dinoflagellates, the presence of trans-spliced transcripts confirms the previously established affinities with the latter. The EST analysis reported herein, together with the recently completed sequence of the <it>P. marinus </it>genome and the development of transfection methodology, should result in improved intervention strategies against dermo disease.</p

PHYSELLA ACUTA, COMPARATIVE IMMUNOLOGY AND EVOLUTIONARY ASPECTS OF GASTROPOD IMMUNE FUNCTION

Gastropod immunobiology has benefitted from investigations focused on the planorbid snail Biomphalaria glabrata, intermediate host for the human parasite Schistosoma mansoni. Though such concentrated efforts have elucidated fascinating aspects of invertebrate immunity, they have not provided full knowledge regarding the evolution of immune function among other gastropod species. This dissertation presents the importance of making strategic choices regarding which organisms to select for comparative immunology. Herein, the choice was made to investigate the immunobiology of Physella acuta, a freshwater snail species of the Physidae, a sister family to Planorbidae to which B. glabrata belongs. Benefiting greatly from the use of next-generation sequencing (NGS), the immunobiology of P. acuta was studied using 454 pyrosequencing, Illumina RNA-seq, experimental infections with Echinostoma paraensei (trematode parasite), and other molecular techniques. These analyses revealed that many components of gastropod immunity have been conserved among physid and planorbid snails. Also, P. acuta displays differences in immune function, such as the use of fibrinogen-related proteins in response to trematode parasite exposure. Remarkably, P. acuta differentially expressed relatively large immune-relevant gene families (CD109/TEP, dermatopin, GTPase IMAP, among others) after exposure to E. paraenesi. Inspection of the individual members that represent these gene families demonstrated complex transcriptional profiles that suggest parasite influence on host immune function and the capacity of a host to maintain homeostasis while supporting parasite development, an extended phenotype of E. paraenesi. These lab-based studies represent the first large scale characterizations of P. acuta immune function. The immune factors described through NGS approaches enable investigations of the ecoimmunology of P. acuta snails collected from the field. This approach uncovered many sequences that are differentially expressed by P. acuta naturally in the field relative to the lab environment. There is variation in the expression of certain antimicrobial factors and genes governing biological processes. Overall, this dissertation has expanded the scope of gastropod immunity and provides resources and insights that are accessible for continued development and understanding of evolutionary and comparative immunology concepts

Specific versus Non-Specific Immune Responses in an Invertebrate Species Evidenced by a Comparative de novo Sequencing Study

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

Genetic markers associated with divergent selection against the parasite Marteilia cochillia in common cockle (Cerastoderma edule) using transcriptomics and population genomics data

The common cockle (Cerastoderma edule) plays an important role in marine ecosystems and represents a valuable socioeconomic resource for coastal communities. In 2012, the cockle beds from Rı́a de Arousa (Galicia, NW Spain) were seriously decimated by the protozoan Marteilia cochillia responsible for marteiliosis. We aimed to identify single nucleotide polymorphisms (SNP) markers potentially associated with resilience to marteiliosis to be used in marker-assisted selection programs for restoring affected cockle beds and recovering their production. For this, we carried out a population genomics approach using 2b-RADseq, where 38 naive samples (before the first detection of M. cochillia in 2012) from two beds of Rı́a de Arousa were compared with 39 affected samples collected in 2018/2019 (after several years of marteiliosis occurring in the area), collected either before (15 non-exposed samples) or during (24 exposed samples) the marteiliosis outbreak. Additionally, 767 differentially expressed genes (DEG) from a previous transcriptomic study addressed during the aforementioned 2018/19 marteiliosis outbreak, were evaluated to identify SNPs showing signals of selection. Using 2b-RADseq, 9,154 SNPs were genotyped and among them, 110 consistent outliers for divergent selection were identified. This set of SNPs was able to discriminate the samples according to their marteiliosis status (naive vs affected; exposed vs non-exposed), while another 123 SNPs were identified linked to DEGs associated with the level of infection across a temporal series. Finally, combining the population genomics and transcriptomics information, we selected the 60 most reliable SNPs associated with marteiliosis resilience. These SNPs were close to or within DEGs, and many of them were related to immune response (phagocytosis and cell adhesion), defence, such as apoptosis, stress, and cellular cycle, among other functions. This set of SNPs will eventually be validated to develop a cost-effective genotyping tool for their application for obtaining cockle-resilient strains for marteiliosis

Winter School on Vistas in Marine Biotechnology

Winter School on Vistas in Marine Biotechnology, 5th to 26th October, 2010 at Marine Biotechnology Division CMFRI, Koch