Insights from Hi-C data regarding the Pacific salmon louse (Lepeophtheirus salmonis) sex-chromosomes.

Salmon lice, Lepeophtheirus salmonis (family Caligidae), are ectoparasites that have negatively impacted the salmon aquaculture industry and vulnerable wild salmon populations. Researchers have studied salmon lice to better understand their biology to develop effective control strategies. In this study, we updated the chromosome-level reference genome assembly of the Pacific subspecies of L. salmonis using Hi-C data. The previous version placed contigs/scaffolds using an Atlantic salmon louse genetic map. By utilizing Hi-C data from Pacific salmon lice, we were able to properly assign locations to contigs/scaffolds previously unplaced or misplaced. This resulted in a more accurate genome assembly and a more comprehensive characterization of the sex-chromosome unique to females (W). We found evidence that the same ZW-ZZ mechanism is common in both Atlantic and Pacific subspecies of salmon lice using PCR assays. The W-chromosome was approximately 800 kbp in size, which is ∼30 times smaller than the Z-chromosome (24 Mbp). The W-chromosome contained 61 annotated genes, including 32 protein-coding genes, 27 long non-coding RNA (lncRNA) genes, and 2 pseudogenes. Among these 61 genes, 39 genes shared homology to genes found on other chromosomes, while 20 were unique to the W-chromosome. Two genes of interest on the W-chromosome, prohibitin-2 and kinase suppressor of ras-2, were previously identified as potential sex-linked markers in the salmon louse. However, we prioritized the 20 unique genes on the W-chromosome as sex-determining candidates. This information furthers our understanding of the biology of this ectoparasite and may help in the development of more effective management strategies.

Genomics of Tenacibaculum Species in British Columbia, Canada.

Tenacibaculum is a genus of Gram-negative filamentous bacteria with a cosmopolitan distribution. The research describing Tenacibaculum genomes stems primarily from Norway and Chile due to their impacts on salmon aquaculture. Canadian salmon aquaculture also experiences mortality events related to the presence of Tenacibaculum spp., yet no Canadian Tenacibaculum genomes are publicly available. Ribosomal DNA sequencing of 16S and four species-specific 16S quantitative-PCR assays were used to select isolates cultured from Atlantic salmon with mouthrot in British Columbia (BC), Canada. Ten isolates representing four known and two unknown species of Tenacibaculum were selected for shotgun whole genome sequencing using the Oxford Nanopore's MinION platform. The genome assemblies achieved closed circular chromosomes for seven isolates and long contigs for the remaining three isolates. Average nucleotide identity analysis identified T. ovolyticum, T. maritimum, T. dicentrarchi, two genomovars of T. finnmarkense, and two proposed novel species T. pacificus sp. nov. type strain 18-2881-AT and T. retecalamus sp. nov. type strain 18-3228-7BT. Annotation in most of the isolates predicted putative virulence and antimicrobial resistance genes, most-notably toxins (i.e., hemolysins), type-IX secretion systems, and oxytetracycline resistance. Comparative analysis with the T. maritimum type-strain predicted additional toxins and numerous C-terminal secretion proteins, including an M12B family metalloprotease in the T. maritimum isolates from BC. The genomic prediction of virulence-associated genes provides important targets for studies of mouthrot disease, and the annotation of the antimicrobial resistance genes provides targets for surveillance and diagnosis in veterinary medicine.

An update of the salmon louse (Lepeophtheirus salmonis) reference genome assembly.

Salmon lice have plagued the salmon farming industry and have negatively impacted salmon populations in the wild. In response, researchers have generated high density genetic maps, genome assemblies, transcriptomes, and whole-genome resequencing data to better understand this parasite. In this study, we used long-read sequencing technology to update the previous genome assemblies of Atlantic Ocean salmon lice with a more contiguous assembly and a more comprehensive gene catalog of Pacific Ocean salmon lice. We were also able to further characterize genomic features previously identified from other studies by using published resequenced genomes of 25 Atlantic and 15 Pacific salmon lice. One example was further characterizing the ZW sex chromosomes. For both the Atlantic and Pacific Ocean salmon lice subspecies, we found that the female W-chromosome is only a small fraction of the Z-chromosome and that the vast majority of the W and Z-chromosome do not contain conserved regions (i.e. pseudoautosomal regions). However, conserved orthologous protein sequences can still be identified between the W- and Z-chromosomes.

Microbiome Profiling Reveals a Microbial Dysbiosis During a Natural Outbreak of Tenacibaculosis (Yellow Mouth) in Atlantic Salmon.

Tenacibaculosis remains a major health issue for a number of important aquaculture species globally. On the west coast of Canada, yellow mouth (YM) disease is responsible for significant economic loss to the Atlantic salmon industry. While Tenacibaculum maritimum is considered to be the primary agent of clinical YM, the impact of YM on the resident microbial community and their influence on the oral cavity is poorly understood. Using a 16s rRNA amplicon sequencing analysis, the present study demonstrates a significant dysbiosis and a reduction in diversity of the microbial community in the YM affected Atlantic salmon. The microbial community of YM affected fish was dominated by two amplicon sequence variants (ASVs) of T. maritimum, although other less abundant ASVs were also found. Interestingly clinically unaffected (healthy) and YM surviving fish also had a high relative abundance of T. maritimum, suggesting that the presence of T. maritimum is not solely responsible for YM. A statistically significant association was observed between the abundance of T. maritimum and increased abundance of Vibrio spp. within fish displaying clinical signs of YM. Findings from our study provide further evidence that YM is a complex multifactorial disease, characterized by a profound dysbiosis of the microbial community which is dominated by distinct ASVs of T. maritimum. Opportunistic taxa, including Vibrio spp., may also play a role in clinical disease progression.

Real-time RT-qPCR assay to detect sequences in the Piscine orthoreovirus-1 genome segment S1 associated with heart and skeletal muscle inflammation in Atlantic salmon.

During the last decade, Piscine orthoreovirus was identified as the main causative agent of heart and skeletal muscle inflammation (HSMI) in Atlantic Salmon, Norway. A recent study showed that PRV-1 sequences from salmonid collected in North Atlantic Pacific Coast (NAPC) grouped separately from the Norwegian sequences found in Atlantic Salmon diagnosed with HSMI. Currently, the routine assay used to screen for PRV-1 in NAPC water and worldwide cannot differentiate between the two groups of PRV-1. Therefore, this study aimed at developing a real-time polymerase chain reaction (RT-qPCR) assay to target the PRV-1 genome segments specific for variants associated with HSMI. The assay was optimized and tested against 71 tissue samples collected from different regions including Norway, Chile and both coast of Canada and different hosts farmed Atlantic Salmon, wild Coho Salmon and escaped Atlantic Salmon collected in British Columbia, West Coast of Canada. This assay has the potential to be used for screening salmonids and non-salmonids that may carry PRV-1 potentially causing HSMI.

Review of infectious agent occurrence in wild salmonids in British Columbia, Canada.

Wild Pacific salmonids (WPS) are economically and culturally important to the Pacific North region. Most recently, some populations of WPS have been in decline. Of hypothesized factors contributing to the decline, infectious agents have been postulated to increase the risk of mortality in Pacific salmon. We present a literature review of both published journal and unpublished data to describe the distribution of infectious agents reported in wild Pacific salmonid populations in British Columbia (BC), Canada. We targeted 10 infectious agents, considered to potentially cause severe economic losses in Atlantic salmon or be of conservation concern for wild salmon in BC. The findings indicated a low frequency of infectious hematopoietic necrosis virus, piscine orthoreovirus, viral haemorrhagic septicaemia virus, Aeromonas salmonicida, Renibacterium salmoninarum, Piscirickettsia salmonis and other Rickettsia-like organisms, Yersinia ruckeri, Tenacibaculum maritimum and Moritella viscosa. No positive results were reported for infestations with Paramoeba perurans in peer-reviewed papers and the DFO Fish Pathology Program database. This review synthesizes existing information, as well as gaps therein, that can support the design and implementation of a long-term surveillance programme of infectious agents in wild salmonids in BC.

Bayesian latent class analysis of ELISA and RT-rPCR diagnostic accuracy for subclinical Renibacterium salmoninarum infection in Atlantic salmon (Salmo salar) broodstock.

Renibacterium salmoninarum infection causes bacterial kidney disease (BKD) in salmonid freshwater and saltwater life stages, with potentially severe financial loss for the aquaculture industry. Preventing vertical transmission, from infected broodstock to eggs, is key to disease management. As there is no perfect reference standard for detecting R. salmoninarum, we used Bayesian latent class analyses to compare real-time reverse transcriptase PCR (RT-rPCR, mRNA target) and enzyme-linked immunosorbent assay (ELISA; p57 antigen target) diagnostic accuracy for detection in Atlantic salmon broodstock from British Columbia, Canada, and assessed ELISA repeatability. In 2016, 4,544 Atlantic salmon broodstock (no clinical signs of BKD or gross lesions) were sampled for ELISA testing of kidney tissue. Two groups of ELISA positives (n = 132) and two groups of a random sample of ELISA negatives (n = 137) were then tested with RT-rPCR, and ELISA testing was repeated. ELISA testing of broodstock provided the best diagnostic sensitivity (DSe; less chance of false-negative results). The use of joint RT-rPCR and ELISA testing improved DSe over that from each test alone, if a sample was considered positive when either test result was positive. Using these testing schemes in combination with management practices can decrease the likelihood of vertical transmission from subclinically infected broodstock.

The immune response of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain: A transcriptomic attempt at identifying molecular actors.

The marine mussel Mytilus edulis, tolerant to a wide range of environmental changes, combines a key role as a sentinel species for environmental monitoring programs and a significant economic importance. Mortality events caused by infective agents and parasites have not been described in mussels, which suggests an efficient immune system. This study aims at identifying the molecular mechanisms involved in the early immune responses M. edulis' hemocytes challenged with Vibrio splendidus LGP32 strain during 2, 4 and 6 h. A total of 149,296 assembled sequences has been annotated and compared to KEGG reference pathways. Several immune related sequences were identified such as Toll-Like receptors (TLRs), transcription factors, cytokines, protease inhibitors, stress proteins and sequences encoding for proteins involved in cell adhesion, phagocytosis, oxidative stress, apoptosis and autophagy. Differential gene expression clustered 10 different groups of transcripts according to kinetics of transcript occurrence. Sequences were assigned to biological process gene ontology categories. Sequences encoding for galectins, fibrinogen-related proteins, TLRs, MyD88, some antimicrobial peptides, lysosomal hydrolases, heat shock proteins and protease inhibitors, as well as proteins of oxidative stress and apoptosis were identified as differently regulated during the exposure to V. splendidus LGP32. The levels of candidate transcripts were quantified in M. edulis' hemocytes exposed to V. splendidus LGP32 and 7SHRW by using branched DNA technology. Transcripts encoding for inhibitor kappa B, inhibitor of apoptosis proteins, tumor protein D54, serine/threonine-proteine kinase SIK2 were identified as up-regulated in hemocytes exposed to both strains.

Functional and molecular responses of the blue mussel Mytilus edulis' hemocytes exposed to cadmium - An in vitro model and transcriptomic approach.

The bivalve mollusk, Mytilus edulis, is used as a sentinel species in several monitoring programs due to its ability to bio-accumulate contaminants. Its immune system consists of hemocytes and humoral components, which constitute the main part of the hemolymph. The present study is aimed at understanding the effects of Cd on the differentially expressed genes involved in the phagocytosis of M. edulis' hemocytes. Our approach focuses on an in vitro model by exposing hemocytes to different concentrations of Cd ranging from 10-9 M to 10-3 M. Phagocytosis and cell viability as functional markers were measured using flow cytometry. The molecular mechanisms regulated by Cd were investigated using RNA-seq and DGE analysis. Results showed that viability and phagocytosis of hemocytes exposed to 10-3 M of Cd were significantly decreased after 21 h of exposure. RNA sequencing data showed that 1112 transcripts (out of 352,976 contigs) were differentially regulated by the highest concentration of Cd. Among these identified transcripts, 1028 and 84 were up and down-regulated respectively. The induction of super oxide dismutase (SOD), glutathion-s-transferase (GST), cytochrome P450 2C8 (CYP2C8), multidrug resistance protein (MRP1) and heat shock protein 70 (HSP70) suggests that Cd can regulate key molecular mechanisms. In addition, several toll-like receptors (TLR) as well as genes involved in phagocytosis (actin and CDC42) and apoptosis (caspase 8 and XIAP/IAP) were induced by Cd. Thus, our model highlights the effect of Cd on the phagocytic function of M. edulis' hemocytes along with the regulation of gene expression involved in innate immunity, detoxification and apoptosis. Further investigations need to be pursued to unravel the effects of Cd on the molecular mechanisms identified in this study.

Correction: Piscine Reovirus: Genomic and Molecular Phylogenetic Analysis from Farmed and Wild Salmonids Collected on the Canada/US Pacific Coast.

[This corrects the article DOI: 10.1371/journal.pone.0141475.].

Copper and hypoxia modulate transcriptional and mitochondrial functional-biochemical responses in warm acclimated rainbow trout (Oncorhynchus mykiss).

To survive in changing environments fish utilize a wide range of biological responses that require energy. We examined the effect of warm acclimation on the electron transport system (ETS) enzymes and transcriptional responses to hypoxia and copper (Cu) exposure in fish. Rainbow trout (Oncorhynchus mykiss) were acclimated to cold (11 °C; control) and warm (20 °C) temperatures for 3 weeks followed by exposure to Cu, hypoxia or both for 24 h. Activities of ETS enzyme complexes I-IV (CI-CIV) were measured in liver and gill mitochondria. Analyses of transcripts encoding for proteins involved in mitochondrial respiration (cytochrome c oxidase subunits 4-1 and 2: COX4-1 and COX4-2), metal detoxification/stress response (metallothioneins A and B: MT-A and MT-B) and energy sensing (AMP-activated protein kinase α1: AMPKα1) were done in liver mitochondria, and in whole liver and gill tissues by RT-qPCR. Warm acclimation inhibited activities of ETS enzymes while effects of Cu and hypoxia depended on the enzyme and thermal acclimation status. The genes encoding for COX4-1, COX4-2, MT-A, MT-B and AMPKα1 were strongly and tissue-dependently altered by warm acclimation. While Cu and hypoxia clearly increased MT-A and MT-B transcript levels in all tissues, their effects on COX4-1, COX4-2 and AMPKα1 mRNA levels were less pronounced. Importantly, warm acclimation differentially altered COX4-2/COX4-1 ratio in liver mitochondria and gill tissue. The three stressors showed both independent and joint actions on activities of ETS enzymes and transcription of genes involved in energy metabolism, stress response and metals homeostasis. Overall, we unveiled novel interactive effects that should not be overlooked in real world situations wherein fish normally encounter multiple stress factors.

Piscine Reovirus: Genomic and Molecular Phylogenetic Analysis from Farmed and Wild Salmonids Collected on the Canada/US Pacific Coast.

Piscine reovirus (PRV) is a double stranded non-enveloped RNA virus detected in farmed and wild salmonids. This study examined the phylogenetic relationships among different PRV sequence types present in samples from salmonids in Western Canada and the US, including Alaska (US), British Columbia (Canada) and Washington State (US). Tissues testing positive for PRV were partially sequenced for segment S1, producing 71 sequences that grouped into 10 unique sequence types. Sequence analysis revealed no identifiable geographical or temporal variation among the sequence types. Identical sequence types were found in fish sampled in 2001, 2005 and 2014. In addition, PRV positive samples from fish derived from Alaska, British Columbia and Washington State share identical sequence types. Comparative analysis of the phylogenetic tree indicated that Canada/US Pacific Northwest sequences formed a subgroup with some Norwegian sequence types (group II), distinct from other Norwegian and Chilean sequences (groups I, III and IV). Representative PRV positive samples from farmed and wild fish in British Columbia and Washington State were subjected to genome sequencing using next generation sequencing methods. Individual analysis of each of the 10 partial segments indicated that the Canadian and US PRV sequence types clustered separately from available whole genome sequences of some Norwegian and Chilean sequences for all segments except the segment S4. In summary, PRV was genetically homogenous over a large geographic distance (Alaska to Washington State), and the sequence types were relatively stable over a 13 year period.

Effects of copper, hypoxia and acute temperature shifts on mitochondrial oxidation in rainbow trout (Oncorhynchus mykiss) acclimated to warm temperature.

Temperature fluctuations, hypoxia and metals pollution frequently occur simultaneously or sequentially in aquatic systems and their interactions may confound interpretation of their biological impacts. With a focus on energy homeostasis, the present study examined how warm acclimation influences the responses and interactions of acute temperature shift, hypoxia and copper (Cu) exposure in fish. Rainbow trout (Oncorhynchus mykiss) were acclimated to cold (11°C; control) and warm (20°C) temperature for 3 weeks followed by exposure to environmentally realistic levels of Cu and hypoxia for 24h. Subsequently, mitochondrial electron transport system (ETS) respiratory activity supported by complexes I-IV (CI-IV), plasma metabolites and condition indices were measured. Warm acclimation reduced fish condition, induced aerobic metabolism and altered the responses of fish to acute temperature shift, hypoxia and Cu. Whereas warm acclimation decelerated the ETS and increased the sensitivity of maximal oxidation rates of the proximal (CI and II) complexes to acute temperature shift, it reduced the thermal sensitivity of state 4 (proton leak). Effects of Cu with and without hypoxia were variable depending on the acclimation status and functional index. Notably, Cu stimulated respiratory activity in the proximal ETS segments, while hypoxia was mostly inhibitory and minimized the stimulatory effect of Cu. The effects of Cu and hypoxia were modified by temperature and showed reciprocal antagonistic interaction on the ETS and plasma metabolites, with modest additive actions limited to CII and IV state 4. Overall, our results indicate that warm acclimation came at a cost of reduced ETS efficiency and increased sensitivity to added stressors.

Alterations in mitochondrial electron transport system activity in response to warm acclimation, hypoxia-reoxygenation and copper in rainbow trout, Oncorhynchus mykiss.

Fish expend significant amounts of energy to handle the numerous potentially stressful biotic and abiotic factors that they commonly encounter in aquatic environments. This universal requirement for energy singularizes mitochondria, the primary cellular energy transformers, as fundamental drivers of responses to environmental change. Our study probed the interacting effects of thermal stress, hypoxia-reoxygenation (HRO) and copper (Cu) exposure in rainbow trout to test the prediction that they act jointly to impair mitochondrial function. Rainbow trout were acclimated to 11 (controls) or 20°C for 2 months. Liver mitochondria were then isolated and their responses in vitro to Cu (0-20µM) without and with HRO were assessed. Sequential inhibition and activation of mitochondrial electron transport system (ETS) enzyme complexes permitted the measurement of respiratory activities supported by complex I-IV (CI-IV) in one run. The results showed that warm acclimation reduced fish and liver weights but increased mitochondrial protein indicating impairment of energy metabolism, increased synthesis of defense proteins and/or reduced liver water content. Whereas acute rise (11→20°C) in temperature increased mitochondrial oxidation rates supported by CI-IV, warm acclimation reduced the maximal (state 3) and increased the basal (state 4) respiration leading to global uncoupling of oxidative phosphorylation (OXPHOS). HRO profoundly inhibited both maximal and basal respiration rates supported by CI-IV, reduced RCR for all except CII and lowered CI:CII respiration ratio, an indication of decreased OXPHOS efficiency. The effects of Cu were less pronounced but more variable and included inhibition of CII-IV maximal respiration rates and stimulation of both CI and CIII basal respiration rates. Surprisingly, only CII and CIII indices exhibited significant 3-way interactions whereas 2-way interactions of acclimation either with Cu or HRO were portrayed mostly by CIV, and those of HRO and Cu were most common in CI and II respiratory indices. Our study suggests that warm acclimation blunts sensitivity of the ETS to temperature rise and that HRO and warm acclimation impose mitochondrial changes that sensitize the ETS to Cu. Overall, our study highlights the significance of the ETS in mitochondrial bioenergetic dysfunction caused by thermal stress, HRO and Cu exposure.

Interactions of copper and thermal stress on mitochondrial bioenergetics in rainbow trout, Oncorhynchus mykiss.

Thermal stress may influence how organisms respond to concurrent or subsequent chemical, physical and biotic stressors. To unveil the potential mechanisms via which thermal stress modulates metals-induced bioenergetic disturbances, the interacting effects of temperature and copper (Cu) were investigated in vitro. Mitochondria isolated from rainbow trout livers were exposed to a range of Cu concentrations at three temperatures (5, 15 and 25 °C) with measurement of mitochondrial complex I (mtCI)-driven respiratory flux indices and uncoupler-stimulated respiration. Additional studies assessed effects of temperature and Cu on mtCI enzyme activity, induction of mitochondrial permeability transition pore (MPTP), swelling kinetics and mitochondrial membrane potential (MMP). Maximal and basal respiration rates, as well as the proton leak, increased with temperature with the Q10 effects being higher at lower temperatures. The effect of Cu depended on the mitochondrial functional state in that the maximal respiration was monotonically inhibited by Cu exposure while low and high Cu concentrations stimulated and inhibited the basal respiration/proton leak, respectively. Importantly, temperature exacerbated the effects of Cu by lowering the concentration of the metal required for toxicity and causing loss of thermal dependence of mitochondrial respiration. Mitochondrial complex I activity was inhibited by Cu but was not affected by incubation temperature. Compared with the calcium (Ca) positive control, Cu-imposed mitochondrial swelling exhibited variable kinetics depending on the inducing conditions, and was highly temperature-sensitive. A partial reversal of the Cu-induced swelling by cyclosporine A was observed suggesting that it is in part mediated by MPTP. Interestingly, the combination of high Cu and high temperature not only completely inhibited mitochondrial swelling but also greatly increased the respiratory control ratio (RCR) relative to the controls. Copper exposure also caused marked MMP dissipation which was reversed by N-acetyl cysteine and vitamin E suggesting a role of reactive oxygen species (ROS) in this response. Taken together, Cu impairs oxidative phosphorylation in part by inhibiting the electron transport chain (ETC), stimulating proton leak, inducing MPTP and dissipating MMP, with high temperature exacerbating these effects. Thus environmental temperature rise due to natural phenomenon or global climate change may sensitize fish to Cu toxicity by exacerbating mitochondrial dysfunction.

Disseminated neoplasia in the soft-shell clam Mya arenaria: membrane lipid composition and functional parameters of circulating cells.

In a previous study we compared lipid composition and functional parameters of circulating cells from Cerastoderma edule affected or not by disseminated neoplasia (neoplastic cells vs hemocytes) (Le Grand et al. Chem Phys Lipids 167:9-20 2013). Neoplastic cells presented morpho-functional modifications concomitant to striking membrane lipid alterations: the proportion of particular plasmalogen molecular species was drastically decreased. We wanted to test whether this pattern was representative of bivalve neoplastic cells. For the purpose, a similar study was conducted on another bivalve species affected by disseminated neoplasia, the soft-shell clam (Mya arenaria). Although total reactive oxygen species production was unaffected, M. arenaria neoplastic cells presented some functional alterations: phagocytosis activity was reduced by 33 %. However, lipid compositions were not drastically altered. Particularly, sterol and plasmalogen levels did not differ between both cell types (about 43 % of membrane lipids and 35 % of phospholipids, respectively in hemocytes and neoplastic cells). This could be related to the fact that disseminated neoplasia was not related to hemolymph cell proliferation in M. arenaria (0.9 ± 0.2 10(6)cell mL(-1), considering both healthy and neoplastic clams, n = 6). Nevertheless this study highlighted minor but specific alterations of membrane lipid composition in M. arenaria neoplastic cells. The only phospholipid subclass in which the fatty acid profile strongly differed between both cell types was serine plasmalogen (PlsSer), with neoplastic cells presenting lower specific enrichment of 20:1n-11 in PlsSer. Such specific alteration of membrane lipid composition strengthened the assumption of an implication of key plasmalogen molecular species in this leukemia-like disease in bivalves.

Functional and molecular responses in Mytilus edulis hemocytes exposed to bacteria, Vibrio splendidus.

This study aims at examining the morphological, functional and molecular responses of Mytilus edulis hemocytes exposed to different strains of Gram-negative bacteria Vibrio splendidus (a virulent strain V. splendidus LGP32, V. splendidus LGP32 Δvsm without metalloprotease and an environmental type strain V. splendidus 7SHRW) at a 1:3 ratio for 2, 4, and 6 h. Our data showed that hemocytes could have a discriminative capacity towards microorganisms. Both V. splendidus LGP32 strains had an effect on hemocyte adhesion, phagocytosis abilities and oxidative burst, whereas the environmental strain 7SHRW induced weak and delayed hemocyte responses. At a molecular level, differential levels of candidate transcripts were measured in M. edulis hemocytes exposed to V. splendidus LGP32-GFP and 7SHRW. Mainly, a down-regulation of defensin was recorded in hemocytes exposed to V. splendidus LGP32. A significant up-regulation of lysozyme and proteasome 26S was observed at 2 h followed by a down-regulation at 4 and 6 h of exposure to the LGP32 strain. Similarly, SOD and GPx genes were up-regulated 2 h post-exposure to LGP32 strain and their expressions decreased after 4 and 6 h post-exposure. Further analysis is however needed in a near future to relate the transcript level variations with the physiological process.

Sequence analysis of a normalized cDNA library of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain.

In the past decades, reports on bivalves' pathogens and associated mortalities have steadily increased. To face pathogenic micro-organisms, bivalves rely on innate defenses established in hemocytes which are essentially based on phagocytosis and cytotoxic reactions. As a step towards a better understanding of the molecular mechanisms involved in the mussel Mytilus edulis innate immune system, we constructed and sequenced a normalized cDNA library specific to M. edulis hemocytes unchallenged (control) and challenged with Vibrio splendidus LGP32 strain for 2, 4 and 6 h. A total of 1,024,708 nucleotide reads have been generated using 454 pyrosequencing. These reads have been assembled and annotated into 19,622 sequences which we believe cover most of the M. edulis hemocytes transcriptome. These sequences were successfully assigned to biological process, cellular component, and molecular function Gene Ontology (GO) categories. Several transcripts related to immunity and stress such as some fibrinogen related proteins and Toll-like receptors, the complement C1qDC, some antioxidant enzymes and antimicrobial peptides have already been identified. In addition, Toll-like receptors signaling pathways and the lysosome and apoptosis mechanisms were compared to KEGG reference pathways. As an attempt for large scale RNA sequencing, this study focuses on identifying and annotating transcripts from M. edulis hemocytes regulated during an in vitro experimental challenge with V. splendidus. The bioinformatic analysis provided a reference transcriptome, which could be used in studies aiming to quantify the level of transcripts using high-throughput analysis such as RNA-Seq.

Transcriptome analysis of neoplastic hemocytes in soft-shell clams Mya arenaria: Focus on cell cycle molecular mechanism.

In North America, a high mortality of soft-shell clams Mya arenaria was found to be related to the disease known as disseminated neoplasia (DN). Disseminated neoplasia is commonly recognized as a tetraploid disorder related to a disruption of the cell cycle. However, the molecular mechanisms by which hemocytes of clams are transformed in the course of DN remain by far unknown. This study aims at identifying the transcripts related to DN in soft shell clams' hemocytes using next generation of sequencing (Illumina HiSeq2000). This study mainly focuses on transcripts and molecular mechanisms involved in cell cycle. Using Illumina next generation of sequencing, more than 95,399,159 reads count with an average length of 45 bp was generated from three groups of hemocytes: (1) a healthy group with less than 10% of tetraploid cells; (2) an intermediate group with tetraploid hemocytes ranging between 10% and 50% and (3) a diseased group with more than 50% of tetraploid cells. After the reads were cleaned by removing the adapters, de novo assembly was performed on the sequences and more than 73,696 contigs were generated with a mean contig length estimated at 585 bp ranging from 189 bp to 14,773 bp. Once a Blastx search against NCBI Non Redundant database was performed and the duplicates removed, 18,378 annotated sequences matched known sequences, 3078 were hypothetical and 9002 were uncharacterized sequences. Fifty percent and 41% of known sequences match sequences from Mollusca and Gastropoda respectively. Among the bivalvia, 33%, 17%, 17% and 15% of the contigs match sequences from Ostreoida, Veneroida, Pectinoida and Mytiloida respectively. Gene ontology analysis showed that metabolic, cellular, transport, cell communication and cell cycle represent 33%, 15%, 9%, 8.5% and 7% respectively of the total biological process. Approximately 70% of the component process is related to intracellular process and 15% is linked to protein and ribonucleoprotein complex. Catalytic activities and binding molecular processes represent 39% and 33% of the total molecular functions. Interestingly, nucleic acid binding represents more than 18% of the total protein class. Transcripts involved in the molecular mechanisms of cell cycle are discussed providing new avenues for future investigations.

Effects of pesticide compounds (chlorothalonil and mancozeb) and benzo[a]pyrene mixture on aryl hydrocarbon receptor, p53 and ubiquitin gene expression levels in haemocytes of soft-shell clams (Mya arenaria).

The aim of this study is to investigate the effects of the pesticides/polycyclic aromatic hydrocarbon mixture on aryl hydrocarbon receptor (AhR), p53 and ubiquitin mRNA level in haemocytes of Mya arenaria exposed to a mixture of chlorothalonil, mancozeb and benzo[a]pyrene (BaP) for 48 and 72 h. AhR, p53 and ubiquitin gene expression levels were quantified using quantitative Real-time PCR. For robust and accurate quantification of transcripts, suitable housekeeping genes were selected from four sets of ribosomal and elongation factors transcripts previously sequenced from Mya arenaria using geNorm open source software. Quantitative Real-time PCR data exhibited a significantly high expression of AhR after 72 h of exposure (P ≤ 0.05). p53 gene expression seems to be up-regulated by the mixture after 48 h, however not significantly; but the level of p53 mRNA is down-regulated by the xenobiotics between 48 and 72 h after exposure. This study postulates that AhR mRNA levels could be used as an indicator of the exposure of clams' haemocytes to a mixture of xenobiotics such as chlorothalonil, mancozeb and BaP. However, further studies have to be pursued in order to unravel the molecular mechanisms involved in the p53 signaling pathway.