Occurrence and seasonal variation of Perkinsus sp. Infection in wild mollusk populations from coastal waters of Qingdao, northern China.

Perkinsosis has been recognized as one of the major threats to natural and farmed bivalve populations, many of which are of commercial as well as environmental significance. Three Perkinsus species have been identified in China, and the Manila clam (Ruditapes philippinarum) was the most frequently infected species in northern China. Although the occurrence and seasonal variation of Perkinsus spp. have previously been examined, the pathological characteristics of these infections in wild Manila clams and sympatric species in China have seldom been reported. In the present study, the prevalence and intensity of Perkinsus infection in wild populations of Manila clams and 10 sympatric species from three sites were investigated by Ray's fluid thioglycolate medium (RFTM) assay seasonally across a single year. Perkinsus infection was only identified in Manila clams, with a high prevalence (274/284 = 96.48 %) and low intensity (89.8 % with a Mackin value ≤ 2, suggesting generally low-intensity infections) throughout the year. Heavily infected clams were mainly identified in Tianheng in January, which displayed no macroscopic signs of disease. An overview of the whole visceral mass section showed that the trophozoites mostly aggregated in gills and connective tissue of the digestive tract, to a lesser extent in the mantle and foot, and even less frequently in adductor muscle and connective tissues of the gonad. PCR and ITS-5.8S rRNA sequencing of 93 representative RFTM-positive samples revealed a 99.69 to 100 % DNA sequence identity to Perkinsus olseni. Unexpectedly, significantly higher infection intensities were usually identified in January and April when the Condition Index (CI) was relatively high. We propose that factors associated with the anthropogenic harvesting pressure and irregular disturbances should be responsible for the uncommon seasonal infection dynamics of perkinsosis observed in the present study.

The Probiotic Bacillus hwajinpoensis Colonizes the Digestive System of Crassostrea gigas Larvae and Protects Them from Vibrio alginolyticus Infection.

The Pacific oyster Crassostrea gigas is one of the most important cultured marine species around the world. Production of Pacific oysters in China has depended primarily on hatchery produced seeds since 2016, with the successful introduction and development of triploid oysters. However, the seed supply of Pacific oysters is threatened by recurring mass mortality events in recent years. Vibriosis is the most commonly encountered disease associated with intensive oyster culture in hatcheries and nurseries. Vibrio alginolyticus and Bacillus hwajinpoensis were the two strains with pathogenic and probiotic effects, respectively, identified during the Pacific oyster larvae production. To monitor their colonization process in Pacific oyster larvae, green fluorescent protein (GFP) and red fluorescent protein (RFP) were labeled to the pathogenic V. alginolyticus and the probiotic B. hwajinpoensis stain, respectively. The pathogenic and probiotic effects of the two strains during the colonization process were then assessed. Stabile expression of GFP and RFP were observed in corresponding stains, and the capabilities of growth, biofilm formation and in vitro adhesion of GFP- and RFP- tagged stains were not significantly different from those of the wild-type strains. Usage of probiotics of 105 CFU/mL significantly inhibited the growth of pathogenic V. alginolyticus and reduced the mortality of D-sharped larvae. Both the pathogenic and probiotic strains employed a similar route to enter and colonize the oyster larvae, which indicates that competing with pathogens for binding and spreading sites were one of the mechanisms of B. hwajinpoensis to provide the probiotic effects to oyster larvae. In summary, employment of fluorescence-tagged pathogenic and probiotic strains simultaneously provides us with an excellent bioassay model to investigate the potential mechanisms of probiotics.

Identification and Characterization of Infectious Pathogens Associated with Mass Mortalities of Pacific Oyster (Crassostrea gigas) Cultured in Northern China.

The Pacific oyster (Crassostrea gigas) aquaculture industry increased rapidly in China with the introduction and promotion of triploid oysters in recent years. Mass mortalities affecting different life stages of Pacific oysters emerged periodically in several important production areas of Northern China. During 2020 and 2021, we conducted a passive two-year investigation of infectious pathogens linked to mass mortality. Ostreid herpesvirus-1 (OsHV-1) was detected to be associated with mass mortalities of hatchery larvae, but not juveniles and adults in the open sea. Protozoan parasites, such as Marteilia spp., Perkinsus spp. and Bonamia spp. were not detected. Bacterial isolation and identification revealed that Vibrio natriegens and Vibrio alginolyticus were the most frequently (9 out of 13) identified two dominant bacteria associated with mass mortalities. Pseudoalteromonas spp. was identified as the dominant bacteria in three mortality events that occurred during the cold season. Further bacteriological analysis was conducted on two representative isolates of V. natriegens and V. alginolyticus, designated as CgA1-1 and CgA1-2. Multisequence analysis (MLSA) showed that CgA1-1 and CgA1-2 were closely related to each other and nested within the Harveyi clade. Bacteriological investigation revealed faster growth, and more remarkable haemolytic activity and siderophore production capacity at 25 °C than at 15 °C for both CgA1-1 and CgA1-2. The accumulative mortalities of experimental immersion infections were also higher at 25 °C (90% and 63.33%) than at 15 °C (43.33% and 33.33%) using both CgA1-1 and CgA1-2, respectively. Similar clinical and pathological features were identified in samples collected during both naturally and experimentally occurring mortalities, such as thin visceral mass, discolouration, and connective tissue and digestive tube lesions. The results presented here highlight the potential risk of OsHV-1 to hatchery production of larvae, and the pathogenic role of V. natriegens and V. alginolyticus during mass mortalities of all life stages of Pacific oysters in Northern China.

A Putative Receptor for Ferritin in Mollusks: Characterization of the Insulin-like Growth Factor Type 1 Receptor.

The ferritin secreted by mammals has been well documented, with the protein capable of localizing to cell membranes and facilitating the delivery of iron to cells through endocytosis. However, the presence of ferritin in the circulatory fluid of mollusks and its functions remain largely unknown. In this study, we aimed to investigate the potential interacting proteins of ferritin in the ark clam (SbFn) through the use of a pull-down assay. Our findings revealed the presence of an insulin-like growth factor type 1 receptor (IGF-1R) in ark clams, which was capable of binding to SbFn and was named SbIGF-1R. SbIGF-1R was found to be composed of two leucine-rich repeat domains (L domain), a cysteine-rich domain, three fibronectin type III domains, a transmembrane domain, and a tyrosine kinase domain. The ectodomain of SbIGF-1R was observed to form a symmetrical antiparallel homodimer in the shape of the letter 'A', with the fibronectin type III domains serving as its 'legs'. The mRNA expression of SbIGF-1R gene was detected ubiquitously in various tissues of the ark clam, with the highest expression levels found in hemocytes, as determined by qRT-PCR. Using a confocal microscopic and yeast two-hybrid assays, the interaction between SbIGF-1R and SbFn was further verified. The results showed that SbFn co-localized with SbIGF-1R on the cell membrane, and their interaction was expected to occur on the FNIII domains of the SbIGF-1R. In conclusion, our findings highlight the identification of a putative receptor, SbIGF-1R, for SbFn, demonstrating the versatility of IGF-1R in ark clams.

Iron Regulatory Protein 1 Inhibits Ferritin Translation Responding to OsHV-1 Infection in Ark Clams, Scapharca Broughtonii.

Elemental iron is an indispensable prosthetic group of DNA replication relative enzymes. The upregulation of ferritin translation by iron regulatory proteins (IRP1) in host cells is a nutritional immune strategy to sequester available iron to pathogens. The efficient replication of Ostreid herpesvirus 1 (OsHV-1), a lethal dsDNA virus among bivalves, depends on available iron. OsHV-1 infection was found to trigger iron limitation in ark clams; however, it is still an enigma how OsHV-1 successfully conducted rapid replication, escaping host iron limitations. In this study, we identified the IRP1 protein (designated as SbIRP-1) in the ark clam (Scapharca broughtonii) and found it could bind to the iron-responsive element (IRE) of ferritin (SbFn) mRNA based on electrophoretic mobility shift assay (EMSA). Knockdown of SbIRP-1 expression (0.24 ± 1.82-fold of that in NC group, p < 0.01) by RNA interference resulted in the accumulation of SbFn in hemocytes (1.79 ± 0.01-fold, p < 0.01) post-24 h of enhanced RNA interference injection. During OsHV-1 infection, SbFn mRNA was significantly upregulated in hemocytes from 24 h to 60 h, while its protein level was significantly reduced from 24 h to 48 h, with the lowest value at 36 h post-infection (0.11 ± 0.01-fold, p < 0.01). Further analysis by RNA immunoprecipitation assays showed that OsHV-1 could enhance the binding of SbIRP-1 with the SbFn IRE, which was significantly increased (2.17 ± 0.25-fold, p < 0.01) at 36 h post-infection. Consistently, SbIRP-1 protein expression was significantly increased in hemocytes from 12 h to 48 h post OsHV-1 infection (p < 0.01). In conclusion, the results suggest that OsHV-1 infection could suppress post-transcriptional translation of SbFn through the regulation of SbIRP-1, which likely contributes to OsHV-1 evasion of SbFn-mediating host iron limitation.

Paired miRNA and RNA sequencing provides a first insight into molecular defense mechanisms of Scapharca broughtonii during ostreid herpesvirus-1 infection.

Ostreid herpesvirus 1 (OsHV-1) infection caused mortalities with relevant economic losses in bivalve aquaculture industry worldwide. Initially described as an oyster pathogen, OsHV-1 can infect other bivalve species, like the blood clam Scapharca broughtonii. However, at present, little is known about the molecular interactions during OsHV-1 infection in the blood clam. We produced paired miRNA and total RNA-seq data to investigate the blood clam transcriptional changes from 0 to 72 h after experimental infection with OsHV-1. High-throughput miRNA sequencing of 24 libraries revealed 580 conserved and 270 new blood clam miRNAs, whereas no genuine miRNA was identified for OsHV-1. Total 88-203 differently expressed miRNAs were identified per time point, mostly up-regulated and mainly targeting metabolic pathways. Most of the blood clam mRNAs, in contrast, were down-regulated up to 60 h post-injection, with the trend analysis revealing the activation of immune genes only when comparing the early and latest stage of infection. Taken together, paired short and long RNA data suggested a miRNA-mediated down-regulation of host metabolic and energetic processes as a possible antiviral strategy during early infection stages, whereas antiviral pathways appeared upregulated only at late infection.

A glimpse on metazoan ZNFX1 helicases, ancient players of antiviral innate immunity.

The human zinc finger NFX1-type containing 1 (ZNFX1) is an interferon-stimulated protein associated to the outer mitochondrial membrane, able to bind dsRNAs and interact with MAVS proteins, promoting type I IFN response in the early stage of viral infection. An N-terminal Armadillo (ARM)-type fold and a large helicase core (P-loop) and zinc fingers confer RNA-binding and ATPase activities to ZNFX1. We studied the phylogenetic distribution of metazoan ZNFX1s, ZNFX1 gene expression trends and genomic and protein signatures during viral infection of invertebrates. Based on 221 ZNFX1 sequences, we obtained a polyphyletic tree with a taxonomy-consistent branching at the phylum-level only. In metazoan genomes, ZNFX1 genes were found either in single copy, with up to some tens of exons in vertebrates, or in multiple copies, with one or a few exons and one of them sometimes encompassing most of the coding sequence, in invertebrates like sponges, sea urchins and mollusks. Structural analyses of selected ZNFX1 proteins showed high conservation of the helicase region (P-loop), an overall conserved region and domain architecture, an ARM-fold mostly traceable, and the presence of intrinsically disordered regions of varying length and position. The remarkable over-expression of ZNFX1 in bivalve and gastropod mollusks infected with dsDNA viruses underscores the antiviral role of ZNFX1, whereas nothing similar was found in virus-infected nematodes and corals. Whether the functional diversification reported in the C. elegans ZNFX1 occurs in other metazoan proteins remains to be established.

Characterization of Host Cell Potential Proteins Interacting with OsHV-1 Membrane Proteins.

The interaction between viral membrane associate proteins and host cellular surface molecules should facilitate the attachment and entry of OsHV-1 into host cells. Thus, blocking the putative membrane proteins ORF25 and ORF72 of OsHV-1 with antibodies that have previously been reported to subdue OsHV-1 replication in host cells, especially ORF25. In this study, prey proteins in host hemocytes were screened by pull-down assay with recombinant baits ORF25 and ORF72, respectively. Gene Ontology (GO) analysis of these prey proteins revealed that most of them were mainly associated with binding, structural molecule activity and transport activity in the molecular function category. The protein-protein interaction (PPI) network of the prey proteins was constructed by STRING and clustered via K-means. For both ORF25 and ORF72, three clusters of these prey proteins were distinguished that were mainly associated with cytoskeleton assembly, energy metabolism and nucleic acid processing. ORF25 tended to function in synergy with actins, while ORF72 functioned mainly with tubulins. The above results suggest that these two putative membrane proteins, ORF25 and ORF72, might serve a role in the transport of viral particles with the aid of a cytoskeleton inside cells.

Viral Decoys: The Only Two Herpesviruses Infecting Invertebrates Evolved Different Transcriptional Strategies to Deflect Post-Transcriptional Editing.

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host's immune response in different virus-host combinations.

Isolation and Characterization of Vibrio kanaloae as a Major Pathogen Associated with Mass Mortalities of Ark Clam, Scapharca broughtonii, in Cold Season.

High temperature is a risk factor for vibriosis outbreaks. Most vibrios are opportunistic pathogens that cause the mortality of aquatic animals at the vibrio optimal growth temperature (~25 °C), whereas a dominant Vibrio kanaloae strain SbA1-1 is isolated from natural diseased ark clams (Scapharca broughtonii) during cold seasons in this study. Consistent symptoms and histopathological features reappeared under an immersion infection with SbA1-1 performed at 15 °C. The pathogenicity difference of SbA1-1 was assessed under different temperatures (15 °C and 25 °C). The cumulative mortality rates of ark clams were significantly higher at the low temperature (15 °C) than at the high temperature (25 °C); up to 98% on 16th day post SbA1-1 infection. While the growth ratio of SbA1-1 was retarded at the low temperature, the hemolytic activity and siderophores productivity of SbA1-1 were increased. This study constitutes the first isolation of V. kanaloae from the natural diseased ark clams (S. broughtonii) in cold seasons and the exposition of the dissimilar pathogenicity of SbA1-1 at a different temperature. All the above indicates that V. kanaloae constitutes a threat to ark clam culture, especially in cold seasons.

Genomic Diversity of the Ostreid Herpesvirus Type 1 Across Time and Location and Among Host Species.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. This is particularly true for pathogens with low per-site mutation rates, such as DNA viruses, that do not exhibit a large amount of evolutionary change among genetic sequences sampled at different time points. However, whole-genome sequencing can reveal the accumulation of novel genetic variation between samples, promising to render most, if not all, microbial pathogens measurably evolving and suitable for analytical techniques derived from population genetic theory. Here, we aim to assess the measurability of evolution on epidemiological time scales of the Ostreid herpesvirus 1 (OsHV-1), a double stranded DNA virus of which a new variant, OsHV-1 µVar, emerged in France in 2008, spreading across Europe and causing dramatic economic and ecological damage. We performed phylogenetic analyses of heterochronous (n = 21) OsHV-1 genomes sampled worldwide. Results show sufficient temporal signal in the viral sequences to proceed with phylogenetic molecular clock analyses and they indicate that the genetic diversity seen in these OsHV-1 isolates has arisen within the past three decades. OsHV-1 samples from France and New Zealand did not cluster together suggesting a spatial structuration of the viral populations. The genome-wide study of simple and complex polymorphisms shows that specific genomic regions are deleted in several isolates or accumulate a high number of substitutions. These contrasting and non-random patterns of polymorphism suggest that some genomic regions are affected by strong selective pressures. Interestingly, we also found variant genotypes within all infected individuals. Altogether, these results provide baseline evidence that whole genome sequencing could be used to study population dynamic processes of OsHV-1, and more broadly herpesviruses.

Mitochondrial genomes of two Polydora (Spionidae) species provide further evidence that mitochondrial architecture in the Sedentaria (Annelida) is not conserved.

Contrary to the early evidence, which indicated that the mitochondrial architecture in one of the two major annelida clades, Sedentaria, is relatively conserved, a handful of relatively recent studies found evidence that some species exhibit elevated rates of mitochondrial architecture evolution. We sequenced complete mitogenomes belonging to two congeneric shell-boring Spionidae species that cause considerable economic losses in the commercial marine mollusk aquaculture: Polydora brevipalpa and Polydora websteri. The two mitogenomes exhibited very similar architecture. In comparison to other sedentarians, they exhibited some standard features, including all genes encoded on the same strand, uncommon but not unique duplicated trnM gene, as well as a number of unique features. Their comparatively large size (17,673 bp) can be attributed to four non-coding regions larger than 500 bp. We identified an unusually large (putative) overlap of 14 bases between nad2 and cox1 genes in both species. Importantly, the two species exhibited completely rearranged gene orders in comparison to all other available mitogenomes. Along with Serpulidae and Sabellidae, Polydora is the third identified sedentarian lineage that exhibits disproportionally elevated rates of mitogenomic architecture rearrangements. Selection analyses indicate that these three lineages also exhibited relaxed purifying selection pressures.

The Enhanced Immune Protection in Small Abalone Haliotis diversicolor Against a Secondary Infection With Vibrio harveyi.

In recent years, more and more studies have shown that early pathogenic bacterial infection in invertebrates can enhance immunity and significantly reduce mortality when reinfected with the same pathogen. There are mechanisms to explain this phenomenon, but they are relatively few. In addition, dose-dependent primary infection is also associated with increased immunity. In the present study, the initial infection dose and mortality of abalone Haliotis diversicolor after reinfection with Vibrio harveyi were recorded, and the mechanism of immune enhancement was investigated by the transcriptomic response of abalone after two successive stimuli with V. harveyi. Priming with different concentrations of pathogen can enhance immunity; however, higher concentration is not always better. Compared with the first exposure, more genes were up-regulated after the second exposure. Among the commonly expressed genes, the immune related genes were significantly or persistently highly expressed after two infections and included pattern recognition receptors as well as immune effectors, such as toll-like receptors, perlucin 4, scavenger receptor class B-like protein, cytochrome P450 1B1-like, glutathione S-transferase 6, lysozyme and so on; in addition, these immune-related genes were mainly distributed in the pathways related to phagocytosis and calcium signaling. Among the specifically expressed genes, compared with the first infection, more genes were involved in the immune, metabolic and digestive pathways after the second infection, which would be more conducive to preventing the invasion of pathogens. This study outlined the mechanism of immune enhancement in abalone after secondary infection at the global molecular level, which is helpful for a comprehensive understanding of the mechanism of immune priming in invertebrates.

Digging into bivalve miRNAomes: between conservation and innovation.

Bivalves are a diverse mollusc group of economic and ecological importance. An evident resilience to pollution, parasites and extreme environments makes some bivalve species important models for studying adaptation and immunity. Despite substantial progress in sequencing projects of bivalves, information on non-coding genes and gene-regulatory aspects is still lacking. Here, we review the current repertoire of bivalve microRNAs (miRNAs), important regulators of gene expression in Metazoa. We exploited available short non-coding RNA (sncRNA) data for Pinctada martensii, Crassostrea gigas, Corbicula fluminea, Tegillarca granosa and Ruditapes philippinarum, and we produced new sncRNA data for two additional bivalves, the Mediterranean mussel Mytilus galloprovincialis and the blood clam Scapharca broughtonii. We found substantial heterogeneity and incorrect annotations of miRNAs; hence, we reannotated conserved miRNA families using recently established criteria for bona fide microRNA annotation. We found 106 miRNA families missing in the previously published bivalve datasets and 89 and 87 miRNA complements were identified in the two additional species. The overall results provide a homogeneous and evolutionarily consistent picture of miRNAs in bivalves and enable future comparative studies. The identification of two bivalve-specific miRNA families sheds further light on the complexity of transcription and its regulation in bivalve molluscs. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Gender Differences in Hemocyte Immune Parameters of Hong Kong Oyster Crassostrea hongkongensis During Immune Stress.

Gender differences in individual immune responses to external stimuli have been elucidated in many invertebrates. However, it is unclear if gender differences do exist in the Hong Kong oyster Crassostrea hongkongensis, one of the most valuable marine species cultivated along the coast of South China. To clarify this, we stimulated post-spawning adult C. hongkongensis with Vibrio harveyi and lipopolysaccharide (LPS). Gender-based differences in some essential functional parameters of hemocytes were studied via flow cytometry. Obvious gender-, subpopulation-, and immune-specific alterations were found in the hemocyte immune parameters of C. hongkongensis. Three hemocyte subpopulations were identified: granulocytes, semi-granulocytes, and agranulocytes. Granulocytes, the chief phagocytes and major producers of esterase, reactive oxygen species, and nitric oxide, were the main immunocompetent hemocytes. Immune parameter alterations were notable in the accumulation of granulocyte esterase activities, lysosomal masses, nitric oxide levels, and granulocyte numbers in male oysters. These results suggest that post-spawning-phase male oysters possess a more powerful immune response than females. Gender and subpopulation differences in bivalve immune parameters should be considered in the future analysis of immune parameters when studying the impact of pathogenic or environmental factors.

Parallel analysis of miRNAs and mRNAs suggests distinct regulatory networks in Crassostrea gigas infected by Ostreid herpesvirus 1.

BACKGROUND: Since 2008, the aquaculture production of Crassostrea gigas was heavily affected by mass mortalities associated to Ostreid herpesvirus 1 (OsHV-1) microvariants worldwide. Transcriptomic studies revealed the major antiviral pathways of the oyster immune response while other findings suggested that also small non-coding RNAs (sncRNA) such as microRNAs might act as key regulators of the oyster response against OsHV-1. To explore the explicit connection between small non-coding and protein-coding transcripts, we performed paired whole transcriptome analysis of sncRNA and messenger RNA (mRNA) in six oysters selected for different intensities of OsHV-1 infection. RESULTS: The mRNA profiles of the naturally infected oysters were mostly governed by the transcriptional activity of OsHV-1, with several differentially expressed genes mapping to the interferon, toll, apoptosis, and pro-PO pathways. In contrast, miRNA profiles suggested more complex regulatory mechanisms, with 15 differentially expressed miRNAs (DE-miRNA) pointing to a possible modulation of the host response during OsHV-1 infection. We predicted 68 interactions between DE-miRNAs and oyster 3'-UTRs, but only few of them involved antiviral genes. The sncRNA reads assigned to OsHV-1 rather resembled mRNA degradation products, suggesting the absence of genuine viral miRNAs. CONCLUSIONS: We provided data describing the miRNAome during OsHV-1 infection in C. gigas. This information can be used to understand the role of miRNAs in healthy and diseased oysters, to identify new targets for functional studies and, eventually to disentangle cause and effect relationships during viral infections in marine mollusks.

In situ hybridization revealed wide distribution of Haliotid herpesvirus 1 in infected small abalone, Haliotis diversicolor supertexta.

Ganglioneuritis was the primary pathologic change in infected abalone associated with Haliotid herpesvirus 1 (HaHV-1) infection, which eventually became known as abalone viral ganglioneuritis (AVG). However, the distribution of HaHV-1 in the other tissues and organs of infected abalone has not been systemically investigated. In the present study, the distribution of HaHV-1-CN2003 variant in different organs of small abalone, Haliotis diversicolor supertexta, collected at seven different time points post experimental infection, was investigated with histopathological examination and in situ hybridization (ISH) of HaHV-1 DNA. ISH signals were first observed in pedal ganglia at 48 h post injection, and were consistently observed in this tissue of challenged abalone. At the same time, increased cellularity accompanied by ISH signals was observed in some peripheral ganglia of mantle and kidney. At the end of infection period, lesions and co-localized ISH signals in infiltrated cells were detected occasionally in the mantle and hepatopancreas. Transmission electron microscope analysis revealed the presence of herpes-like viral particles in haemocyte nuclei of infected abalone. Our results indicated that, although HaHV-1-CN2003 was primarily neurotropic, it could infect other tissues including haemocytes.

Influence of temperature on the pathogenicity of Ostreid herpesvirus-1 in ark clam, Scapharca broughtonii.

OsHV-1 is an epidemic pathogen of molluscs, and temperature has been recognized as a decisive environmental factor in its pathogenicity. In recent years, ark clam, Scapharca broughtonii, emerged as a host for OsHV-1. In the north of China, massive summer mortalities of ark clams infected with OsHV-1 have been continuously reported since 2012. However, the interaction between temperature and the pathogenicity of OsHV-1 was unknown in ark clams. In this study, the effect of temperature (10 °C to 18 °C stepped by 2 °C) on the occurrence of OsHV-1 disease in ark clams was analyzed. OsHV-1 infection led to gill erosion but not below the critical low temperature (between 12 °C and 14 °C). However, OsHV-1 persisted for more than 2 weeks at 12 °C post inoculation and replication was reactivated when the temperature was elevated to 18 °C. No significant reduction of OsHV-1 DNA load was found when the temperature descended to 12 °C from 18 °C, while the gill erosion remained unchanged. Ark clams failed to show the capability of effective clearance of OsHV-1 below the critical low temperature. Our results demonstrated that the pathogenicity of OsHV-1 was influenced significantly by temperature. Moreover, high temperature favored infection, which could provide more information to understand summer mortality of ark clams.

A-to-I editing of Malacoherpesviridae RNAs supports the antiviral role of ADAR1 in mollusks.

BACKGROUND: Adenosine deaminase enzymes of the ADAR family are conserved in metazoans. They convert adenine into inosine in dsRNAs and thus alter both structural properties and the coding potential of their substrates. Acting on exogenous dsRNAs, ADAR1 exerts a pro- or anti-viral role in vertebrates and Drosophila. RESULTS: We traced 4 ADAR homologs in 14 lophotrochozoan genomes and we classified them into ADAD, ADAR1 or ADAR2, based on phylogenetic and structural analyses of the enzymatic domain. Using RNA-seq and quantitative real time PCR we demonstrated the upregulation of one ADAR1 homolog in the bivalve Crassostrea gigas and in the gastropod Haliotis diversicolor supertexta during Ostreid herpesvirus-1 or Haliotid herpesvirus-1 infection. Accordingly, we demonstrated an extensive ADAR-mediated editing of viral RNAs. Single nucleotide variation (SNV) profiles obtained by pairing RNA- and DNA-seq data from the viral infected individuals resulted to be mostly compatible with ADAR-mediated A-to-I editing (up to 97%). SNVs occurred at low frequency in genomic hotspots, denoted by the overlapping of viral genes encoded on opposite DNA strands. The SNV sites and their upstream neighbor nucleotide indicated the targeting of selected adenosines. The analysis of viral sequences suggested that, under the pressure of the ADAR editing, the two Malacoherpesviridae genomes have evolved to reduce the number of deamination targets. CONCLUSIONS: We report, for the first time, evidence of an extensive editing of Malacoherpesviridae RNAs attributable to host ADAR1 enzymes. The analysis of base neighbor preferences, structural features and expression profiles of molluscan ADAR1 supports the conservation of the enzyme function among metazoans and further suggested that ADAR1 exerts an antiviral role in mollusks.

Chromosomal-level assembly of the blood clam, Scapharca (Anadara) broughtonii, using long sequence reads and Hi-C.

BACKGROUND: The blood clam, Scapharca (Anadara) broughtonii, is an economically and ecologically important marine bivalve of the family Arcidae. Efforts to study their population genetics, breeding, cultivation, and stock enrichment have been somewhat hindered by the lack of a reference genome. Herein, we report the complete genome sequence of S. broughtonii, a first reference genome of the family Arcidae. FINDINGS: A total of 75.79 Gb clean data were generated with the Pacific Biosciences and Oxford Nanopore platforms, which represented approximately 86× coverage of the S. broughtonii genome. De novo assembly of these long reads resulted in an 884.5-Mb genome, with a contig N50 of 1.80 Mb and scaffold N50 of 45.00 Mb. Genome Hi-C scaffolding resulted in 19 chromosomes containing 99.35% of bases in the assembled genome. Genome annotation revealed that nearly half of the genome (46.1%) is composed of repeated sequences, while 24,045 protein-coding genes were predicted and 84.7% of them were annotated. CONCLUSIONS: We report here a chromosomal-level assembly of the S. broughtonii genome based on long-read sequencing and Hi-C scaffolding. The genomic data can serve as a reference for the family Arcidae and will provide a valuable resource for the scientific community and aquaculture sector.