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.

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.

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.

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.

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.

Dual Transcriptomic Analysis Reveals a Delayed Antiviral Response of Haliotis diversicolor supertexta against Haliotid Herpesvirus-1.

Haliotid herpesvirus-1 (HaHV-1) is the first identified gastropod herpesvirus, causing a highly lethal neurologic disease of abalone species. The genome of HaHV-1 has been sequenced, but the functions of the putative genes and their roles during infection are still poorly understood. In the present study, transcriptomic profiles of Haliotis diversicolor supertexta at 0, 24 and 60 h post injection (hpi) with HaHV-1 were characterized through high-throughput RNA sequencing. A total of 448 M raw reads were obtained and assembled into 2.08 × 105 unigenes with a mean length of 1486 bp and an N50 of 2455 bp. Although we detected increased HaHV-1 DNA loads and active viral expression at 24 hpi, this evidence was not linked to significant changes of host transcriptomic profiles between 0 and 24 hpi, whereas a rich immune-related gene set was over-expressed at 60 hpi. These results indicate that, at least at the beginning of HaHV-1 infection, the virus can replicate with no activation of the host immune response. We propose that HaHV-1 may evolve more effective strategies to modulate the host immune response and hide during replication, so that it could evade the immune surveillance at the early stage of infection.

RNA-seq of HaHV-1-infected abalones reveals a common transcriptional signature of Malacoherpesviruses.

Haliotid herpesvirus-1 (HaHV-1) is the viral agent causative of abalone viral ganglioneuritis, a disease that has severely affected gastropod aquaculture. Although limited, the sequence similarity between HaHV-1 and Ostreid herpesvirus-1 supported the assignment of both viruses to Malacoherpesviridae, a Herpesvirales family distantly related with other viruses. In this study, we reported the first transcriptional data of HaHV-1, obtained from an experimental infection of Haliotis diversicolor supertexta. We also sequenced the genome draft of the Chinese HaHV-1 variant isolated in 2003 (HaHV-1-CN2003) by PacBio technology. Analysis of 13 million reads obtained from 3 RNA samples at 60 hours post injection (hpi) allowed the prediction of 51 new ORFs for a total of 117 viral genes and the identification of 207 variations from the reference genome, consisting in 135 Single Nucleotide Polymorphisms (SNPs) and 72 Insertions or Deletions (InDels). The pairing of genomic and transcriptomic data supported the identification of 60 additional SNPs, representing viral transcriptional variability and preferentially grouped in hotspots. The expression analysis of HaHV-1 ORFs revealed one putative secreted protein, two putative capsid proteins and a possible viral capsid protease as the most expressed genes and demonstrated highly synchronized viral expression patterns of the 3 infected animals at 60 hpi. Quantitative reverse transcription data of 37 viral genes supported the burst of viral transcription at 30 and 60 hpi during the 72 hours of the infection experiment, and allowed the distinction between early and late viral genes.

Susceptibility of two abalone species, Haliotis diversicolor supertexta and Haliotis discus hannai, to Haliotid herpesvirus 1 infection.

Abalone viral ganglioneuritis (AVG), caused by Haliotid herpesvirus-1 (HaHV-1) infection, has been reported as the main cause of mortality and heavy losses of wild and cultivated abalone in Taiwan and Australia since 2003. HaHV-1 DNA has also been reported in diseased abalone collected in early 2000s in China. However, no data is available about the susceptibility, disease process and pathological changes of HaHV-1 infection in the primary cultivated abalone species in China. In the present study, two cultivated abalone species, Haliotis diversicolor supertexta and Haliotis discus hannai, were challenged with HaHV-1-CN2003 collected in 2003 in China using three different methods. Results showed that H. diversicolor supertexta was highly susceptible to HaHV-1-CN2003 infection and suffered acute mortality using all three challenge methods. H. discus hannai was not susceptible to the viral infection. Histopathology combined with transmission electron microscopy and quantitative PCR analysis revealed that the tropism of HaHV-1-CN2003 includes both neural tissue and haemocytes.

Long-range PCR and high-throughput sequencing of Ostreid herpesvirus 1 indicate high genetic diversity and complex evolution process.

Ostreid herpesvirus 1 (OsHV-1) is an important pathogen associated with mass mortalities of cultivated marine mollusks worldwide. Since no cell line allows OsHV-1 replication in vitro, it is difficult to isolate enough high-purity viral DNA for High-Throughput Sequencing (HTS). We developed an efficient approach for the enrichment of OsHV-1 DNA for HTS with long-range PCR. Twenty-three primer pairs were designed to cover 99.3% of the reference genome, and their performances were examined on ten OsHV-1 infected samples. Amplicon mixtures from six successfully amplified samples were sequenced with Illumina platform, and one of them (ZK0118) was also sequenced with the PacBio platform. PacBio reads were assembled into 2 scaffolds compared to 9-68 scaffolds assembled from the Illumina reads. Genomic comparison confirmed high genetic diversity among OsHV-1 variants. Phylogenetic analysis revealed that OsHV-1 evolution was mainly impacted by its host species rather than spatial segregation.

Dual transcriptomic analysis of Ostreid herpesvirus 1 infected Scapharca broughtonii with an emphasis on viral anti-apoptosis activities and host oxidative bursts.

The ark shell, Scapharca (Anadara) broughtonii, is an economically important marine shellfish species in Northwestern Pacific. Mass mortalities of ark shell adults related to Ostreid herpesvirus-1 (OsHV-1) infection have occurred frequently since 2012. However, due to the lack of transcriptomic resource of ark shells, the molecular mechanisms underpinning the virus-host interaction remains largely undetermined. In the present study, we resolved the dual transcriptome changes of OsHV-1 infected ark shell with Illumina sequencing. A total of 44 M sequence reads were generated, of which 67,119 reads were mapped to the OsHV-1 genome. De novo assembly of host reads resulted in 276,997 unigenes. 74,529 (26.90%), 47,653 (17.20%) and 19, 611 (7.07%) unigenes were annotated into GO, KOG and KEGG database, respectively. According to RSEM expression values, we identified 2998 differentially expressed genes (DEGs) between control and challenged groups, which included 2065 up-regulated unigenes and 933 down-regulated unigenes. Further analysis of functional pathways indicated that OsHV-1 could inhibit host cell apoptosis mainly by the up-regulation of inhibitor of apoptosis protein (IAP), and thus facilitating its successful replication. While host hemoglobins could induce oxidative burst by suppressing its peroxidase activity, and thus defense against OsHV-1 infection. Although we reported a narrow expression of the OsHV-1 genome compared to Crassostrea gigas infection, we highlighted several common viral genes highly expressed in the two hosts, suggesting an important functional role. This study offers insights into the pathogenesis mechanisms of OsHV-1 infection in bivalve mollusks of the Arcidae family.