AgBiome (United States)

We recently developed a rat whole exome sequencing (WES) panel and used it to evaluate early somatic mutations in archival liver tissues from F344/N rats exposed to the hepatocarcinogen, Aflatoxin B1 (AFB1), a widely studied, potent mutagen and hepatocarcinogen associated with hepatocellular carcinoma (HCC). Rats were exposed to 1-ppm AFB1 in feed for 14, 90, and 90 days plus a recovery 60-day, non-exposure period (150-day) timepoint. Isolated liver DNA was exome sequenced. We identified 172 sequence variants across all timepoints, of which 101 were non-synonymous variants. Well-annotated genes carried a diverse set of 29 non-synonymous mutations at 14 days, increasing to 39 mutations at 90 days and then decreasing to 33 mutations following the 60-day recovery. Gene Set Enrichment Analysis conducted on previously reported, available RNA expression data of the same exome sequenced archival samples identified altered transcripts in pathways associated with malignant transformation. These included HALLMARK gene sets associated with cell proliferation (MYC Targets Version 1 and Version 2, E2F targets), cell cycle (G2M checkpoint, mitotic spindle), cell death (apoptosis), and DNA damage (DNA repair, UV response Up, Reactive oxygen species) pathways. DriverNet Impact analysis integrated exome-seq and expression data to reveal somatic mutations in Mcm8, Bdp1, and Cct6a that may drive cancer formation. Connectivity with transcript expression changes identified these genes as the top-ranked candidate driver genes associated with hepatocellular transformation. In conclusion, exome sequencing revealed early somatic mutations that may play a role in cancer cell transformation that are translatable to aflatoxin-induced HCC.

The etiology of sea star wasting syndrome is hypothesized to be caused by a densovirus, sea star-associated densovirus (SSaDV), that has previously been reported on the Pacific and Atlantic Coasts of the United States. In this study, we reevaluated the presence of SSaDV among sea stars from the North American Atlantic Coast and in doing so discovered a novel densovirus that we have named Asterias forbesi-associated densovirus (AfaDV), which shares 78% nucleotide pairwise identity with SSaDV. In contrast to previous studies, SSaDV was not detected in sea stars from the North American Atlantic Coast. Using a variety of PCR-based techniques, we investigated the tissue tropism, host specificity, and prevalence of AfaDV among populations of sea stars at five locations along the Atlantic Coast. AfaDV was detected in three sea star species (Asterias forbesi, Asterias rubens, and Henricia sp.) found in this region and was highly prevalent (>80% of individuals tested; n = 134), among sampled populations. AfaDV was detected in the body wall, gonads, and pyloric caeca (digestive gland) of specimens but was not detected in their coelomic fluid. A significant difference in viral load (copies mg-1) was found between tissue types, with the pyloric caeca having the highest viral loads. Further investigation of Asterias forbesi gonad tissue found germ line cells (oocytes) to be virus positive, suggesting a potential route of vertical transmission. Taken together, these observations show that the presence of AfaDV is not an indicator of sea star wasting syndrome because AfaDV is a common constituent of these animals' microbiome, regardless of health.IMPORTANCE Sea star wasting syndrome is a disease primarily observed on the Pacific and Atlantic Coasts of North America that has significantly impacted sea star populations. The etiology of this disease is unknown, although it is hypothesized to be caused by a densovirus, SSaDV. However, previous studies have not found a correlation between SSaDV and sea star wasting syndrome on the North American Atlantic Coast. This study suggests that this observation may be explained by the presence of a genetically similar densovirus, AfaDV, that may have confounded previous studies. SSaDV was not present in sea stars screened in this study, and instead, AfaDV was commonly found in sea star populations across the New England region, with no apparent signs of disease. These results suggest that sea star densoviruses may be common constituents of the animals' microbiome, and the diversity and extent of these viruses among wild populations may be greater than previously recognized.

Abstract Viral metagenomes prepared from tissues from Forbes’ sea star (Asterias forbesi) led to the discovery of a complete genome of a novel sea star densovirus (AfaDV). The genome organization of AfaDV and phylogenetic analysis place this virus among the Ambidensovirus genus in the subfamily Densoviridae, family Parvoviridae. AfaDV shares 78% nucleotide pairwise identity to the sea star associated densovirus (SSaDV), previously described as the putative causative agent of Sea Star Wasting Syndrome among sea stars from the Northwest Pacific. SSaDV was not found in specimens collected in this study, and the discovery of AfaDV might explain previous reports of SSaDV among sea stars from the Atlantic Coast. A qPCR assay was designed to assess tissue tropism, host specificity, and prevalence of AfaDV among wild populations of sea stars at five locations on the North American Atlantic Coast. AfaDV was detected in all three common sea star species (Asterias forbesi, Asterias rubens, and Henricia sp.) found in the region and was highly prevalent (80-100% of individuals tested, n=134), among populations collected at disparate sites 7 years apart. AfaDV was detected in the body wall, gonads, and pyloric caeca (digestive gland) of specimens but was not detected in their coelomic fluid. A significant difference in viral load was found between tissue types with the pyloric caeca having the highest viral load suggesting it is the primary site of viral replication in the animal. Further investigation of Asterias forbesi gonad tissue found germline cells (oocytes) to be virus positive suggesting a potential route of vertical transmission. Taken together, these observations show that the presence AfaDV is not an indicator of Sea Star Wasting Syndrome because AfaDV is a common constituent of these animals’ microbiome, regardless of health. These results broaden the understanding of echinoderm densoviruses outside the context of disease that suggest these viruses might form commensal or mutualistic relationships with their hosts.

Sea stars are among the most important predators in benthic ecosystems worldwide which is partly attributed to their unique gastrointestinal features and feeding behaviors. Despite their ecological importance, the microbiome of these animals and its influence on adult host health and development largely remains unknown. To begin to understand such interactions we sought to understand what bacteria are associated with these animals, how the microbiome is partitioned across regions of the body and how seawater influences their microbiome. We analyzed the microbiome composition of a geographically and taxonomically diverse set of sea star taxa by using 16S rRNA gene amplicon sequencing and compared microorganisms associated with different regions of their body and to their local environment. In addition, we estimated the bacterial and coelomocyte abundance in the sea star coelomic fluid and bacterioplankton abundance in the surrounding seawater via epifluorescence microscopy. The average bacterial cell abundance observed in the coelomic fluid was one to two orders of magnitude lower than the bacterioplankton abundance in the surrounding seawater suggesting a selection against the presence of microorganisms in the coelomic fluid. The sea star microbiome was also significantly different from seawater with relatively few shared microbial taxa. Microbial communities were found to be significantly different between the pyloric caeca, gonads, coelomic fluid, and body wall of the animals. The most noticeable difference between anatomical sites was the greater relative abundance of Spirochaetae and Tenericutes found in hard tissues (gonads, pyloric caeca, and body wall) than in the coelomic fluid. The microbiome of sea stars thus appears to be anatomically partitioned, distinct from the microbial community of seawater and contains a relatively low abundance of bacteria within the coelomic cavity.