**Project Completed**

Project PIs:
Ian Hewson (Department of Microbiology)
Lars Rudstam (Department of Natural Resources)

Several years of research into invertebrate-associated viruses have highlighted the occurrence of a class of small, circular, replication initiator protein-encoding single-stranded DNA (CRESS-DNA) viruses in marine and freshwater arthropods, including the water flea Daphnia spp. (Hewson et al., 2013), copepods Labidocera aestiva and Acartia tonsa (Dunlap et al., 2013) and amphipod Diporeia sp. (Hewson et al., 2013). The viruses are both active in replication, and present at high prevalence in natural populations. In Daphnia mendotae, prevalence was significantly correlated to mortality (r2 = 0.79) over seasonal cycles of abundance (Hewson et al., 2013). In cell cultures of experimentally infected Spodoptera frugifera (sf9) cells, circoviruses caused enhanced mortality and slower cell growth than in uninfected cells.

Despite our observations of circoviruses across a range of crustaceans, we know remarkably little about how they affect their hosts, and moreover how these effects may alter their host’s roles in biogeochemical cycles or food webs. In this project, our focus is on amphipods, targeting the freshwater taxon Diporeia sp in the laurentian Great Lakes, and the kelp amphipod Peramphithoe femorata. Both organisms are important to aquatic habitats for different reasons. Diporeia is a major consumer of settled spring bloom materials, and has experienced significant decline in geographic range in the last 20 years for reasons that are not totally clear. It is a major food source for larval fish. Peramphithoe on the other hand is a major consumer of detrital giant kelps on the US West Coast, which are threatened due to nutrient pollution and ocean-scale climatic phenomena, like el nino.

Peramphithoe femorata

This project was the first to gauge how viruses affect the ecological and biogeochemical roles that amphipods play in their respective habitats. We first examined the prevalence and viral load of both species amongst variable populations to understand their relationship to host food quality (C:N ratio, lipid content, etc). We then performed inoculation trials with their viruses in mesocosms to understand their impacts on key biogeochemical process, including NH4+ flux into overlying waters, decomposition of plant material, bacterial production, and inorganic nutrient transformation.

Significant Results:

This project has generated considerable new knowledge about the ecology and biogeochemistry of CRESS-DNA viruses in amphipods (and isopods). All project data has been deposited at BCO-DMO at http://www.bco-dmo.org/project/557561 and all sequence data generated has been acceded to NCBI.

By the end of Y1,we had analyzed metaviromes prepared from 18 contemporary amphipod samples (7 from the Great Lakes and 11 from marine habitats), and confirmed the presence of circoviruses amongst viruses inhabiting their tissues. Significant proportions of metavirome contiguous sequences (contigs) did not explicitly identify as viral (Table 1).  The majority of viral contigs identify as CRESS-DNA viruses or baculoviruses (arthropod associated). Reads from metaviromes predominantly map back to a small subset of CRESS-DNA viral contigs (LM29173, LM19063, Michigan_A3_122c). Furthermore, these specific viral genomes recruit reads from SRA libraries, particularly from Michigan and Ontario ballast water, which may represent a mechanism of transmission of CRESS-DNA viruses between Great Lake populations.

Additionally, in Y1 and Y2, we identified that CRESS-DNA viral prevalence and load (LM29173) had a pattern that was distinct but adjacent Diporeia populations. A similar pattern of viral prevalence/load was not observed for CRESS-DNA virus genotypes A3_122 or A4_481. This may implicate LM29173 as the most likely candidate CRESS-DNA pathogen in Diporeia spp. Michigan exhibits greatest prevelance and load of LM29173, followed by Lakes Huron and Superior, respectively. This data coincides with patterns of Diporiea population decline. Greater viral prevalence/load in Michigan samples are reflected in correlations between viral infection and latitude or depth (eg. Lake Michigan is situated north of Lakes Superior/Huron) (Fig. 5). These patterns were also evident in trends in dreissenid mussel density, lake DOC content, and collection depth, indicating covariation in multiple parameters.

In Y2 and Y3, mesocosm experiments were performed to assess the relationship between CRESS-DNA viral infection and host biogeochemical function. Artificial inoculation of amphipods with virus-sized material (<0.2 μm) from infected conspecifics did not elicit major changes in host mortality in mesocosm-based studies. Likewise, viral inoculation did not significantly alter host behavior (detritivory, herbivory, bioturbation), as detected by mesocosm bacterial abundance and metabolism.

In Y2 and Y3, we explored the nutritional quality of hosts as a function of CRESS-DNA viral infection (Hypothesis 3). Diporeia from lake Michigan possess greater C:N, %lipid content, and maintain both greater LM29173 prevalence/load. No clear correlation between C:N or % lipid content and CRESS-DNA viral infection was observed across all lakes. However, nutritional quality of hosts varied between lakes independent of CRESS-DNA viral prevalence/load. These results stimulated investigation into the population identity of amphipods inhabiting the different lakes (Diporeia spp.) since viral load corresponded most closely with lake geography rather than any biogeochemical pattern. Hence, we performed cytochrome oxidase I sequencing of these, and several other crustacean samples (to establish phylogenetic context).  The results of this survey indicated that Diporeia inhabiting different lakes comprised distinct haplotypes , and CRESS-DNA viral load corresponded most closely with haplotype present. The consequence of this result is that early observations (Hewson et al., 2013) of LM29173 load/prevalence correspondence with population decline was likely spurious and reflected host genetic population.

In Y3 and Y4 (1^st NCE), we sought to examine the relationship between LM29173 load/prevalence and gene transcription since mesocosm based studies performed in Y2 indicated that CRESS-DNA virus presence does not significantly alter the behavior of the amphipod, nor the impact of the amphipod on co-occurring bacterial metabolism. Therefore, results indicated that these pervasive CRESS-DNA viruses may have a subtle effect on co-occurring metazoans. In order to investigate these effects, we performed comparative transcriptomics on individual amphipods to identify genes and gene pathways that are differentially expressed in the presence of CRESS-DNA viral genotype, LM29173 in the Great Lakes. These efforts identified 2,208 significantly differentially expressed transcripts in amphipods with relative high average LM29173 load. The greatest proportion of annotated, differentially expressed genes were associated with functions including: (1) replication, recombination and repair, (2) cell wall/membrane/envelope biogenesis, and (3) post-translational modification, protein turnover, and chaperones. Transcriptome analysis and quantification (RT-qPCR) of three transcripts (nonmuscular myosin heavy chain, β-actin, ubiquitin-conjugating enzyme E2) indicated that Lake Michigan and Lake Superior amphipods with high LM29173 load exhibit opposite trends in gene expression. Contigs associated with innate immunity displayed no consistent pattern of expression, though several transcripts were significantly overexpressed in amphipods with high viral load. Further qPCR analyses of a structural (b-actin) and constituitively expressed gene (ubiquitin) genes confirmed that LM29173 prevalence/load was directly tied to host haplotype.

A fundamental limitation of pre-existing descriptions of invertebrate-associated CRESS-DNA viruses was an absence of explicit evidence that these viruses directly infected metazoan tissues, as opposed to co-occurring prokaryotic symbionts/parasites. Therefore, to strengthen the validity of pending publications, this research was supplemented by localizing LM29173 within Diporeia tissues using a single oligonucleotide-sensitive variant of fluorescent in-situ hybridization (CARD-FISH) in Y4. This analysis demonstrates that Diporeia likely harbor viral ssDNA in hepatopancreatic and cephalothoracic muscular tissue, providing initial targets to further explore CRESS-DNA virus tropism in crustaceans. Additionally, this assessment may provide a mechanism to investigate tissue-specific impacts of CRESS-DNA virus infection on crustacean physiology.

Finally, in light of haplotype-specificity of LM29173, we examined the phylogeny and biogeography of CRESS-DNA viruses in microcrustacea generally by targeted study of prevalence/load in geographically-and habitat-distinct samples in Y5 (2^nd NCE). We examined the host specificity, transience, and phylogeography of a CRESS-DNA virus in a microcrustacean (Idotea spp.), allowing approximate generalization of findings concerning CRESS-DNA virus ecology in distinctly different (limnic vs. coastal marine) ecosystems. The targeted viral genotype was also remarkable as it exhibited sequence homology and genomic features similar to both ssRNA and ssDNA viral taxa, and was therefore classified a member of the newly described Cruciviridae family. This investigation aided in resolving the phylogenetic position of the Cruciviridae and offered insight into the biogeography, specificity, and potential host of a “chimeric” viral genome.

In addition, DNA viral assemblages associated with 31 microcrustacean populations from disparate aquatic habitats were sequenced to compare viral phylogeography on a multi-ecosystem scale, enabling identification of 215 unique circular replication-protein encoding ssDNA (CRESS-DNA) viral genotypes. Due to rapid evolution and non-conserved genomic architecture, taxonomic demarcation and ecological inferences pertaining to these novel CRESS-DNA viruses pose particular challenges. We applied non-alignment based methods to compare viral genomes, indicating that microcrustacean-associated viruses share common oligomer signatures with other invertebrate-associated ssDNA viruses. This provided taxonomic delineation and the potential for host-virus pairing. Likewise, this analysis of genomic content indicated that microcrustacean-associated CRESS-DNA viruses exhibit limited codon repertoires and significantly bias towards greater %GC content. We determined that overall measures of genomic content correlate with biogeographic and host-specific parameters, perhaps suggesting host- and habitat-driven selection. Structural open reading frames (ORFs) contained the majority of genomic variants (relative to conserved nonstructural, replication-associated ORFs), indicating that selective pressures likely permit greater variation in intergenic or capsid-encoding regions. Furthermore, discovery of endogenized viral elements within crustacean genomes revealed a paleovirological record of arthropod infection by CRESS-DNA viruses and specific conservation of the nonstructural replication-associated gene (rep), facilitating our understanding of microcrustacean-virus interactions over time.

Work that was performed on this project can be found via the links below:

R/V Blue Heron Chief Scientist Training Cruise 2017

Salish Sea Research Cruise 2016

Australia Echinoderm Expedition 2015

Research Expedition: Santa Catalina Island, January 2015

Research Expedition: The BATS Validation Cruise BVAL-98, October 2014

Research Expedition: Great Lakes Environmental Monitoring Cruise, June – August 2014

Publications arising from this work are below:

Gezon N, Hawick D, Hewson I, Strychar K (2020) “Circular Rep encoding single stranded (CRESS) DNA virus-like sequences detected in quagga mussels (Dreissena rostriformis bugensis) and sediments from the central Lake Michigan benthos” Journal of Great Lakes Research 46:302-310

Hewson I (2019) “Technical pitfalls that bias comparative microbial community analyses of aquatic diseases” Diseases of Aquatic Organisms. 137: 109-124

Hewson I, Bistolas KSI, Button JB, Jackson EW (2018) “Occurrence and seasonal dynamics of RNA viral genotypes in three contrasting temperate lakes” PLoS One 13: e0194419

Bistolas, KSI, Besemer RM, Rudstam LG, Hewson I (2017) “Distribution and Inferred Evolutionary Characteristics of a Chimeric ssDNA Virus Associated with Intertidal Marine Isopods” Viruses 9: 361

Bistolas KSI., Rudstam L.G., Hewson I., (2017) “Gene expression of benthic amphipods (genus: Diporeia) in relation to a circular ssDNA virus across two Laurentian Great Lakes” PeerJ, 5:e3810

Bistolas KSI, Jackson EW, Watkins JM, Rudstam LG, Hewson I (2017) “Distribution of circular single stranded viruses associated with benthic amphipods of the genus Diporeia in the Laurentian Great Lakes”. Freshwater Biology. 62:1220-1237

Bistolas KSI, Button JB, Jackson EWJ, Hewson I (2016) “Circoviruses of Crustaceans” pp. 419-421 in Kibenge SB and Godoy M “Aquaculture Virology” Elsevier, New York, 549pp.

Jackson EW, Bistolas KSI, Button JBB, Hewson I (2016) “Novel circular single-stranded DNA viruses among an asteroid, echinoid and holothurian (Phylum: Echinodermata)”. PLoS One. DOI:10.1371/journal.pone.0166093

Hewson I, Eaglesham JB, Hook TO, LaBarre BA, Sepulveda MS, Thompson PD, Watkins JM, Rudstam LG (2013) “Investigation of viruses associated with Diporeia spp. from the Laurentian Great Lakes and Owasco Lake, NY as a potential stressor of declining populations” Journal of Great Lakes Research 39: 499-506