Molecular and Microscopic Evidence of Viruses in Marine Copepods

25-35 percent of copepod mortality cannot be accounted for by predation alone, suggesting that nonpredatory factors such as the role of marine viruses may be contributing to the population dynamics of this dominant mesozooplanktonic group. This seems to be the first study to investigate potential viral infection of the marine mesozooplankton community.

Viral metagenomics and seasonality in the copepods Acartia tonsa and Labidocera aestiva were investigated, as well as potential environmental virus reservoirs. Virus particles from copepod homogenate were isolated and viral DNA subsequently extracted, fragmented, amplified and analysed. Viral metagenome sequences with high similarity to the Circoviridae virus family were amplified by PCR to construct a maximum likelihood phylogenetic tree. Transmission electron microscopy of copepod tissue was performed.

Circoviridae-like viral genomes were found in L. aestiva (LaCopCV) and A. tonsa (AtCopCV); both showed weak amino acid sequence similarities to the Rep protein in Circoviridae and Circo-like characteristics including: 2 non-overlapping (but differently orientated) ORFs and a distinct nine nucleotide DNA loop. LaCopCV had weak capsid-encoding ORF similarities to Circoviruses. Previously Circoviruses were known only as vertebrate pathogens, but they may be important as invertebrate pathogens.

Both Circo-like genomes had similar Rep-encoding ORFs to those conserved in eukaryotic Rep-encoding circular ssDNA viruses; AtCopCV had highest BLASTp similarity to a circo-like virus isolated from bat faeces and LaCopCV had highest BLASTp similarity to a circo-like virus from Chesapeake Bay virioplankton. Although phylogenetically clustered with other circo-like viruses from environmental samples, AtCopCV and LaCopCV diverge from known circoviruses as a novel marine invertebrate infecting group.

LaCopCV average viral load in L. aestiva was 1.13x10⁵ per copepod and 100 % of copepods were infected in 3 of 4 locations sampled. Most arthropod infection by DNA virus work has been done on white spot syndrome virus; LaCopCV viral load of L. aestiva was on an order of magnitude similar to that of WSSV in the commercially important prawn genus, Penaeus.

LaCopCV transcription was detected, indicating that it was actively replicating in copepod tissue. AtCopCV seasonality was unclear, weakly suggesting fluctuations correlating with copepod population changes. Neither virus was found in other zooplankton species or the virioplankton, but sediments were found to be a LaCopCV reservoir. TEM displayed virus particles, suggesting viral replication in the tissues of both copepod species, rather than in copepod-associated microorganisms; however the significantly larger size of these particles than those of known eukaryotic circoviruses indicates that they may not be LaCopCV or AtCopCV. More detailed copepod viriome sampling is required, which may reveal other potential viral sources of these particles.

This study provides definitive evidence of copepod population viral infection and a new Rep-encoding circular ssDNA virus of marine invertebrates. Viral roles may be imperative towards understanding zooplankton ecology. Further work exploring mesoplankton virus pathology, infection and ecological influence is a necessary new direction.

Dunlap, D. S., Ng, T. F. F., Rosario, K., Barbosa, J. G., Greco, A. M., Breitbart, M., & Hewson, I. (2013). Molecular and microscopic evidence of viruses in marine copepods. Proceedings of the National Academy of Sciences, 110(4), 1375-1380.