Viruses found in marine copepods

Copepods are one of the main key players in the mesoplankton communities of our oceans, they play a significant role in food web ecology and biogeochemical cycling. There have been many studies in terms of population dynamics and ecology of marine copepods but little is still known of their mortality. It has been shown that only 65-75% of copepod mortality is a result of predation, leaving 35-25% of death unaccounted for. Suspected mortality mechanisms include the products of harmful algal blooms, environmental stressors, parasites and disease. Until now viral infection has been an un-investigated contributor to mesoplankton mortality. While copepods have been shown to transmit viruses to other organisms, no work has been done on the infection of copepods themselves. Dunlap et al., reports for the first time significant microscopic and molecular evidence for viral activity in marine copepod, Acartia tonsa (Dana) and Labidocera aestiva (Wheeler), populations of Tampa Bay, Florida.

Copepods were collected from varying locations in Tampa Bay, throughout the year of study and purified in culture in the laboratory before experimentation took place. Metagenomic analysis of the copepods revealed individual viruses similar to the Circoviriadae family associated with both species. Both found viruses, named Acartia tonsa copepod circo-like virus (AtCopCV) and Labidocera aestiva copepod circo-like virus (LaCopCV), had similar amino acid sequences with small, circular, single-stranded DNA genomes as previously seen in circoviruses. It is worth noting that this methodology shows slight bias towards these sorts of ssDNA, however this process allows for sufficient DNA recovery of samples. Both genomes were also shown to have two non-overlapping open reading frames (ORF), agreeing with circovirus genome. Traditionally it has been shown circoviruses ORF’s to be orientated in opposite directions, however, in both copepod sequences they were found to be orientated in the same direction. Until 2011, circoviruses were though to only infect vertebrates, however they have recently been found to infect dragonflies amongst other terrestrial and marine invertebrates. This orientation difference has been previously reported in some environmental and faecal metagenomes, suggesting that the copepod viruses to be an understudied branch of the Circoviradae family.

Rep-encoding ORFs were sequenced and searched with BLASTp to find similar sequences. AtCopCV was shown to have highest similarity to circo-like viruses found in bat faeces in China, while LaCopCV virus showed most similarity to a free viroplankton in the Chesapeake Bay. However, phylogenetic analysis of the Reps of both copepod viruses showed high divergence from previously studied circoviruses, suggesting they are both unique ssDNA viral lineages infecting marine invertebrates. In terms of the putative capsid in the viruses the LaCopCV showed weak similarity to previously studied circoviruses whereas AtCopCV showed none. However, both viruses do show the characteristic circovirus stem-loop acting as the point of rolling circle of replication.

Quantitative PCR was used to examine the summer viral load of the copepods. In three out of four of the L. aestiva individuals there was evidence of infection of an average of 1.13 × 10⁵ copies per individual copepod. Which is a high prevalence in these wild animals that compared well with similar studies in post-larval Fenneropenaeus chinensis and P. trituberculatus larvae. Viral transcription was also examined in spring time which showed consistent circo-like viruses in 11 out of the 14 examined copepods. AtCopCV was detected throughout most the year in A. tonsa pools of up to 500 copepods with much less prevalence in the peak of summer and winter months. Viruses found in these copepods were unique to these species of copepods, they were not found in any other type of zooplankton. However, LaCopCV were found in sediment analysis suggesting it to be a reservoir for this viruses in the sediments.

Transmission election microscopy further quantified virus like particles in A.tonsa and L.aestiva. In both species virus like particles were seen in connective tissues, showing propagation of the viruses directly into the copepods. These virus-like particles were 39.5 ± 7.2 nm in L. aestival and 37 ± 4.9 nm in A. tonsa, showing much larger values than known eukaryotic circoviruses. This brings uncertainty in the identity of these virus like particles seen under the microscope, suggesting that a less bias method may be needed to reveal the identity of these visualised VLP’s.

This study shows the first definitive evidence for viral particles infecting calanoid copepods and is a big step into the investigation of non-predatory mortality in marine mesoplankton. This is important for understanding the potential role of viruses in the mortality of marine copepods, however it is evident that the study itself is a first step as while there is a lot of speculation throughout the report, there is no classification of these VLP’s. More work would be required to understand the route of infection and mode of action of these VLP’s. Further comparisons to other marine invertebrate viruses may also reveal further the identity of these studied viruses.

Dunlap D. S., Ng T. F., Rosario K., Barbosa J. G., Greco A. M., Breitbart M., Hewson I. (2013) PNAS, 110, 4, 1375–1380.