Molecular and microscopic evidence of viruses in marine copepods

Abstract

As dominant members of marine mesozooplankton communities, copepods play critical roles in oceanic food webs and biogeochemical cycling. Despite the ecological significance of copepods, little is known regarding the causes of copepod mortality, and up to 35% of total copepod mortality cannot be accounted for by predation alone. Viruses have been established as ecologically important infectious agents in the oceans; however, viral infection has not been investigated in mesozooplankton communities. Here we used molecular and microscopic techniques to document viral infection in natural populations of the calanoid copepods Acartia tonsa (Dana) and Labidocera aestiva (Wheeler) in Tampa Bay, FL. Viral metagenomics revealed previously undocumented viruses in each species, named Acartia tonsa copepod circo-like virus (AtCopCV) and Labidocera aestiva copepod circo-like virus (LaCopCV). LaCopCV was found to be extremely prevalent and abundant in L. aestiva populations, with up to 100% prevalence in some samples and average viral loads of 1.13 × 10(5) copies per individual. LaCopCV transcription was also detected in the majority of L. aestiva individuals, indicating viral activity. AtCopCV was sporadically detected in A. tonsa populations year-round, suggesting temporal variability in viral infection dynamics. Finally, virus-like particles of unknown identity were observed in the connective tissues of A. tonsa and L. aestiva by transmission electron microscopy, demonstrating that viruses were actively proliferating in copepod connective tissue as opposed to infecting gut contents, parasites, or symbionts. Taken together, these results provide strong independent lines of evidence for active viral infection in dominant copepod species, indicating that viruses may significantly influence mesozooplankton ecology.

Fig. 1.

Genome organization and phylogenetic placement of the circo-like viruses identified in L. aestiva (LaCopCV) and A. tonsa (AtCopCV). Both genomes have a stem-loop with a conserved nonanucleotide motif at the apex and two nonoverlapping ORFs encoding putative replication initiator (Rep) (gray) and capsid (light gray) proteins oriented in the same direction. ORFs with no hits in the database are shown in white. A maximum likelihood phylogenetic tree of Rep amino acid sequences from these viruses along with viral and associated satellite members of the viral Rep family (PF02407) and other circular Rep-encoding ssDNA viruses from various environmental sources shows that the copepod viruses are only distantly related to known and proposed members of the Circoviridae family (i.e., circoviruses and cycloviruses) and are more closely related to circo-like virus sequences assembled from environmental viral metagenomes. The collapsed circovirus and cyclovirus clade represents Reps from 43 genomes, whereas the alphasatellite clade represents 17 genomes.

Fig. 2.

Transmission electron micrographs (Left, broad view; Right, close-up) showing virus-like particles in connective tissue of A. tonsa (Upper) and L. aestiva (Lower).