Hidden diversity and evolution of viruses in market fish

Abstract

Aquaculture is the fastest growing industry worldwide. Aquatic diseases have had enormous economic and environmental impacts in the recent past and the emergence of new aquatic pathogens, particularly viruses, poses a continuous threat. Nevertheless, little is known about the diversity, abundance and evolution of fish viruses. We used a meta-transcriptomic approach to help determine the virome of seemingly healthy fish sold at a market in Sydney, Australia. Specifically, by identifying and quantifying virus transcripts we aimed to determine (i) the abundance of viruses in market fish, (ii) test a key component of epidemiological theory that large and dense host populations harbour a greater number of viruses compared to their more solitary counterparts and (iii) reveal the relative roles of virus-host co-divergence and cross-species transmission in the evolution of fish viruses. The species studied comprised both shoaling fish-eastern sea garfish (Hyporhamphus australis) and Australasian snapper (Chrysophrys auratus)-and more solitary fish-eastern red scorpionfish (Scorpaena jacksoniensis) and largetooth flounder (Pseudorhombus arsius). Our analysis identified twelve potentially novel viruses, eight of which were likely vertebrate-associated across four viral families and that exhibited frequent cross-species transmission. Notably, the most solitary of the fish species studied, the largetooth flounder, harboured the least number of viruses while eastern sea garfish, a densely shoaling fish, had the highest number of viruses. These results support the emerging view that fish harbour a large and largely uncharacterised virome.

Keywords: fish; meta-transcriptomics; phylogenetics; virome; virus evolution.

Figure 1.

(a) Relative abundance of viral contigs within each RNA sequencing library for each fish species and tissue type, falling across nine viral families. The relative abundance of the host reference gene, ribosomal protein S13, is also included for each library. The proportion of viral contigs (including those that are unclassified) in each tissue type is shown below in pie charts. (b) Total relative abundance of viral contigs within each tissue type falling into each viral family.

Figure 2.

Relative abundance of viral contigs within each RNA sequencing library for each fish species and tissue type, normalized for each viral family.

Figure 3.

Phylogenetic relationships of likely vertebrate-associated viruses discovered from assembled contigs: (a) Astroviridae, (b) Picornaviridae, (c) Flaviviridae and (d) Hepadnaviridae. The maximum likelihood phylogenetic trees show the topological position of the newly discovered potential viruses (bold text), in the context of their closest relatives (major genera are labelled). Fish viruses are coloured to correspond to host order, as indicated in the fish order phylogeny. All branches are scaled to the number of amino acid substitutions per site and trees were mid-point rooted for clarity only. An asterisk indicates node support of >70% bootstrap support.

Figure 4.

Tanglegrams of rooted phylogenetic trees for each virus family of the vertebrate-associated fish viruses described here, constructed using TreeMap3 v3.0 (48). Viruses identified in this study are indicated in red, while all other viruses have previously been identified in fish. The ‘untangle’ function was used to maximise the congruence between the host (left) and virus (right) phylogenies. Below each tanglegram, reconciliation analysis of each virus family using Jane (47) illustrates the range of the proportion of possible events. The ‘event costs’ associated with incongruences between trees were conservative towards co-divergence and defined here as: 0 for co-divergence, 1 for duplication, 1 for host-jumping and 1 for extinction. An asterisk on the virus trees indicates node support of >70% bootstrap support.

Figure 5.

Phylogenetic relationships of likely invertebrate-associated viruses discovered from assembled contigs: (a) Nodaviridae, (b) Bunyaviridae-like, (c) Picornaviridae-like and (d) Rhabdoviridae. The maximum likelihood phylogenetic trees show the topological position of the newly discovered potential viruses (blue), in the context of their closest relatives. All branches are scaled to the number of amino acid substitutions per site and trees were mid-point rooted for clarity only. An asterisk indicates node support of >70% bootstrap support.