Virome Analysis of Signal Crayfish (Pacifastacus leniusculus) along Its Invasion Range Reveals Diverse and Divergent RNA Viruses

Abstract

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader's dispersal.

Keywords: RNA viruses; high-throughput sequencing; invasion range; invasive alien species; signal crayfish virome.

Figure 1

Position of sampling locations and differences in crayfish (Pacifastacus leniusculus and Pontastacus leptodactylus) presence and abundance (CPUE; catch per unit effort) along the signal crayfish invasion range in the Korana River in 2018. Sampling was performed at both upstream (UF) and downstream (DF) invasion fronts and upstream (UC) and downstream (DC) invasion cores.

Figure 2

Heatmap showing average read coverage resulting from mapping of normalized read datasets from individual locations to selected putative invertebrate virus-like contigs together with the dendrograms clustering the sampling locations (top) and viral contigs (left) according to average read coverage values. Heatmap and dendrograms were constructed using decimal logarithm transformed values, however, non-transformed average read coverage numbers are plotted on the heatmap.

Figure 3

Gel electrophoresis of PCR amplicons originating from newly identified signal crayfish-associated viruses. Four primer pairs were used for amplifying app. 200 nts long regions of signal crayfish-associated reo-like virus 1 (V1), signal crayfish-associated hepe-like virus 1 (V2), signal crayfish-associated toti-like virus 1 (V3), and signal crayfish-associated picorna-like virus 1 (V4) genomes. Samples from different locations (UF—upstream front, UC—upstream core, DC—downstream core, DF—downstream front) were analyzed. Unmarked wells represent no template controls of each amplification reaction.

Figure 4

Phylogenetic relationships and genome organization of signal crayfish-associated reo-like virus 1. (A) Phylogenetic tree built with maximum likelihood approach, based on the alignment of the conserved segment of RNA-dependent RNA polymerase domain of signal crayfish-associated reo-like virus 1 (virus-like contig 4) and representative selected sequences of phylogenetically related viruses. The numbers on the branches represent bootstrap support values (>50% shown), the branch length represents the average number of amino acid substitutions per site (B) The predicted partial genome organization of the novel signal crayfish-associated reo-like virus 1, with positions and length of the open reading frames (ORF) indicated with corresponding arrow length.

Figure 5

Phylogenetic relationships and genome organization of signal crayfish-associated hepe-like virus 1. (A) Phylogenetic tree built with maximum likelihood approach, based on the alignment of the conserved segment of RNA-dependent RNA polymerase domain of signal crayfish-associated hepe-like virus 1 (virus-like contig 139) and representative selected sequences of phylogenetically related viruses. The numbers on the branches represent bootstrap support values (>50% shown), the branch length represents the average number of amino acid substitutions per site. (B) The predicted genome organization of the novel signal crayfish-associated hepe-like virus 1, with positions and lengths of the open reading frames (ORF) indicated with corresponding arrow lengths.

Figure 6

Phylogenetic relationships and genome organization of signal crayfish-associated toti-like virus 1. (A) Phylogenetic tree built with maximum likelihood approach, based on the alignment of the conserved segment of RNA-dependent RNA polymerase domain of signal crayfish-associated toti-like virus 1 (virus-like contig 141) and representative selected sequences of phylogenetically related viruses. The numbers on the branches represent bootstrap support values (>50% shown), the branch length represents the average number of amino acid substitutions per site. (B) The predicted genome organization of the novel signal crayfish-associated toti-like virus 1 with positions and lengths of the open reading frames (ORF) indicated with corresponding arrow lengths.

Figure 7

Phylogenetic relationships and genome organization of signal crayfish-associated picorna-like virus 1. (A) Phylogenetic tree built with maximum likelihood approach, based on the alignment of the conserved segment of RNA-dependent RNA polymerase domain of signal crayfish-associated picorna-like virus 1 (virus-like contig 1) and representative selected sequences of phylogenetically related viruses. The numbers on the branches represent bootstrap support values (>50% shown), the branch length represents the average number of amino acid substitutions per site. (B) The predicted partial genome organization of the novel signal crayfish-associated picorna-like virus 1, with positions and length of the open reading frame (ORF) indicated with corresponding arrow length.

Figure 8

Nucleotide diversity values (π) calculated for signal crayfish-associated hepe-like virus 1, signal crayfish-associated toti-like virus 1, and signal crayfish-associated reo-like virus 1 at different locations (UF—upstream front, UC—upstream core, DC—downstream core, DF—downstream front) along the invasion range of the signal crayfish in the Korana River, Croatia.