Ever-increasing viral diversity associated with the red imported fire ant Solenopsis invicta (Formicidae: Hymenoptera)

Abstract

Background: Advances in sequencing and analysis tools have facilitated discovery of many new viruses from invertebrates, including ants. Solenopsis invicta is an invasive ant that has quickly spread worldwide causing significant ecological and economic impacts. Its virome has begun to be characterized pertaining to potential use of viruses as natural enemies. Although the S. invicta virome is the best characterized among ants, most studies have been performed in its native range, with less information from invaded areas.

Methods: Using a metatranscriptome approach, we further identified and molecularly characterized virus sequences associated with S. invicta, in two introduced areas, U.S and Taiwan. The data set used here was obtained from different stages (larvae, pupa, and adults) of S. invicta life cycle. Publicly available RNA sequences from GenBank's Sequence Read Archive were downloaded and de novo assembled using CLC Genomics Workbench 20.0.1. Contigs were compared against the non-redundant protein sequences and those showing similarity to viral sequences were further analyzed.

Results: We characterized five putative new viruses associated with S. invicta transcriptomes. Sequence comparisons revealed extensive divergence across ORFs and genomic regions with most of them sharing less than 40% amino acid identity with those closest homologous sequences previously characterized. The first negative-sense single-stranded RNA virus genomic sequences included in the orders Bunyavirales and Mononegavirales are reported. In addition, two positive single-strand virus genome sequences and one single strand DNA virus genome sequence were also identified. While the presence of a putative tenuivirus associated with S. invicta was previously suggested to be a contamination, here we characterized and present strong evidence that Solenopsis invicta virus 14 (SINV-14) is a tenui-like virus that has a long-term association with the ant. Furthermore, based on virus sequence abundance compared to housekeeping genes, phylogenetic relationships, and completeness of viral coding sequences, our results suggest that four of five virus sequences reported, those being SINV-14, SINV-15, SINV-16 and SINV-17, may be associated to viruses actively replicating in the ant S. invicta.

Conclusions: The present study expands our knowledge about viral diversity associated with S. invicta in introduced areas with potential to be used as biological control agents, which will require further biological characterization.

Keywords: Bunyavirales; Metatranscriptome; Mononegavirales; Red imported fire ant; S. invicta; Tenuivirus; Virome; Virus diversity.

Fig. 1

Genome organization of five putative new viruses associated with S. invicta transcriptomes. Predicted open reading frames (ORFs) encoding proteins homologous to known viral proteins or conserved domain that they encode are represented and colored according to their orientation in the genome (dark blue: those encoded on positive strand and light blue: those encoded on negative strand). Graphic representation of A segmented negative single-strand RNA genome S. invicta virus 14 (SINV-14) and B conserved consensus sequence located at the ends of tenuivirus genome segment (5′-ACACAAAGU and ACUUUGAGU-3′) detected in SINV-14 genome. Dashed lines indicate incomplete sequence. Nucleotides highlighted in red differ from consensus sequence. C Negative-sense single-strand RNA genome S. invicta virus 15 (SINV-15), and positive-sense single-strand RNA genomes D S. invicta virus 16 (SINV-16) and E S. invicta virus 17 (SINV-17) are shown. F Partial double-strand DNA genome of S. invicta-associated densovirus (SINaDNV) is represented. RdRp and L: RNA-dependent RNA polymerase; G: glycoprotein; N: nucleocapsid protein; p3: unknown; NS4: non-structural protein 4; NCP: non-nucleocapsid protein; P: phosphoprotein; M: matrix protein; CP: capsid protein; H: Helicase; V: viral methyltransferase; F: Ftsj-like methyltransferase; NS1and NS2: non-structural protein 1 and 2, respectively

Fig. 2

Evolutionary history inferred for different genomic segments of S. invicta virus 14 (SINV-14). Midpoint-rooted Bayesian phylogenetic trees based on A full-length amino-acid sequences of RNA-dependent RNA polymerase (RdRp), B amino-acid sequence of conserved glycoprotein domain, C full-length amino-acid sequence of movement protein and D full-length amino-acid sequence of nucleocapsid protein. Bayesian posterior probabilities are shown at the nodes. Nodes with posterior probability values between > 0.50 and ≤ 85 are indicated by empty circles, > 0.85 and < 0.95 are indicated by half-filled circles and those with values ≥ 0.95 are indicated by filled circles. The scale bar at the bottom indicates the number of amino acid substitutions per site. Branches highlighted in red indicate virus characterized in this study. Genera are indicated at the right side. Proposed species unaccepted yet by International Committee on Taxonomy of Viruses (ICTV) are followed by an asterisk. Proteins sequences encoded by cognates components for a given virus that do not have conserved domain according to Conserved Domain Database (CDD) were not included

Fig. 3

Evolutionary history of new viruses associated to S. invicta transcriptome based on Bayesian inference. Midpoint-rooted phylogenetic trees were constructed based on A conserved domain (pfam00946) of RNA-dependent RNA polymerase (RdRp) of S. invicta virus 15 (SINV-15) and representative species of the order Mononegavirales, B conserved domain (pfam00680) of RdRp of S. invicta virus 16 (SINV-16) and representative species of the families Iflaviridae and Dicistroviridae and C full-length amino-acid sequences of RdRp of S. invicta virus 17 (SINV-17) and representative species of the proposed new genus Negevirus (unassigned to any family) and families Virgaviridae and Kitaviridae, are shown. D Bayesian phylogenetic tree based on full-length amino-acid sequences of non-structural gene 1 (NS1) of S. invicta-associated densovirus (SINaDNV) and representative species of the family Parvoviridae. Bayesian posterior probabilities are shown at the nodes. Nodes with posterior probability values between > 0.65 and ≤ 85 are indicated by empty circle, > 0.85 and < 0.95 are indicated by half-filled circles and those with values ≥ 0.95 are indicated by filled circles. The scale bar at the bottom indicates the number of amino acid substitutions per site. Branches highlighted in red indicates viruses characterized in this study. Genera/Families/Subfamilies are shown at right side

Fig. 4

Boxplot of housekeeping genes and viruses abundance. A Abundance among housekeeping genes from all libraries were compared. Abundance was calculated as percentage of housekeeping or viruses reads per total number of reads in each library. B Abundance between viruses and housekeeping genes were compared. Statistical test was performed among virus and housekeeping genes abundance from the same libraries. C Normalized abundance of S. invicta virus 14 (SINV-14) segments significantly differ among them. Normalized abundance was calculated by dividing the abundance of each segment by the total abundance of the entire genome. Box plots with different letters indicate significant differences between groups according to non-parametric Kruskal–Wallis test followed by post hoc multiple comparison test using Fisher’s least significant difference (p < 0.05). Box plots show the first and third quartiles as a box, horizontal line corresponds to the median and whiskers correspond to 1.5 times the inter-quartile distance (IQR) from the difference between first and third quartiles

Fig. 5

Compositional biases of Solenopsis invicta virus 14 (SINV-14) differ from plant tenuivirus and tenui-like viruses associated with flies. Principal Components Analysis (PCA) of synonymous codon usage (A–D) and dinucleotide bias (E–H) of SINV-14 and other viruses that infect plants and animals (vertebrate and invertebrate) are shown. Scatterplots show the first two principal components that account for the largest variance (shown in parenthesis) in the data set with ellipses representing each group and dots individuals. Viruses were divided into two main groups: plant viruses include those that replicate in plants and insect vectors belonging to the genera Tenuivirus and Orthotospovirus and non-plant viruses including those that do not replicate in plants belonging to the genera Orthobunyavirus and representatives within family Phenuiviridae. For detailed information of the data set see Additional file 4: Table S4. For NS4 protein, comparison was performed only for SINV-14, Fitzroy Crossing tenui-like virus1 (FCTenV1) and tenuiviruses, as this gene is not shared by other groups. WhHV, Wuhan horsefly virus