Background
Insects are the most abundant and diverse group of animals on earth [
1]. High throughput sequencing has led to huge advances in revealing previously unknown diversity of insect viruses, significantly contributing to filling deep phylogenetic gaps along evolutionary history within the most diverse viral lineages [
2‐
4]. Nonetheless, like insect diversity, the diversity of viruses associated with insects is far from clear [
1,
3]. While many studies have focused on viromes of arbovirus-transmitting insects, especially those involved in transmission of medically important viruses, such as mosquitoes [
5‐
8], other groups that are important pests either impacting agricultural or natural ecosystems, such as invasive ants, have been less studied [
9‐
11]. In addition to contributing to better understanding of basic aspects of virus ecology and evolution, these studies may contribute to new opportunities to use viruses as tools to develop more sustainable insect control methods [
12‐
14].
The red imported fire ant,
Solenopsis invicta, is an invasive pest causing significant ecological impact and economic loss in invaded areas [
15,
16]. Originating from South America,
S. invicta was accidentally introduced into the southern region of the United States (U.S.) almost a century ago, becoming a serious problem [
17]. Since then, it has spread throughout the southeastern U.S. and more recently into Oklahoma, New Mexico, Arizona, and California [
18]. Limited introduction events, likely associated with small founder populations, led to a significant reduction in natural enemies and enemy diversity associated with
S. invicta in introduced areas [
19‐
21]. Therefore,
S. invicta populations may reach sizes even greater than those observed in its native range, making control difficult and even more costly [
22]. High densities observed in
S. invicta populations in the U.S. have facilitated its dispersal across the world, contributing to repeated introduction events in several countries, such as China, Taiwan, and Australia [
18]. Morrison et al. [
23] demonstrated, based on predictive models, that most tropical and subtropical regions worldwide are potentially appropriate for
S. invicta infestation. Highly competitive ability, generalist feeding habits and high populations densities make
S. invicta a successful invasive species causing huge disturbance in biodiversity by displacing native ants and other arthropods in introduced regions [
15]. Currently, chemical insecticides are the most common control strategy used against
S. invicta [
24]. Low efficacy due to temporary effects of chemical applications, high cost in extensive areas, and off-target effects harmful to beneficial and other native species are substantial impairments to addressing invasive ant damage and expansion. Therefore, establishment of management strategies that are both environmentally friendly and self-sustainable are necessary.
Classical biological control has been one strategy used in an attempt to control this pest in the U.S., with viruses considered a promising resource to be used as biopesticides [
24‐
26]. Over the last decade, a great effort has been made in characterizing the
S. invicta virome pertaining to potential use in biological control [
11,
27‐
30]. To date, the
S. invicta virome is composed of mainly positive-sense single-strand RNA (+ ssRNA) viruses in the order
Picornavirales. These include eleven viruses from families
Dicistroviridae,
Polycipiviridae,
Iflaviridae,
Soliniviridae and two unclassified viruses [
11,
31]. Additionally, one double-strand RNA (dsRNA) and one single-strand DNA (ssDNA) of the families
Totiviridae and
Parvoviridae, respectively, have been characterized [
11,
32]. While most of these viruses have been reported associated with
S. invicta in its native range, only the species
Solenopsis invicta virus 1,
Solenopsis invicta virus 2,
Solenopsis invicta virus 3,
Solenopsis invicta virus 6 and
Nylanderia fulva virus 1 have been reported in the United States [
11,
31]. Although great progress has been reached in identification and molecular characterization of the
S. invicta virome, the effect of these viruses and their potential use in biological control will require additional investigation. Interactions among
S. invicta and Solenopsis invicta virus 1 (SINV-1), SINV-2 and SINV-3, have been the only ones previously characterized [
25,
33,
34]. SINV-1 was shown to affect claustral queen weight making them lighter than uninfected ones, whereas SINV-2 directly affected fitness of queens by reducing their reproductive output [
33]. SINV-3 was shown to be the most aggressive virus, causing significant mortality in
S. invicta colonies, with greatest potential as a biological control agent [
25,
35].
Here using a metatranscriptome approach, we identified and molecularly characterized five new virus genome sequences associated with
S. invicta in two introduced areas, U.S and Taiwan. These investigations utilized existing publicly available RNA sequences deposited in NCBI GenBank as Sequence Read Archive (SRA) data files (Table
1). Five new virus sequences were found associated with
S. invicta, with the first negative-sense single-stranded RNA (-ssRNA) virus sequences included in the orders
Bunyavirales and
Mononegavirales reported.
In addition, two + ssRNA viruses sequences, included in the family
Iflaviridae and an unassigned species, and a partial ssDNA virus sequence, were also characterized.
Table 1
Information summary of S. invicta transcriptome dataset analyzed in this study
A | SRX3035962 | Larvae | Whole body | Mississippi, USA | March 2016 | Polygynous | |
B | SRX3035961 | Larvae | Whole body | Mississippi, USA | March 2016 | Polygynous | |
C | SRX3035960 | Larvae | Whole body | Mississippi, USA | March 2016 | Polygynous | |
X | SRX3035959 | Pupa | Whole body | Mississippi, USA | March 2016 | Polygynous | |
Y | SRX3035964 | Pupa | Whole body | Mississippi, USA | March 2016 | Polygynous | |
Z | SRX3035963 | Pupa | Whole body | Mississippi, USA | March 2016 | Polygynous | |
Q2 | DRX037806 | Adults | Whole body | Texas, USA | September 2012 | Polygynous, queen | |
W2 | DRX037809 | Adults | Whole body | Texas, USA | September 2012 | Polygynous, worker | |
W3 | DRX037810 | Adults | Whole body | Texas, USA | September 2012 | Polygynous, worker | |
Y05 | SRX5464977 | Adults | Brain | Florida, USA | May 2014 | Queen | –* |
K05 | SRX5464984 | Adults | Brain | Florida, USA | May 2014 | Queen | – |
CA01 | SRX5464990 | Adults | Brain | Florida, USA | May 2014 | Queen | – |
2-small | SRX5822389 | Adults | Whole body | Taiwan | May 2014 | A pool of two virgin queens, lab colony | |
Discussion
S. invicta is a ground-dwelling ant that feeds on a broad diet that may include plant and insect exudates, prey, and decaying matter. Thus, the opportunities for exchange of virus particles from the environment, whether native or novel, are many. The social nature of ant colonies where exchanges of biological fluids among colony members of all castes and life stages must occur also facilitates distribution of virus within the colony. Distinguishing clearly between different host/microbe relationships and forms of virus transmission (horizontal and vertical) will be challenging with invasive ant systems.
Using a transcriptome approach, we searched for RNA viruses in the SRA data collected from
S. invicta. Curiously, while a great diversity of positive and negative sense viruses have been reported from ants [
9,
10] and other arthropods [
2], the
S. invicta virome has been composed mainly of ssRNA viruses in the order
Picornavirales, with no negative sense RNA viruses previously reported. One factor that could be responsible for this bias is the selection of polyadenylated RNA for library preparation in previous studies [
11,
30]. The utilization of unbiased library preparation, using ribosomal depletion methods, besides the detection of + ssRNA viruses, has enabled the discovery of many viruses with non-polyadenylated genome, especially within order
Bunyavirales [
2]. The fact of some of the libraries used here were prepared using this approach [
36] allowed us to characterize the first bunyavirus reported from ants. In addition, we also reported for the first time another negative sense virus within the order
Mononegavirales associated with
S. invicta. Although we did not perform any amplification step of viruses genomes characterized here, the presence of untruncated ORFs carrying intact functional domains (Additional file
3: Table S3), the high abundance of viral reads, and the similar organization and genomes sizes compared to other closely related viruses strongly suggest that we obtained the correct full-length or near full-length genomes sequences (Fig.
1). The only exception is SINaDNV, due to the linear single strand DNA genome, only active transcriptional units were sampled using our transcriptome approach and further investigation is needed to reveal its full-length genome structure.
Our analysis of RNA obtained from geographically and temporally different samples suggests shifts in the virome of native and invasive fire ants [
11]. In established exotic populations of the
S. invicta Yang et al. [
21] identified SINV-1 and SINV-2 and hypothesized that while these two viruses may persist, the more virulent virus, SINV-3 arrived with founders but caused high host mortality resulting in individual carriers of the virus being rapidly eliminated. Nine additional viruses were identified in
S. invicta RNA samples from the native South American range of the species [
11]. The viruses described here show distribution and composition that varied according to geographic location and
S. invicta stage (Tables
1 and
2). To date, four of five viruses previously reported associated with
S. invicta in introduced areas have also been described to occur in its native origin [
11,
21]. Although viral diversity associated with
S. invicta has been well studied in Argentina, the viruses reported in this study have not been detected there yet [
11,
30], suggesting that these viruses may not be present in the sampled area in Argentina, and even that new host-virus associations may have occurred in introduced areas.
While the origins of viruses that replicate in plant and insect vectors remains unknown, such as those within order
Bunyavirales and
Mononegavirales, the discovery of possible intermediate forms has been suggested [
2,
51]. Whereas tenuiviruses are known to be plant viruses that replicate in the insect vector tenui-like viruses have been reported from non-plant vectors. Li, Shi [
2] reported the first tenui-like virus,
Horsefly horwuvirus (virus WhHV), associated with a pool of horseflies (family
Tabanidae), and proposed that it may represent a transitional form between plant-infecting virus and arthropod-specific viruses. In addition, a partial genome sequence of another tenui-like, FCTenV1, has been reported associated to
Culex annulirostris [
51]. While these two tenui-like viruses have been reported associated with flies (Order Diptera), we characterized a new tenui-like virus sequence closely related to the FCTenV1, associated with
S. invicta transcriptome (Order Hymenoptera). Interestingly, in contrast with WhHV, which lacks an ambisense coding strategy, the SINV-14 genome sequence exactly mirrors the genomic structure of typical plant tenuivirus, predicted to encode proteins using ambisense strategy, and also has the conserved sequences at the ends of all four segments identical to those found in tenuivirus genomes (Fig.
1A, B). While these virus sequences may represent different steps of transitional viruses forms between tenui-infecting plant and those insects-specific viruses, the direction of the process, whether they come from plant to insect or otherwise, remains unknown.
Phylogenetic incongruence observed between the nucleocapsid protein compared to the other proteins strongly suggests that SINV-14 may have a recombinant/reassortment origin, where the RNA3 or part of it was acquired from a divergent phenuivirus. While it could be argued that this might be an artefact due to assembling a segmented genome using a transcriptome approach, the fact that we did not find any other contigs related to phenuiviruses, the high read abundance, the constant association between these four segments across different libraries and the presence of the conserved tenuivirus sequence located at the ends of genome segments (Fig.
1B), suggest that they are part of a unique genome, rather than being an artefact. In addition, phylogenetic congruence among different segments indicate that they are in an intimate codivergence process (Additional file
5: Figure S1).
Valles et al. [
52] using an expressed sequence tag (EST) library from
S. invicta, detected a short sequence (approximately 750 nt, GenBank access: EF409991) related to plant tenuiviruses. Further identity analysis showed that sequence is 99.8% identical to SINV14 RNA4. They suggested that the sequence would be likely a contamination due to the ant diet feeding either plant or infected insect. Tenuiviruses are typical plant viruses that replicate in the insect vector [
53,
54], and the high abundance of SINV-14 compared to housekeeping genes is strong evidence of active replication in
S. invicta. Furthermore, the highest virus abundance was found in a sample prepared from a dissected ant brain, which rules out the possibility of contamination due to feeding on plants or association with insect vectors infected with a tenuivirus. The Maize stripe virus (MSpV), a typical plant tenuivirus, was detected in the brain of its vector
Peregrinus maidis, providing evidence of replication of tenuiviruses in this tissue [
55]. Tenuiviruses replicate in diverse tissues of their insect vectors and are transovarially transmitted between generations suggesting that SINV-14, and other tenui-like viruses, could be sustained through vertical transmission in their insect host [
54]. Additionally, significant asymmetric abundance sequences of different components of SINV-14 suggest a very specific and active interaction. Asymmetric accumulation in multipartite viruses has been shown and seems to be common trait, shared by RNA and DNA multipartite viruses infecting plants and animals [
56,
57]; this has been suggested to be involved in control of gene expression allowing fast virus adaptation [
58]. Although this has not yet been shown for any phenuivirus and may be host dependent [
56], our results suggest that this may occur, at least for SINV-14, and more experiments will be necessary to confirm.
While SINV-14 is evolutionarily closely related to insect tenui-like viruses and plant tenuivirus, the synonymous codon usage and dinucleotide analyses demonstrate a distinct compositional bias compared to FCTenV1 and WhHV, and all other viruses analyzed here, indicating that the virus may be actively replicating in ants rather than plants and other insects. The active replication may have driven the virus genome to distinct compositional bias at the nucleotide level, while maintaining protein integrity at the amino acid level and close relationship with those of other tenui-like viruses, as observed through phylogenetic analysis. The fact that most sequences examined here are probably from viruses that replicate in plant or vertebrate hosts and also in the insect vector could be the reason driving such difference between SINV-14, most likely associated only with ant, compared to other viruses. Furthermore, phylogenetic congruence across different segments that mirror the genetic structure of invasive S. invicta, suggest an intimate and long-term codivergence process between virus-host.
We presented strong evidence that the sequences from SINV-14 may be from a virus that has been associated long-term and may actively replicate in ants. However, the possibility of this virus, and other tenui-like viruses, replicating in plants is unknown. The presence of the protein carrying an NS4 domain, that is related to cell-to-cell and long-distance movement in plants [
59] in insect viruses is puzzling. Solenopsis invicta virus 14 NS4 is highly divergent showing 19.85 to 24.4% of identity compared to other tenuivirus (Additional file
6: Figure S2). In addition, mutation in most sites associated with cell-to-cell and long-distance movement (Additional file
6: Figure S2), suggests that this protein might have lost the capacity to move viral genome in plant, whereas its maintenance in insect viruses may be related to another role acquired through functional diversification. The possible function of the plant virus movement protein from insect viruses is of significant interest, and its role in insects and plants remains to be addressed.
Altogether, based on virus abundance compared to housekeeping genes, abundant viruses with viral reads higher than 0.01%, phylogenetic relationship, complete viral coding sequence regions recovered, and compositional bias for SINV-14, our results suggest that four out five viruses reported here, those being SINV-14, SINV-15, SINV-16 and SINV-17 are truly replicating in S. invicta. Our results suggest fluid shifts in the virome of this invasive species. Further research describing this virome in native and invasive regions and ecosystems could provide insight on virus evolution and invasion mechanics.
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