Solenopsis richteri (Hymenoptera: Formicidae) alates infected with deformed wing virus display wing deformity with altered mobility.

Deformed wing virus (DWV) has long been identified as a critical pathogen affecting honeybees, contributing to colony losses through wing deformities, neurological impairments, and reduced lifespan. Since DWV also affects other pollinators, it poses a significant threat to global pollination networks. While honeybees have been the focal point of DWV studies, emerging research indicates that this RNA virus is not host-specific but rather a generalist pathogen capable of infecting a wide range of insect species, including other bee species such as bumblebees and solitary bees, as well as wasps and ants. This expands the potential impact of DWV beyond honeybees to broader ecological communities. The black imported fire ant, Solenopsis richteri, is an economically important invasive ant species. In this study, we describe deformed wing (DW) symptoms in S. richteri. DW alates were found in three of nine (33%) laboratory colonies. The symptoms ranged from severely twisted wings to a single crumpled wing tip. Additionally, numerous symptomatic alates also displayed altered mobility, ranging from an ataxic gait to an inability to walk. Viral replication of DWV was confirmed using a modified strand-specific RT-PCR. Our results suggest that S. richteri can be an alternative host for DWV, expanding our understanding of DWV as a generalist pathogen in insects. However, additional research is required to determine whether DWV is the etiological agent responsible for DW syndrome in S. richteri.

Two +ssRNA mycoviruses cohabiting the fungal cultivar of leafcutter ants.

Leafcutter ants are dominant herbivores in the Neotropics and rely on a fungus (Leucoagaricus gongylophorus) to transform freshly gathered leaves into a source of nourishment rather than consuming the vegetation directly. Here we report two virus-like particles that were isolated from L. gongylophorus and observed using transmission electron microscopy. RNA sequencing identified two +ssRNA mycovirus strains, Leucoagaricus gongylophorus tymo-like virus 1 (LgTlV1) and Leucoagaricus gongylophorus magoulivirus 1 (LgMV1). Genome annotation of LgTlV1 (7401 nt) showed conserved domains for methyltransferase, endopeptidase, viral RNA helicase, and RNA-dependent RNA polymerase (RdRp). The smaller genome of LgMV1 (2636 nt) contains one open reading frame encoding an RdRp. While we hypothesize these mycoviruses function as symbionts in leafcutter farming systems, further study will be needed to test whether they are mutualists, commensals, or parasites.

Varying impact of neonicotinoid insecticide and acute bee paralysis virus across castes and colonies of black garden ants, Lasius niger (Hymenoptera: Formicidae).

Pesticides and pathogens are known drivers of declines in global entomofauna. However, interactions between pesticides and viruses, which could range from antagonistic, over additive to synergistic, are poorly understood in ants. Here, we show that in ants the impact of single and combined pesticide and virus stressors can vary across castes and at the colony level. A fully-crossed laboratory assay was used to evaluate interactions between a sublethal dose of the neonicotinoid thiamethoxam and Acute bee paralysis virus (ABPV) in black garden ants, Lasius niger. After monitoring colonies over 64 weeks, body mass, neonicotinoid residues and virus titres of workers and queens, as well as worker behavioural activity were measured. ABPV, but not thiamethoxam, reduced activity of workers. Neonicotinoid exposure resulted in reduced body mass of workers, but not of queens. Further, thiamethoxam facilitated ABPV infections in queens, but not in workers. Overall, virus exposure did not compromise detoxification and body mass, but one colony showed high virus titres and worker mortality. Although the data suggest additive effects at the level of individuals and castes, co-exposure with both stressors elicited antagonistic effects on colony size. Our results create demand for long-term holistic risk assessment of individual stressors and their interactions to protect biodiversity.

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

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.

Deformed Wing Virus in Two Widespread Invasive Ants: Geographical Distribution, Prevalence, and Phylogeny.

Spillover of honey bee viruses have posed a significant threat to pollination services, triggering substantial effort in determining the host range of the viruses as an attempt to understand the transmission dynamics. Previous studies have reported infection of honey bee viruses in ants, raising the concern of ants serving as a reservoir host. Most of these studies, however, are restricted to a single, local ant population. We assessed the status (geographical distribution/prevalence/viral replication) and phylogenetic relationships of honey bee viruses in ants across the Asia-Pacific region, using deformed wing virus (DWV) and two widespread invasive ants, Paratrechina longicornis and Anoplolepis gracilipes, as the study system. DWV was detected in both ant species, with differential geographical distribution patterns and prevenance levels between them. These metrics, however, are consistent across the geographical range of the same ant species. Active replication was only evident in P. longicornis. We also showed that ant-associated DWV is genetically similar to that isolated from Asian populations of honey bees, suggesting that local acquisition of DWV by the invasive ants may have been common at least in some of our sampled regions. Transmission efficiency of DWV to local arthropods mediated by ant, however, may vary across ant species.

Complete genome sequences of two novel dicistroviruses detected in yellow crazy ants (Anoplolepis gracilipes).

We report two novel RNA viruses from yellow crazy ants, (Anoplolepis gracilipes) detected using next-generation sequencing. The complete genome sequences of the two viruses were 10,662 and 8,238 nucleotides in length, respectively, with both possessing two open reading frames with three conserved protein domains. The genome organization is characteristic of members of the genus Triatovirus in the family Dicistroviridae. The two novel viruses were tentatively named "Anoplolepis gracilipes virus 1" and "Anoplolepis gracilipes virus 2" (AgrV-1 and AgrV-2). Phylogenetic analyses based on amino acid sequences of the non-structural polyprotein (ORF1) suggest that the two viruses are triatovirus-like viruses. This is the first report on the discovery of novel triatovirus-like viruses in yellow crazy ants with a description of their genome structure (two ORFs and conserved domains of RNA helicase, RNA-dependent RNA polymerase, and capsid protein), complete sequences, and viral prevalence across the Asia-Pacific region.

Genetic Strain Diversity of Multi-Host RNA Viruses that Infect a Wide Range of Pollinators and Associates is Shaped by Geographic Origins.

Emerging viruses have caused concerns about pollinator population declines, as multi-host RNA viruses may pose a health threat to pollinators and associated arthropods. In order to understand the ecology and impact these viruses have, we studied their host range and determined to what extent host and spatial variation affect strain diversity. Firstly, we used RT-PCR to screen pollinators and associates, including honey bees (Apis mellifera) and invasive Argentine ants (Linepithema humile), for virus presence and replication. We tested for the black queen cell virus (BQCV), deformed wing virus (DWV), and Kashmir bee virus (KBV) that were initially detected in bees, and the two recently discovered Linepithema humile bunya-like virus 1 (LhuBLV1) and Moku virus (MKV). DWV, KBV, and MKV were detected and replicated in a wide range of hosts and commonly co-infected hymenopterans. Secondly, we placed KBV and DWV in a global phylogeny with sequences from various countries and hosts to determine the association of geographic origin and host with shared ancestry. Both phylogenies showed strong geographic rather than host-specific clustering, suggesting frequent inter-species virus transmission. Transmission routes between hosts are largely unknown. Nonetheless, avoiding the introduction of non-native species and diseased pollinators appears important to limit spill overs and disease emergence.

Foodborne Transmission and Clinical Symptoms of Honey Bee Viruses in Ants Lasius spp.

Emerging infectious diseases are often the products of host shifts, where a pathogen jumps from its original host to a novel species. Viruses in particular cross species barriers frequently. Acute bee paralysis virus (ABPV) and deformed wing virus (DWV) are viruses described in honey bees (Apis mellifera) with broad host ranges. Ants scavenging on dead honey bees may get infected with these viruses via foodborne transmission. However, the role of black garden ants, Lasius niger and Lasius platythorax, as alternative hosts of ABPV and DWV is not known and potential impacts of these viruses have not been addressed yet. In a laboratory feeding experiment, we show that L. niger can carry DWV and ABPV. However, negative-sense strand RNA, a token of virus replication, was only detected for ABPV. Therefore, additional L. niger colonies were tested for clinical symptoms of ABPV infections. Symptoms were detected at colony (fewer emerging workers) and individual level (impaired locomotion and movement speed). In a field survey, all L. platythorax samples carried ABPV, DWV-A and -B, as well as the negative-sense strand RNA of ABPV. These results show that L. niger and L. platythorax are alternative hosts of ABPV, possibly acting as a biological vector of ABPV and as a mechanical one for DWV. This is the first study showing the impact of honey bee viruses on ants. The common virus infections of ants in the field support possible negative consequences for ecosystem functioning due to host shifts.

The detection of honey bee (Apis mellifera)-associated viruses in ants.

Interspecies virus transmission involving economically important pollinators, including honey bees (Apis mellifera), has recently sparked research interests regarding pollinator health. Given that ants are common pests within apiaries in the southern U.S., the goals of this study were to (1) survey ants found within or near managed honey bee colonies, (2) document what interactions are occurring between ant pests and managed honey bees, and 3) determine if any of six commonly occurring honey bee-associated viruses were present in ants collected from within or far from apiaries. Ants belonging to 14 genera were observed interacting with managed honey bee colonies in multiple ways, most commonly by robbing sugar resources from within hives. We detected at least one virus in 89% of the ant samples collected from apiary sites (n = 57) and in 15% of ant samples collected at non-apiary sites (n = 20). We found that none of these ant samples tested positive for the replication of Deformed wing virus, Black queen cell virus, or Israeli acute paralysis virus, however. Future studies looking at possible virus transmission between ants and bees could determine whether ants can be considered mechanical vectors of honey bee-associated viruses, making them a potential threat to pollinator health.

Season- and caste-specific variation in RNA viruses in the invasive Argentine ant European supercolony.

The Argentine ant (Linepithema humile, Mayr) is a highly invasive species. Recently, several RNA viruses have been identified in samples from invasive Argentine ant colonies. Using quantitative PCR, we investigated variation in the levels of these viruses in the main European supercolony over the course of a year. We discovered that virus prevalence and amounts of viral RNA were affected by season and caste: ants had more virus types during warm versus cold months, and queens had more virus types and higher virus prevalence than did workers or males. This seasonal variation was largely due to the appearance of positive-strand RNA viruses in the summer and their subsequent disappearance in the winter. The prevalences of positive-strand RNA viruses were positively correlated with worker foraging activity. We hypothesise that during warmer months, ants are more active and more numerous and, as a result, they have more conspecific and heterospecific interactions that promote virus transmission.

A new polycipivirus identified in Colobopsis shohki.

A new polycipivirus was identified in the arboreal ant Colobopsis shohki. The viral RNA was 11,855 nt in length with five 5'-proximal open reading frames (ORFs) encoding structural proteins and a long 3' ORF encoding the replication polyprotein. The protein sequences of these ORFs had significant similarity to those of the polycipiviruses Lasius niger virus 1 and Solenopsis invicta virus 2. The results of phylogenetic analysis and its genome organization suggested that this virus belongs to the genus Sopolycivirus in the family Polycipiviridae. The name "Colobopsis shohki virus 1" (CshV1) is proposed for the new virus.

Influence of temperature on the pathogenicity of Solenopsis invicta virus 3.

Field evaluations assessing the prevalence of Solenopsis invicta virus 3 (SINV-3) have shown that the virus exhibits a distinct seasonal phenology in the host, Solenopsis invicta, that is negatively correlated with warmer temperatures. Active SINV-3 infections were established in Solenopsis invicta colonies, which were subsequently maintained at 19.1, 22.2, 25.5, 27.7, and 29.3 °C. The quantity of brood declined in all SINV-3-treated colonies regardless of temperature over the initial 30 days. However, the quantity of brood in colonies held at 29.3 °C began increasing (recovering) in the next 40 days until they were statistically equivalent to untreated control colonies. Meanwhile, the quantity of brood continued to decline in colonies held at 19.1, 22.2, 25.5, and 27.7 °C for the duration of the test (81days). By the end of the test, these colonies were in poor health as indicated by decreased brood. Conversely, the amount of brood for colonies held at 29.3 °C increased to above 3, indicating healthy vigorous growth. Worker ants from SINV-3-treated colonies maintained at 19.1, 22.2, and 25.5 °C showed strong production of the VP2 capsid protein by Western blotting; 100% of the colonies sampled (n = 3) showed production of VP2. However, VP2 was detected in only 33% of colonies maintained at 27.7 °C, and the VP2 response was nearly undetectable in all colonies maintained at 29.3 °C. These results indicate that virus assembly does not appear to be occurring efficiently at the higher temperatures. Also, the quantity of SINV-3 detected in queens was significantly lower in those maintained at 29.3 °C compared with the lower temperature treatments. These results indicate that warm summer temperatures combined with fire ant thermoregulatory behavior and perhaps behavioral fevers may explain the low prevalence of SINV-3 in fire ant colonies during the summer.

Different bacterial and viral pathogens trigger distinct immune responses in a globally invasive ant.

Invasive species populations periodically collapse from high to low abundance, sometimes even to extinction. Pathogens and the burden they place on invader immune systems have been hypothesised as a mechanism for these collapses. We examined the association of the bacterial pathogen (Pseudomonas spp.) and the viral community with immune gene expression in the globally invasive Argentine ant (Linepithema humile (Mayr)). RNA-seq analysis found evidence for 17 different viruses in Argentine ants from New Zealand, including three bacteriophages with one (Pseudomonas phage PS-1) likely to be attacking the bacterial host. Pathogen loads and prevalence varied immensely. Transcriptomic data showed that immune gene expression was consistent with respect to the viral classification of negative-sense, positive-sense and double-stranded RNA viruses. Genes that were the most strongly associated with the positive-sense RNA viruses such as the Linepithema humile virus 1 (LHUV-1) and the Deformed wing virus (DWV) were peptide recognition proteins assigned to the Toll and Imd pathways. We then used principal components analysis and regression modelling to determine how RT-qPCR derived immune gene expression levels were associated with viral and bacterial loads. Argentine ants mounted a substantial immune response to both Pseudomonas and LHUV-1 infections, involving almost all immune pathways. Other viruses including DWV and the Kashmir bee virus appeared to have much less immunological influence. Different pathogens were associated with varying immunological responses, which we hypothesize to interact with and influence the invasion dynamics of this species.

Apoptosis as a primary defense mechanism in response to viral infection in invasive fire ant Solenopsis invicta.

Apoptosis is considered an innate defense mechanism of insect hosts at the early stage of pathogen infection. The present study attempts to determine whether apoptosis is involved in defending the fire ant, Solenopsis invicta from a natural viral pathogen Solenopsis invicta virus 1 (SINV-1). Results of TEM examination and TUNEL assay both revealed the signature of apoptosis in the midgut epithelium of SINV-1-infected fire ant larvae. A time-course study was conducted to monitor changes in the dynamics of SINV-1 viral titers and apoptosis levels in the midgut epithelium of SINV-1-infected larvae. We found that the viral titer significantly decreases as apoptosis level increases, suggesting that the apoptotic epithelium constitutes a barrier against dissemination of SINV-1. These findings serve as the very first empirical evidence for the virus-induced apoptosis in ants and also help explain some previously observed mortality patterns and behavioral alterations associated with SINV-1 in fire ants.

ICTV Virus Taxonomy Profile: Polycipiviridae.

Polycipiviridae is a family of picorna-like viruses with non-segmented, linear, positive-sense RNA genomes of approximately 10-12 kb. Unusually for viruses within the order Picornavirales, their genomes are polycistronic, with four (or more) consecutive 5'-proximal open reading frames (ORFs) encoding structural (and possibly other) proteins and a long 3' ORF encoding the replication polyprotein. Members of species within the family have all been detected in ants or via arthropod transcriptomic datasets. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Polycipiviridae, which is available at www.ictv.global/report/polycipiviridae.

Nine new RNA viruses associated with the fire ant Solenopsis invicta from its native range.

The red imported fire ant (Solenopsis invicta) escaped its natural enemies when it was introduced into North America in the 1930s from South America. US efforts have focused on discovery of natural enemies, like viruses, to provide sustainable control of the ant. Nine new virus genomes were sequenced from the invasive fire ant Solenopsis invicta using metagenomic RNA sequencing. The virus genomes were verified by Sanger sequencing and random amplification of cDNA ends reactions. In addition to the nine new virus genomes, the previously described Solenopsis viruses were also detected, including Solenopsis invicta virus 1 (SINV-1), SINV-2, SINV-3, SINV-4, SINV-5, and Solenopsis invicta densovirus. The virus sequences came from S. invicta workers, larvae, pupae, and dead workers taken from midden piles collected from across the ant's native range in Formosa, Argentina. One of the new virus genomes (Solenopsis invicta virus 6) was also detected in populations of North American S. invicta. Phylogenetic analysis of the RNA dependent RNA polymerase, the entire nonstructural polyprotein, and genome characteristics were used to tentatively taxonomically place these new virus genome sequences; these include four new species of Dicistroviridae, one Polycipiviridae, one Iflaviridae, one Totiviridae, and two genome sequences that were too taxonomically divergent to be placed with certainty. The S. invicta virome is the best characterized from any ant species and includes 13 positive-sense, single-stranded RNA viruses (Solenopsis invicta virus 1 to Solenopsis invicta virus 13), one double-stranded RNA virus (Solenopsis midden virus), and one double-stranded DNA virus (Solenopsis invicta densovirus). These new additions to the S. invicta virome offer potentially new classical biological control agents for S. invicta.

Discovery of three RNA viruses using ant transcriptomic datasets.

Three novel RNA viruses, named Formica fusca virus 1 (GenBank accession no. MH477287), Lasius neglectus virus 2 (MH477288) and Myrmica scabrinodis virus 2 (MH477289), were discovered in ants collected in Cambridge, UK. The proposed virus names were given based on the hosts in which they were identified. The genome sequences were obtained using de novo transcriptome assembly of high-throughput RNA sequencing reads and confirmed by Sanger sequencing. Phylogenetic analysis showed that Formica fusca virus 1 grouped within the family Nyamiviridae, Lasius neglectus virus 2 grouped within the family Rhabdoviridae and Myrmica scabrinodis virus 2 belongs to the family Dicistroviridae. All three viruses are highly divergent from previously sequenced viruses.

Viral infections in fire ants lead to reduced foraging activity and dietary changes.

Despite the presence of conserved innate immune function, many insects have evolved a variety of mechanical, chemical, and behavioral defensive responses to pathogens. Illness-induced anorexia and dietary changes are two behavioral defensive strategies found in some solitary insects, but little is known regarding the role of such behaviors in social insects, especially in ants. In the present study we examined if such reduced foraging activity exists for a social insect, the invasive fire ant Solenopsis invicta, and its viral pathogen, Solenopsis invicta virus 1 (SINV-1). Virus-free fire ant colonies were split into two colony fragments, one of which subsequently was inoculated with SINV-1. Four food resources with different macronutrient ratios were presented to both colony fragments. SINV-1-inoculated colony fragments consistently displayed reduced foraging performance (e.g., foraging intensity and recruitment efficiency), a decline in lipid intake, and a shift in dietary preference to carbohydrate-rich foods compared with virus-free fragments. These findings provide the first evidence for virus-induced behavioral responses and dietary shifts in shaping the host-pathogen interactions in fire ants. The findings also suggest a possible mechanism for how fire ant colonies respond to viral epidemics. Potential implications of these behavioral differences for current management strategies are discussed.

Viruses of invasive Argentine ants from the European Main supercolony: characterization, interactions and evolution.

The Argentine ant (Linepithema humile) is a highly invasive pest, yet very little is known about its viruses. We analysed individual RNA-sequencing data from 48 Argentine ant queens to identify and characterisze their viruses. We discovered eight complete RNA virus genomes - all from different virus families - and one putative partial entomopoxvirus genome. Seven of the nine virus sequences were found from ant samples spanning 7 years, suggesting that these viruses may cause long-term infections within the super-colony. Although all nine viruses successfully infect Argentine ants, they have very different characteristics, such as genome organization, prevalence, loads, activation frequencies and rates of evolution. The eight RNA viruses constituted in total 23 different virus combinations which, based on statistical analysis, were non-random, suggesting that virus compatibility is a factor in infections. We also searched for virus sequences from New Zealand and Californian Argentine ant RNA-sequencing data and discovered that many of the viruses are found on different continents, yet some viruses are prevalent only in certain colonies. The viral loads described here most probably present a normal asymptomatic level of infection; nevertheless, detailed knowledge of Argentine ant viruses may enable the design of viral biocontrol methods against this pest.

Prospecting for viral natural enemies of the fire ant Solenopsis invicta in Argentina.

Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant, Solenopsis invicta Buren in its native range (i.e., Formosa, Argentina) with the ultimate goal of testing and releasing new viral pathogens into U.S. S. invicta populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182 S. invicta colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove S. invicta genes, known S. invicta virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled de novo yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family Dicistroviridae. Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that S. invicta serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with S. invicta. This study expands knowledge of the RNA virome diversity found within S. invicta populations.