An invasive ant increases deformed wing virus loads in honey bees.

The majority of invasive species are best known for their effects as predators. However, many introduced predators may also be substantial reservoirs for pathogens. Honey bee-associated viruses are found in various arthropod species including invasive ants. We examined how the globally invasive Argentine ant (Linepithema humile), which can reach high densities and infest beehives, is associated with pathogen dynamics in honey bees. Viral loads of deformed wing virus (DWV), which has been linked to millions of beehive deaths around the globe, and black queen cell virus significantly increased in bees when invasive ants were present. Microsporidian and trypanosomatid infections, which are more bee-specific, were not affected by ant invasion. The bee virome in autumn revealed that DWV was the predominant virus with the highest infection levels and that no ant-associated viruses were infecting bees. Viral spillback from ants could increase infections in bees. In addition, ant attacks could pose a significant stressor to bee colonies that may affect virus susceptibility. These viral dynamics are a hidden effect of ant pests, which could have a significant impact on disease emergence in this economically important pollinator. Our study highlights a perhaps overlooked effect of species invasions: changes in pathogen dynamics.

Can immune gene silencing via dsRNA feeding promote pathogenic viruses to control the globally invasive Argentine ant?

Pest control methods that can target pest species with limited environmental impacts are a conservation and economic priority. Species-specific pest control using RNA interference is a challenging but promising avenue in developing the next generation of pest management. We investigate the feasibility of manipulating a biological invader's immune system using double-stranded RNA (dsRNA) in order to increase susceptibility to naturally occurring pathogens. We used the invasive Argentine ant as a model, targeting the immunity-associated genes Spaetzle and Dicer-1 with dsRNA. We show that feeding with Spaetzle dsRNA can result in partial target gene silencing for up to 28 days in the laboratory and 5 days in the field. Dicer-1 dsRNA only resulted in partial gene knockdown after 2 days in the laboratory. Double-stranded RNA treatments were associated with significant gene expression disruptions across immune pathways in the laboratory and to a lower extent in the field. In total, 12 viruses and four bacteria were found in these ant populations. Some changes in viral loads in dsRNA-treated groups were observed. For example, Linepithema humile Polycipivirus 2 (LhuPCV2) loads increased after 2 days of treatment with Spaetzle and Dicer-1 dsRNA treatments in the laboratory. After treatment with the dsRNA in the field, after 5 days the virus Linepithema humile toti-like virus 1 (LhuTLV1) was significantly more abundant. However, immune pathway disruption did not result in a consistent increase in microbial infections, nor did it alter ant abundance in the field. Some viruses even declined in abundance after dsRNA treatment. Our study explored the feasibility of lowering a pest's immunity as a control tool. We demonstrate that it is possible to alter immune gene expression of pest species and pathogen loads, although in our specific system the affected pathogens did not appear to influence pest abundance. We provide suggestions on future directions for dsRNA-mediated immune disruption in pest species, including potential avenues to improve dsRNA delivery as well as the importance of pest and pathogen biology. Double-stranded RNA targeting immune function might be especially useful for pest control in systems in which viruses or other microorganisms are prevalent and have the potential to be pathogenic.

Viral communities in the parasite Varroa destructor and in colonies of their honey bee host (Apis mellifera) in New Zealand.

The parasitic mite Varroa destructor is a leading cause of mortality for Western honey bee (Apis mellifera) colonies around the globe. We sought to confirm the presence and likely introduction of only one V. destructor haplotype in New Zealand, and describe the viral community within both V. destructor mites and the bees that they parasitise. A 1232 bp fragment from mitochondrial gene regions suggests the likely introduction of only one V. destructor haplotype to New Zealand. Seventeen viruses were found in bees. The most prevalent and abundant was the Deformed wing virus A (DWV-A) strain, which explained 95.0% of the variation in the viral community of bees. Black queen cell virus, Sacbrood virus, and Varroa destructor virus 2 (VDV-2) played secondary roles. DWV-B and the Israeli acute paralysis virus appeared absent from New Zealand. Ten viruses were observed in V. destructor, with > 99.9% of viral reads from DWV-A and VDV-2. Substantially more variation in viral loads was observed in bees compared to mites. Where high levels of VDV-2 occurred in mites, reduced DWV-A occurred in both the mites and the bees co-occurring within the same hive. Where there were high loads of DWV-A in mites, there were typically high viral loads in bees.

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.

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.

Behavioural variation and plasticity along an invasive ant introduction pathway.

Once established in new areas, introduced species may exhibit changes in their biology due to phenotypic plasticity, novel selection pressures and genetic drift. Moreover, the introduction process itself has been hypothesised to act as a selective filter for traits that promote invasiveness. We tested the hypothesis that behaviours thought to promote invasiveness-such as increased foraging activity and aggression-are selected for during invasion by comparing traits among native and introduced populations of the widespread Argentine ant (Linepithema humile). We studied Argentine ant populations in the native range in Argentina and in three invaded regions along an introduction pathway: California, Australia and New Zealand. In each region, we set up 32 experimental colonies to measure foraging activity and interspecific aggression in a subset of the study regions. These colonies were subject to experimental manipulation of carbohydrate availability and octopamine, a biogenic amine known to modulate behaviour in insects, to measure variation in behavioural plasticity. We found variation in foraging activity among populations, but this variation was not consistent with selection on behaviour in relation to the invasion process. We found that colonies with limited access to carbohydrates exhibited unchanged exploratory behaviour, but higher exploitation activity and lower aggression. Colonies given octopamine consistently increased foraging behaviour (both exploration and exploitation), as well as aggression when also sugar-deprived. There was no difference in the degree of behavioural response to our experimental treatments along the introduction pathway. We did not find support for selection of behavioural traits associated with invasiveness along the Argentine ant's introduction pathway or clear evidence for an association between the introduction process and variation in behavioural plasticity. These results indicate that mechanisms promote behavioural variation in a similar fashion both in native and introduced ranges. Our results challenge the assumption that introduced populations always perform better in key behavioural traits hypothesised to be associated with invasion success.

Ergonomics of load transport in the seed harvesting ant Messor barbarus: morphology influences transportation method and efficiency.

We studied in the field the load transport behavior of workers of the polymorphic Mediterranean seed harvester ant Messor barbarus Individual ants used two different methods to transport food items: carrying and dragging. The probability of dragging instead of carrying varied significantly with both the mass of the item transported and its linear dimension. Moreover, the values of item mass and length at which dragging began to occur increased with increasing size of the workers. However, larger ants began dragging at decreasing values of the relative mass represented by the items transported, which reflects different biomechanical constraints resulting from allometric relationships between the different parts of their body. Transport rate was significantly higher in large ants but varied in the same way for workers of different sizes with the relative mass of the item transported. Nevertheless, although large ants were individually more efficient than small ants in transporting food items, the relative transport rate, defined as the ratio of transport rate to the mass of the ant, was higher for small ants than for large ants. Colonies should thus have a greater benefit in investing in small ants than in large ants for the transport of food items. This may explain why the proportion of large ants is so small on the foraging columns of M. barbarus and why large ants are most often employed in colonies for tasks other than transporting food items.

Learning and discrimination of cuticular hydrocarbons in a social insect.

Social insect cuticular hydrocarbon (CHC) mixtures are among the most complex chemical cues known and are important in nest-mate, caste and species recognition. Despite our growing knowledge of the nature of these cues, we have very little insight into how social insects actually perceive and discriminate among these chemicals. In this study, we use the newly developed technique of differential olfactory conditioning to pure, custom-designed synthetic colony odours to analyse signal discrimination in Argentine ants, Linepithema humile. Our results show that tri-methyl alkanes are more easily learned than single-methyl or straight-chain alkanes. In addition, we reveal that Argentine ants can discriminate between hydrocarbons with different branching patterns and the same chain length, but not always between hydrocarbons with the same branching patterns but different chain length. Our data thus show that biochemical characteristics influence those compounds that ants can discriminate between, and which thus potentially play a role in chemical signalling and nest-mate recognition.