Positive selection has shaped the evolution of Argentine ant immune genes both in native and introduced supercolonies.

The highly invasive Argentine ant (Linepithema humile) started its colonisation from the species' native range in South America approximately 150 years ago and has since become one of the major pests in the world. We investigated how the shifts into new ranges have affected the evolution of Argentine ants' immune genes. To the best of our knowledge, this is the first broadscale population genetic study focusing on ants' immune genes. We analysed comprehensive targeted-seq data of immune and non-immune genes containing 174 genes from 18 Argentine ant supercolonies covering the species' native and introduced ranges. We predicted that the immune gene evolution of introduced supercolonies differs from that of the native supercolonies and proposed two different, non-mutually exclusive hypotheses for this: 1) the enemy release hypothesis and 2) the higher pathogen pressure hypothesis - both of which seem to explain the observed evolutionary patterns on their behalf. Our results show that the introduced supercolonies were targeted by weaker selection than natives, but positive selection was evident among supercolonies of both ranges. Moreover, in some cases, such as the antiviral RNAi genes, introduced range supercolonies harboured a higher proportion of positively selected genes than natives. This observation was striking, knowing the recent demographic history and the detected generally lower selection efficacy of introduced supercolonies. In conclusion, it is evident that pathogen pressure is ubiquitous and strongly affects the immune gene evolution in Argentine ants.

Antiviral immune response reveals host-specific virus infections in natural ant populations.

Hosts can carry many viruses in their bodies, but not all of them cause disease. We studied ants as a social host to determine both their overall viral repertoire and the subset of actively infecting viruses across natural populations of three subfamilies: the Argentine ant (Linepithema humile, Dolichoderinae), the invasive garden ant (Lasius neglectus, Formicinae) and the red ant (Myrmica rubra, Myrmicinae). We used a dual sequencing strategy to reconstruct complete virus genomes by RNA-seq and to simultaneously determine the small interfering RNAs (siRNAs) by small RNA sequencing (sRNA-seq), which constitute the host antiviral RNAi immune response. This approach led to the discovery of 41 novel viruses in ants and revealed a host ant-specific RNAi response (21 vs. 22 nt siRNAs) in the different ant species. The efficiency of the RNAi response (sRNA/RNA read count ratio) depended on the virus and the respective ant species, but not its population. Overall, we found the highest virus abundance and diversity per population in Li. humile, followed by La. neglectus and M. rubra. Argentine ants also shared a high proportion of viruses between populations, whilst overlap was nearly absent in M. rubra. Only one of the 59 viruses was found to infect two of the ant species as hosts, revealing high host-specificity in active infections. In contrast, six viruses actively infected one ant species, but were found as contaminants only in the others. Disentangling spillover of disease-causing infection from non-infecting contamination across species is providing relevant information for disease ecology and ecosystem management.

Genetic modification of the ant Lasius niger using CRISPR-Cas9 technology.

CRISPR-Cas9 has become one of the most prominent gene editing tools available and it has been utilized in various organisms from bacteria to fungi, plants, and animals. In this study, we developed a CRISPR-Cas9 protocol for the black garden ant Lasius niger, a common and easily available study species for lab and field experiments. To create indel mutations using CRISPR-Cas9 in L. niger, we targeted three different locations in a well-studied eye pigmentation gene cinnabar, generating several mutations that disrupt the ommochrome biosynthesis pathway and result in the lack of the pigment and therefore, abnormal eye coloration in adult workers. We also developed a protocol to collect L. niger eggs, inject them with CRISPR-Cas9 construct, and rear the eggs into mature adult workers with the assistance of nursing workers. We demonstrated for the first time in L. niger that CRISPR-Cas9 is an excellent tool to create targeted mutations for this species. Our protocol can be referred to when developing similar studies for other species of ants and eusocial insects.

Identification and characterisation of common glow-worm RNA viruses.

The common glow-worms (Lampyris noctiluca) are best known for emission of green light by their larvae and sexually active adult females. However, both their DNA and RNA viruses remain unknown. Glow-worms are virologically interesting, as they are non-social and do not feed as adults, and hence their viral transmission may be limited. We identified viral sequences from 11 different virus taxa by the RNA-sequencing of two Finnish populations of adult glow-worms. The viruses represent nine different virus families and have negative, positive, or double-stranded RNA genomes. We also found a complete retroviral genome. Similar viral sequences were found from the sequencing data of common eastern firefly of North America, a species belonging to the same family (Lampyridae) as that of the common glow-worm. On average, an individual glow-worm had seven different RNA virus types and most of them appeared to establish a stable infection since they were found from glow-worms during two consecutive years. Here we present the characterization of load, prevalence, and interactions for each virus. Most of the glow-worm RNA viruses seem to be transmitted vertically, which may reflect the biology of glow-worms as non-social capital breeders, i.e., they invest stored resources in reproduction.

Social environment affects the transcriptomic response to bacteria in ant queens.

Social insects have evolved enormous capacities to collectively build nests and defend their colonies against both predators and pathogens. The latter is achieved by a combination of individual immune responses and sophisticated collective behavioral and organizational disease defenses, that is, social immunity. We investigated how the presence or absence of these social defense lines affects individual-level immunity in ant queens after bacterial infection. To this end, we injected queens of the ant Linepithema humile with a mix of gram+ and gram- bacteria or a control solution, reared them either with workers or alone and analyzed their gene expression patterns at 2, 4, 8, and 12 hr post-injection, using RNA-seq. This allowed us to test for the effect of bacterial infection, social context, as well as the interaction between the two over the course of infection and raising of an immune response. We found that social isolation per se affected queen gene expression for metabolism genes, but not for immune genes. When infected, queens reared with and without workers up-regulated similar numbers of innate immune genes revealing activation of Toll and Imd signaling pathways and melanization. Interestingly, however, they mostly regulated different genes along the pathways and showed a different pattern of overall gene up-regulation or down-regulation. Hence, we can conclude that the absence of workers does not compromise the onset of an individual immune response by the queens, but that the social environment impacts the route of the individual innate immune responses.

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.

Evolutionary genetics of insect innate immunity.

Patterns of evolution in immune defense genes help to understand the evolutionary dynamics between hosts and pathogens. Multiple insect genomes have been sequenced, with many of them having annotated immune genes, which paves the way for a comparative genomic analysis of insect immunity. In this review, I summarize the current state of comparative and evolutionary genomics of insect innate immune defense. The focus is on the conserved and divergent components of immunity with an emphasis on gene family evolution and evolution at the sequence level; both population genetics and molecular evolution frameworks are considered.

Social insect genomes exhibit dramatic evolution in gene composition and regulation while preserving regulatory features linked to sociality.

Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ∼4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared with Drosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor-binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the "socio-genomes" of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations.

Draft genome of the globally widespread and invasive Argentine ant (Linepithema humile).

Ants are some of the most abundant and familiar animals on Earth, and they play vital roles in most terrestrial ecosystems. Although all ants are eusocial, and display a variety of complex and fascinating behaviors, few genomic resources exist for them. Here, we report the draft genome sequence of a particularly widespread and well-studied species, the invasive Argentine ant (Linepithema humile), which was accomplished using a combination of 454 (Roche) and Illumina sequencing and community-based funding rather than federal grant support. Manual annotation of >1,000 genes from a variety of different gene families and functional classes reveals unique features of the Argentine ant's biology, as well as similarities to Apis mellifera and Nasonia vitripennis. Distinctive features of the Argentine ant genome include remarkable expansions of gustatory (116 genes) and odorant receptors (367 genes), an abundance of cytochrome P450 genes (>110), lineage-specific expansions of yellow/major royal jelly proteins and desaturases, and complete CpG DNA methylation and RNAi toolkits. The Argentine ant genome contains fewer immune genes than Drosophila and Tribolium, which may reflect the prominent role played by behavioral and chemical suppression of pathogens. Analysis of the ratio of observed to expected CpG nucleotides for genes in the reproductive development and apoptosis pathways suggests higher levels of methylation than in the genome overall. The resources provided by this genome sequence will offer an abundance of tools for researchers seeking to illuminate the fascinating biology of this emerging model organism.

The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle.

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.

Draft genome of the red harvester ant Pogonomyrmex barbatus.

We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.

Rapid evolution of immune proteins in social insects.

The existence of behavioral traits connected to defense against pathogens manifests the importance of pathogens in the evolution of social insects. However, very little is known about how pathogen pressure has affected the molecular evolution of genes involved in their innate immune system. We have studied the sequence evolution of several immune genes in ants and honeybees. The results show high rates of evolution in both ants and honeybees as measured by the ratio of amino acid changes to silent nucleotide changes, the ratio being clearly higher than in Drosophila immune genes or in nonimmunity genes of bees. This conforms to our expectations based on high pathogen pressure in social insects. The codon-based likelihood method found clear evidence of positive selection only in one ant gene, even though positive selection has earlier been found in both ant and termite immune genes. There is now indication that selection on the amino acid composition of the immune-related genes has been an important part in the fight against pathogens by social insects. However, we cannot distinguish in all the cases whether the high observed d(N)/d(S) ratio results from positive selection within a restricted part of the studied genes or from relaxation of purifying selection associated with effective measures of behaviorally based colony-level defenses.

Selection on an antimicrobial peptide defensin in ants.

Ants live in crowded nests with interacting individuals, which makes them particularly prone to infectious diseases. The question is, how do ants cope with the increased risk of pathogen transmission due to sociality? We have studied the molecular evolution of defensin, a gene encoding an antimicrobial protein, in ants. Defensin sequences from several ant species were analyzed with maximum likelihood models of codon substitution to infer selection. Positive selection was detected in the mature region of defensin, whereas the signal and pro regions seem to be evolving neutrally. We also found a significantly higher rate of nonsynonymous substitutions in some phylogenetic lineages, as well as dN/dS >1, suggesting varying selection pressures in different lineages. Earlier studies on the molecular evolution of insect antimicrobial peptide genes have focused on termites and dipteran species, and detected positive selection only in duplicated termicin genes in termites. These findings, together with our present results, provide an indication that the immune systems of social insects (ants and termites) and dipteran insects may have responded differently to the selection pressure caused by microbial pathogens.

Wolbachia transmission dynamics in Formica wood ants.

BACKGROUND: The role of Wolbachia endosymbionts in shaping the mitochondrial diversity of their arthropod host depends on the effects they have on host reproduction and on the mode of transmission of the bacteria. We have compared the sequence diversity of wsp (Wolbachia surface protein gene) and the host mtDNA in a group of Formica ant species that have diverged approximately 0.5 million years ago (MYA). The aim was to study the relationship of Wolbachia and its ant hosts in terms of vertical and horizontal transmission of the bacteria. RESULTS: All studied ant species were doubly infected with two Wolbachia strains (wFex1 and wFex4) all over their geographical distribution area in Eurasia. The most common haplotypes of these strains were identical with strains previously described from a more distantly related Formica ant, with an estimated divergence time of 3.5 - 4 MYA. Some strain haplotypes were associated to the same or closely related mtDNA haplotypes as expected under vertical transmission. However, in several cases the wsp haplotypes coexisted with distant mtDNA haplotypes, a pattern which is more compatible with horizontal transmission of the bacteria. CONCLUSION: Two lines of evidence suggest that the sharing of Wolbachia strains by all F. rufa species is rather due to horizontal than vertical transmission. First, the fact that endosymbiont strains identical to those of F. rufa ants have been found in another species that diverged 3.5-4 MYA strongly suggests that horizontal transfer can and does occur between Formica ants. Second, the frequent sharing of identical Wolbachia strains by distant mitochondrial lineages within the F. rufa group further shows that horizontal transmission has occurred repeatedly. Nevertheless, our dataset also provides some evidence for longer-term persistence of infection, indicating that Wolbachia infection within this host clade has been shaped by both horizontal and vertical transmission of symbionts. The fact that all the ants were infected irrespective of the family structure of their societies gives no support to the proposed hypotheses that the spreading of Wolbachia in ants might be associated to the types of their societies.

Exceptionally high density of NUMTs in the honeybee genome.

The available genome sequences of 4 insects (the fruit fly, the African malaria mosquito, the flour beetle, and the honeybee) are used to compare the amount of mitochondrial DNA transferred to the nuclear genome (NUMTs). The data from the beetle and the bee show frequent transfer of NUMTs, whereas NUMTs in the 2 other insects are rare. The density of NUMTs in the honeybee (>1.0 bp transferred DNA per 1 kb of the nuclear sequence) is the highest in any animal studied, about ten times higher than in humans and comparable to the densities in plant genomes. The density of NUMTs in the beetle (0.056 bp/kb) is of the same order of magnitude as that in humans. The analysis of the honeybee genome indicates that NUMTs originate from all parts of the mitochondrial genome, that about two-thirds of the nuclear copies result from secondary transpositions within the nuclear genome, that the copies are significantly associated to "mariner" type transposons, and that the NUMTs consist mainly of short and fragmented copies.