The invasive white-footed ant Technomyrmex difficilis has emerged as a rising pest in several regions, yet its invasion dynamics remain underexplored. This species outcompetes native ants and causes agricultural losses by tending pest insects, including aphids and mealybugs. This study provides the first integrated analysis of the species' behavioral, chemical, and genetic variation across Texas and Florida populations. Observations suggest that the recently discovered Texas population of white-footed ants originated from Florida. Microsatellite and mitochondrial DNA analyses revealed low genetic diversity in both populations, with a shared haplotype consistent with the Texas population originating from Florida. STRUCTURE analysis further supported genetic clustering between the two regions. Despite similar within-colony coefficients of relatedness for workers, the populations differed in reproductive strategy: Florida colonies showed signs of inbreeding and high inter-colony aggression, whereas Texas colonies exhibited potential localized outbreeding, low aggression, and more uniform cuticular hydrocarbon profiles. Aggression was positively correlated with chemical divergence but not with genetic differentiation. This study establishes a foundational understanding and highlights the importance of integrating multiple types of data to understand the invasion biology of Technomyrmex difficilis.

Cuticular hydrocarbons (CHCs) are ubiquitous among insects where they form an outer wax layer that helps maintain water balance and prevent desiccation. In social insects, CHCs were subsequently co-opted as semiochemicals in many contexts, including nestmate recognition, which maintains boundaries among competing colonies by ousting non-nestmates. In some ant populations, workers do not discriminate against non-nestmates. This leads to the development of supercolonies, a large network of interconnected nests exchanging unrelated individuals. In this study, we investigate CHC production by workers and their resistance to desiccation in the ant Nylanderia fulva, which exhibits supercolonial behavior within its invasive range in the USA. We found reduced CHC production by workers and increased susceptibility toward desiccation compared to other invasive ants of similar body size. This CHC-poor chemical profile sheds light on the susceptibility of this species to abiotic stress through desiccation with implications for its potential distribution and its development of large supercolonies in its invasive range by impairing nestmate recognition.

Cuticular hydrocarbons (CHCs) are ubiquitous among insects where they form an outer wax layer that helps maintain water balance and prevent desiccation. In social insects, CHCs were subsequently co-opted as semiochemicals in many contexts, including nestmate recognition, which maintains boundaries among competing colonies by ousting non-nestmates. In some ant populations, workers do not discriminate against non-nestmates. This leads to the development of supercolonies, a large network of interconnected nests exchanging unrelated individuals. In this study, we investigate CHC production by workers and their resistance to desiccation in the ant Nylanderia fulva, which exhibits supercolonial behavior within its invasive range in the USA. We found greatly reduced CHC production by workers and increased susceptibility toward desiccation compared to other invasive ants of similar body size. This relative absence of CHCs sheds light on the susceptibility of this species to abiotic stress through desiccation with implications for its potential distribution and its development of large supercolonies in its invasive range by impairing nestmate recognition.

Most population genetic studies concern spatial genetic differentiation, but far fewer aim at analyzing the temporal genetic changes that occur within populations. Vector species, including mosquitoes and biting midges, are often characterized by oscillating adult population densities, which may affect their dispersal, selection, and genetic diversity over time. Here, we used a population of Culicoides sonorensis from a single site in California to investigate short-term (intra-annual) and long-term (inter-annual) temporal variation in genetic diversity over a 3 yr period. This biting midge species is the primary vector of several viruses affecting both wildlife and livestock, thus a better understanding of the population dynamics of this species can help inform epidemiological studies. We found no significant genetic differentiation between months or years, and no correlation between adult populations and the inbreeding coefficient (F-IS). However, we show that repeated periods of low adult abundance during cooler winter months resulted in recurring bottleneck events. Interestingly, we also found a high number of private and rare alleles, which suggests both a large, stable population, as well as a constant influx of migrants from nearby populations. Overall, we showed that the high number of migrants maintains a high level of genetic diversity by introducing new alleles, while this increased diversity is counterbalanced by recurrent bottleneck events potentially purging unfit alleles each year. These results highlight the temporal influences on population structure and genetic diversity in C. sonorensis and provide insight into factors effecting genetic variation that may occur in other vector species with fluctuating populations.

Group genetic diversity is usually associated with a reduced risk of disease outbreak and a slower rate of pathogen transmission. In social insects, multiple mating by queens (polyandry) evolved several times albeit reducing worker’s inclusive fitness. One major hypothesis suggests that polyandry has been selected for to mitigate the risk of outbreak thanks to increased genetic diversity within colonies. We investigated this hypothesis in the ant Cataglyphis mauritanica, in which nestmate workers are produced by several clonal, single-mated queens. Using natural colonies, we correlated genetic diversity with worker survival to a fungal entomopathogen. We further tested whether workers from different paternal lineages (but a common maternal genome) show differential resistance in experimentally single- or multiple-patriline groups, and whether an increased number of patrilines in a group improved disease resistance. We show that workers from distinct patrilines vary in their resistance to a pathogen in single-patriline colonies, but the difference among patrilines disappears when they are mixed in multiple-patriline colonies. Furthermore, pathogen resistance was affected by the number of patrilines in a group, with two- and three-patriline groups being more resistant than single-patriline groups. However, resistance did not differ between groups made of two and three patrilines; similarly, it was not associated with genetic diversity in natural colonies. Overall, our results suggest that collective disease defenses might homogenize workers’ resistance from different patrilines and, thereby, stabilize colony resistance. The occurrence of multiple breeders in insect societies has been hypothesized to be selected for because increased within-colony genetic diversity reduces the risk of severe outbreaks. We show that nestmate workers from distinct paternal lineages vary in their resistance to pathogens when reared in single-patriline groups. However, this difference disappears when workers are mixed in multiple-patriline groups. These results suggest that multiple mating by queens dilutes the deleterious consequences of a single patriline producing only susceptible offspring, rather than directly enhancing pathogen resistance.

In insects, ecological competition has often resulted in phenotypic changes and modifications to foraging areas. In termites—and social insects as a whole—colonies cannot easily escape competition through the relocation of their colony. In these species, the outcomes of inter and intra-specific competition are influenced by different life history traits, such as colony size, breeding system (number and types of reproductives), food preference, tunneling patterns, nest site selection, and antagonism between colonies. Here, we investigated variation in breeding system and spatial distribution among colonies of a higher termite Amitermes parvulus and a subterranean termite Reticulitermes flavipes within an urban landscape. We first developed microsatellite markers as a tool to study these life history traits in A. parvulus . Second, we assessed competitive exclusion or tolerance of A. parvulus and R. flavipes colonies by determining their fine-scale distribution using monitoring stations on a grid site, and their large-scale distribution across an urban landscape. Third, we investigated the breeding system of A. parvulus colonies. We showed that the numerous colonies of R. flavipes inhabiting a restricted area contrast with the few, but spatially expansive colonies of A. parvulus, suggesting these species face different degrees of intra-specific competition. We showed that colonies of A. parvulus frequently merged together, and all of them were likely headed by inbred neotenic reproductives, two characteristics rarely observed in higher termites. Overall, our study revealed drastic differences in colony structure, breeding systems and foraging ranges between the two species. These differences may reflect differences in food preference and food availability between the two species allowing their co-existence within the same urban environment.

BACKGROUND: A frequent goal of pest management strategies targeting social insects is total colony elimination. Insecticidal baits are highly effective at controlling social insect pests, although their ability to provide total colony elimination has only been well studied in a few species. Genetically testing colony elimination in many urban pest ants can be challenging due to indistinct colony boundaries observed in unicolonial, invasive species; however, some pest ants, such as the dark rover ant (Bra-chymyrmex patagonicus), maintain strict colony borders through aggression towards non-nestmates. Each of these distinct colonies can be identified using molecular markers, allowing for the tracking of individual colonies pre- and post-treatment to measure colony density. While counting the number of foraging workers to assess treatment efficacy may suffice in some cases, it offers little insight into the colony-level impacts of a treatment. RESULTS: Using microsatellite markers, distinct rover ant colonies were identified and tracked around residential structures before and after the application of an imidacloprid bait. The number of foraging ants at the treated structures was reduced by an average of 83.0% over a 28-day observation period. Baiting also significantly reduced the total number of colonies present. At the treatment structures, only similar to 25% of the original colonies remained at the end of the study. Colonies with foraging trails <1.5 m from a bait station had a higher chance of being eliminated. CONCLUSION: Using insecticidal baits against B. patagonicus can be highly effective at colony elimination; however, with such small foraging ranges and high colony densities, proper placement is required to ensure enough bait is properly positioned to treat all colonies affecting a structure. (C) 2022 Society of Chemical Industry.

Low dispersal, occurrence of asexual reproduction and geographic discontinuity increase genetic differentiation between populations, which ultimately can lead to speciation. In this work, we used a multidisciplinary framework to characterize the genetic and phenotypic differentiation between and within two cryptic ant species with restricted dispersal, Cataglyphis cursor and C. piliscapa and used behavioral experiments to test for reproductive isolation. Their distribution is segregated by the Rhône River and they have been traditionally distinguished only by hair numbers, although a statistical assessment is still lacking. We found strong genetic (microsatellites, nuclear and mitochondrial sequences), morphological (number of hairs, tibia length, male genitalia) and chemical (cuticular hydrocarbons) differentiation not only between species but also among localities within species. However, inter-specific differentiation was slightly higher than intra-specific differentiation for most markers. Overall, this pattern could either reflect reproductive isolation or could result from a longer period of geographic isolation between species than among localities within species without necessarily involving reproductive isolation. Interestingly, our behavioral experiments showed an absence of mating between species associated to a higher aggressiveness of workers towards heterospecific males. This suggests that sexual selection may, at least partially, fuel reproductive isolation. We also showed that cuticular hydrocarbons, mtDNA sequences and number of hairs provide reliable criteria allowing species discrimination. Overall, this species complex offers a case study to further investigate varying stages of a speciation continuum by estimating reproductive isolation between pairs of localities varying by their level of genetic differentiation.

Social insect colonies are characterized by an efficient division of labor, allowing high-value individuals (i.e., reproductives and brood) to be sheltered from tasks associated with increased risk of pathogen exposure, such as foraging or corpse disposal. This social organization helps limit the transmission of disease throughout the colony. Further, individuals can actively respond to imminent disease threats by altering their behaviors as a means of social immunity. In subterranean termites, although workers typically avoid detected pathogens, they can be attracted to pathogen cues when a nestmate is infected. Infected termites are usually groomed, but they may instead be cannibalized if the infection has already become lethal. The mechanisms governing these changes in behavior are unclear. We set out to examine immediate changes in individual behaviors, investigating the role that the infected individual plays in communicating its infection status to nestmates. We also assessed gradual changes in social organization after the re-introduction of an infected termite to the colony. Our results reveal that infected termites likely do not signal their infection status to nestmates through shaking behaviors and reduced movements, suggesting the occurrence of other mechanisms used in communicating infection. We also found that infected termites do not self-isolate and may travel to the densest part of the colony, where they can potentially benefit from grooming by large groups of nestmates. These results provide new insights into how individual changes in immune behaviors contribute to overall colony health, highlighting that, at early stages of infection, termites favor a rescuing strategy rather than isolation and/or cannibalization.

Social life is usually associated with enhanced propagule pressure, which increases the chance of introducing several individuals during a single introduction event. Social insects are therefore among the most successful invasive species, benefiting from rapid establishment and increased foundation success in new habitats. In termites, propagule pressure may also be increased by the development of reproductive individuals from a small group of foraging workers. This suggests that enhanced exploration activity may increase propagule pressure through an elevated chance of transporting isolated groups of foragers. Here, we analyzed the exploration behavior of three termite species of the Reticulitermes genus, comparing the invasive species Reticulitermes flavipes (testing both native and introduced populations) to the native species Reticulitermes grassei and Reticulitermes lucifugus . Different features representative of the exploration capacity were measured during 48 h, including: the number of tunnels, the length of tunnels, the number of foragers, and the interindividual distance of foragers in a straight line or through tunnels. Our results show that compared to the native Reticulitermes species, R. flavipes foragers from both populations dug more tunnels with a longer total length, and individuals were more spatially dispersed and covered a larger exploration zone. These findings suggest that the enhanced exploration ability of R. flavipes may have played a role in its invasion success, by increasing its propagule pressure through a higher chance of human-mediated transport. In addition, the absence of differences between the native and introduced populations of R. flavipes suggests that the exploration behaviors facilitating the worldwide invasion of this species originated in its native range.

In insects, ecological competition has often resulted in phenotypic changes and modifications to foraging areas. In termites - and social insects as a whole - colonies cannot easily escape competition through the relocation of their colony. In these species, the outcomes of inter and intra-specific competition are influenced by different life history traits, such as colony size, breeding system (number and types of reproductives), food preference, tunneling patterns, nest site selection, and antagonism between colonies. Here, we investigated variation in breeding system and spatial distribution among colonies of a higher termite Amitermes parvulus and a subterranean termite Reticulitermes flavipes within an urban landscape. We first developed microsatellite markers as a tool to study these life history traits in A. parvulus . Second, we assessed competitive exclusion or tolerance of A. parvulus and R. flavipes colonies by determining their fine-scale distribution using monitoring stations on a grid site, and their large-scale distribution across an urban landscape. Third, we investigated the breeding system of A. parvulus colonies. We showed that the numerous colonies of R. flavipes inhabiting a restricted area contrast with the few, but spatially expansive colonies of A. parvulus , suggesting these species face different degrees of intra-specific competition. We showed that colonies of A. parvulus frequently merged together, and all of them were headed by inbred neotenic reproductives, two characteristics rarely observed in higher termites. Overall, our study revealed drastic differences in colony structure, breeding systems and foraging ranges between the two species. These differences may reflect differences in food preference and food availability between the two species allowing their co-existence within the same urban environment.

Social life and lifelong partner commitments are expected to favor thorough partner choice, as an ill-suited partnership may have long-term consequences, adversely affecting the parents and spanning several cohorts of offspring. Here, we used ~1400 termite incipient colonies to estimate the short- and long-term costs of inbreeding upon the survival of the parents over a 15-month period, their productivity, and the resistance of their offspring toward pathogen pressure. We observed that foundation success was not influenced by the relatedness of partners, but by their levels of microbial load. We showed faster growth in inbred colonies with low levels of microbial load, revealing a potential tradeoff between pathogen defense and offspring production. Yet, inbreeding takes its toll later in colony development when offspring from incipient colonies face pathogen pressure. Although the success of a lifetime partnership is initially determined by the partner’s health, the cost of inbreeding in incipient colonies favors outbred colonies reaching maturity.

Summary After humans, social insects represent one of the most complex groups of social organisms, relying on a well-organized communication system among colony members. The transfer of information among individuals is primarily based on cuticular hydrocarbons (CHC). These chemical compounds, produced by all insects, initially evolved to prevent water loss 1 . They were subsequently co-opted as semiochemicals to communicate various types of information. This includes nestmate recognition in social insects 2,3 , enabling different colonies to partition resources by ousting conspecific competitors. In this study, we report the near complete loss of CHC production by workers of the ant Nylanderia fulva . This absence of CHCs is a double-edged sword. It represents a causative agent in the ecological success of this ant species — enabling the development of a large supercolony in its invasive range through limited ability to differentiate nestmates— but increases the risk of suffering ecological stress through desiccation.

Proper vector surveillance relies on the ability to identify species of interest accurately and efficiently, though this can be difficult in groups containing cryptic species. Culicoides Latreille is a genus of small biting flies responsible for the transmission of numerous pathogens to a multitude of vertebrates. Regarding pathogen transmission, the C. variipennis species complex is of particular interest in North America. Of the six species within this group, only C. sonorensis Wirth & Jones is a proven vector of bluetongue virus and epizootic hemorrhagic disease virus. Unfortunately, subtle morphological differences, cryptic species, and mitonuclear discordance make species identification in the C. variipennis complex challenging. Recently, single-nucleotide polymorphism (SNP) analysis enabled discrimination between the species of this group; however, this demanding approach is not practical for vector surveillance. The aim of the current study was to develop a reliable and affordable way of distinguishing between the species within the C. variipennis complex, especially C. sonorensis. Twenty-five putative microsatellite markers were identified using the C. sonorensis genome and tested for amplification within five species of the C. variipennis complex. Machine learning was then used to determine which markers best explain the genetic differentiation between species. This led to the development of a subset of four and seven markers, which were also tested for species differentiation. A total of 21 microsatellite markers were successfully amplified in the species tested. Clustering analyses of all of these markers recovered the same species-level identification as the previous SNP data. Additionally, the subset of seven markers was equally capable of accurately distinguishing between the members of the C. variipennis complex as the 21 microsatellite markers. Finally, one microsatellite marker (C508) was found to be species-specific, only amplifying in the vector species C. sonorensis among the samples tested. These microsatellites provide an affordable way to distinguish between the sibling species of the C. variipennis complex and could lead to a better understanding of the species dynamics within this group. Additionally, after further testing, marker C508 may allow for the identification of C. sonorensis with a single-tube assay, potentially providing a powerful new tool for vector surveillance in North America.

The inability to distinguish between species can be a serious problem in groups responsible for pathogen transmission. Culicoides biting midges transmit many pathogenic agents infecting wildlife and livestock. In North America, the C. variipennis species complex contains three currently recognized species, only one of which is a known vector, but limited species-specific characters have hindered vector surveillance. Here, genomic data were used to investigate population structure and genetic differentiation within this species complex. Single nucleotide polymorphism data were generated for 206 individuals originating from 17 locations throughout the United States and Canada. Clustering analyses suggest the occurrence of two additional cryptic species within this complex. All five species were significantly differentiated in both sympatry and allopatry. Evidence of hybridization was detected in three different species pairings indicating incomplete reproductive isolation. Additionally, COI sequences were used to identify the hybrid parentage of these individuals, which illuminated discordance between the divergence of the mitochondrial and nuclear datasets.

We investigated the use of termite baiting, a proven system of targeted colony elimination, in an overall area-wide control strategy against subterranean termites. At two field sites, we used microsatellite markers to estimate the total number of Reticulitermes colonies, their spatial partitioning, and breeding structure. Termite pressure was recorded for two years before and after the introduction of Trelona® (active ingredient novaluron) to a large area of one of the sites. Roughly 70% of the colonies in the treatment site that were present at the time of baiting were not found in the site within two months after the introduction of novaluron. Feeding activity of the remaining colonies subsequently ceased over time and new invading colonies were unable to establish within this site. Our study provides novel field data on the efficacy of novaluron in colony elimination of Reticulitermes flavipes, as well as evidence that an area-wide baiting program is feasible to maintain a termite-free area within its native range.

Native to eastern Asia, the Formosan subterranean termite Coptotermes formosanus (Shiraki) is recognized as one of the 100 worst invasive pests in the world, with established populations in Japan, Hawaii and the southeastern United States. Despite its importance, the native source(s) of C. formosanus introductions and their invasive pathway out of Asia remain elusive. Using ~22,000 SNPs, we retraced the invasion history of this species through approximate Bayesian computation and assessed the consequences of the invasion on its genetic patterns and demography. We show a complex invasion history, where an initial introduction to Hawaii resulted from two distinct introduction events from eastern Asia and the Hong Kong region. The admixed Hawaiian population subsequently served as the source, through a bridgehead, for one introduction to the southeastern US. A separate introduction event from southcentral China subsequently occurred in Florida showing admixture with the first introduction. Overall, these findings further reinforce the pivotal role of bridgeheads in shaping species distributions in the Anthropocene and illustrate that the global distribution of C. formosanus has been shaped by multiple introductions out of China, which may have prevented and possibly reversed the loss of genetic diversity within its invasive range.

The level of gene flow between diverging lineages ultimately determines the outcome of a speciation event. If secondary contact occurs before this process is complete, reproductive isolation barriers must exist or evolve to prevent hybridization. The selective pressures facilitating and maintaining genetic divergence do not always involve an observable phenotypic response, thus cryptic species form. The inability to distinguish between sibling species can be a particularly serious problem in groups responsible for pathogen transmission. Culicoides biting midges occur almost world-wide and vector many disease-causing pathogens that affect wildlife and livestock. In North America, the C. variipennis species complex contains three currently recognized species, only one of which is a vector, and limited molecular and morphological differences have hindered vector surveillance. Here, genomic methods were used to investigate speciation and genetic structure within this complex. Single nucleotide polymorphism (SNP) data were generated using ddRAD sequencing for 206 individuals originating from 17 locations throughout the United States and Canada. Clustering analyses consistently suggest the occurrence of five putative species with significant differentiation occurring in both sympatric and allopatric populations. Evidence of hybridization was detected in three different species pairings, indicating a lack of pre-zygotic reproductive isolation within the complex. Mitochondrial genes were used to trace the hybrid parentage of these individuals, which illuminated discordance with the SNP data. In this study, we highlight the potential role of geographic, ecological, and behavioral isolation in speciation and in maintaining species boundaries, despite hybridization and long range dispersal.

Biological invasions are becoming more prevalent due to the rise of global trade and expansion of urban areas. Ants are among the most prolific invaders, with many exhibiting a multi-queen colony structure, dispersal through budding and a lack of inter-nest aggression. Although these characteristics are generally associated with the invasions of exotic ants, they may also facilitate the spread of native ants into novel habitats (e.g., urban areas). Native to North American forests, the odorous house ant Tapinoma sessile has become abundant in urban environments throughout the United States. Forest-dwelling colonies typically have a small workforce, inhabit a single nest, and are headed by a single queen, whereas urban colonies tend to be several orders of magnitude larger, inhabit multiple nests and are headed by multiple queens. Here, we explore and compare the population genetic and breeding structure of T. sessile within and between urban and natural environments in several localities across its distribution range. We found the social structure of a colony to be a plastic trait in both habitats, although extreme polygyny (i.e., nests with multiple queens) was confined to urban habitats. Additionally, polydomous colonies (i.e., nests lacking genetic differentiation and behavioral antagonism) were only present in urban habitats, suggesting T. sessile can only achieve unicoloniality within urbanized areas. Finally, we identified strong differentiation between urban and natural populations in each locality and continent-wide, indicating cities may restrict gene flow and exert intense selection pressure. Overall, our study highlights urbanization’s influence in charting the evolutionary course for species.

The production of royal pheromones by reproductives (queens and kings) enables social insect colonies to allocate individuals into reproductive and non-reproductive roles. In many termite species, nestmates can develop into neotenics when the primary king or queen dies, which then inhibit the production of additional reproductives. This suggests that primary reproductives and neotenics produce royal pheromones. The cuticular hydrocarbon heneicosane was identified as a royal pheromone in Reticulitermes flavipes neotenics. Here, we investigated the presence of this and other cuticular hydrocarbons in primary reproductives and neotenics of this species, and the ontogeny of their production in primary reproductives. Our results revealed that heneicosane was produced by most neotenics, raising the question of whether reproductive status may trigger its production. Neotenics produced six additional cuticular hydrocarbons absent from workers and nymphs. Remarkably, heneicosane and four of these compounds were absent in primary reproductives, and the other two compounds were present in lower quantities. Neotenics therefore have a distinct ‘royal’ blend from primary reproductives, and potentially over-signal their reproductive status. Our results suggest that primary reproductives and neotenics may face different social pressures. Future studies of these pressures should provide a more complete understanding of the mechanisms underlying social regulation in termites.