Hybridization and invasiveness in social insects - The good, the bad and the hybrid.

Hybridization may help drive biological invasions by reducing Allee effects, increasing genetic variation, and generating novel adaptive genotypes/phenotypes. Social insects (ants, bees, wasps, and termites) are among the world's worst invasive species. In this review, we study the relationship between hybridization and invasiveness in social insects. We examine three types of hybridization based on the reproductive characteristics of first-generation hybrids. We discuss several examples of the association between hybridization and invasiveness, which are predominantly found in bees and termites. However, hybridization also occurs in several non-invasive species, and highly invasive species are not consistently associated with hybridization events, indicating that hybridization is not a main driver of invasiveness in social insects. We discuss why hybridization is not more commonly seen in invasive social insects.

Nest composition, stable isotope ratios and microbiota unravel the feeding behaviour of an inquiline termite.

Termites are eusocial insects having evolved several feeding, nesting and reproductive strategies. Among them, inquiline termites live in a nest built by other termite species: some of them do not forage outside the nest, but feed on food stored by the host or on the nest material itself. In this study, we characterized some dimensions of the ecological niche of Cavitermes tuberosus (Termitidae: Termitinae), a broad-spectrum inquiline termite with a large neotropical distribution, to explain its ecological success. We used an integrative framework combining ecological measures (physico-chemical parameters, stable isotopic ratios of N and C) and Illumina MiSeq sequencing of 16S rRNA gene to identify bacterial communities and to analyse termites as well as the material from nests constructed by different termite hosts (the builders). Our results show that (1) nests inhabited by C. tuberosus display a different physico-chemical composition when compared to nests inhabited by its builder alone; (2) stable isotopic ratios suggest that C. tuberosus feeds on already processed, more humified, nest organic matter; and (3) the gut microbiomes cluster by termite species, with the one of C. tuberosus being much more diverse and highly similar to the one of its main host, Labiotermes labralis. These results support the hypothesis that C. tuberosus is a generalist nest feeder adapted to colonize nests built by various builders, and explain its ecological success.

Widespread occurrence of asexual reproduction in higher termites of the Termes group (Termitidae: Termitinae).

BACKGROUND: A decade ago, the mixed reproductive strategy Asexual Queen Succession (AQS) was first described in termites. In AQS species, the workers, soldiers and dispersing reproductives are produced through sexual reproduction, while non-dispersing (neotenic) queens arise through automictic thelytokous parthenogenesis, replace the founding queen and mate with the founding king. As yet, AQS has been documented in six species from three lineages of lower (Rhinotermitidae) and higher (Termitinae: Termes group and Syntermitinae) termites. Independent evolution of the capacity of thelytoky as a preadaptation to AQS is supported by different mechanisms of automixis in each of the three clades. These pioneering discoveries prompt the question on the extent of thelytoky and AQS in the diversified family of higher termites. RESULTS: Here, we investigated the capacity of thelytoky and occurrence of AQS in three species from the phylogenetic proximity of the neotropical AQS species Cavitermes tuberosus (Termitinae: Termes group): Palmitermes impostor, Spinitermes trispinosus, and Inquilinitermes inquilinus. We show that queens of all three species are able to lay unfertilized eggs, which undergo thelytokous parthenogenesis (via gamete duplication as in C. tuberosus) and develop through the transitional stage of aspirants into replacement neotenic queens. CONCLUSIONS: The breeding system in P. impostor is very reminiscent of that described in C. tuberosus and can be characterized as AQS. In the remaining two species, our limited data do not allow classifying the breeding system as AQS; yet, also in these species the thelytokous production of neotenic females appears to be a systematic element of reproductive strategies. It appears likely that the capacity of thelytokous parthenogenesis evolved once in the Termes group, and may ultimately be found more widely, well beyond these Neotropical species.

Bacteriome-associated Wolbachia of the parthenogenetic termite Cavitermes tuberosus.

Wolbachia has deeply shaped the ecology and evolution of many arthropods, and interactions between the two partners are a continuum ranging from parasitism to mutualism. Non-dispersing queens of the termite Cavitermes tuberosus are parthenogenetically produced through gamete duplication, a mode of ploidy restoration generally induced by Wolbachia. These queens display a bacteriome-like structure in the anterior part of the mesenteron. Our study explores the possibility of a nutritional mutualistic, rather than a parasitic, association between Wolbachia and C. tuberosus. We found a unique strain (wCtub), nested in the supergroup F, in 28 nests collected in French Guiana, the island of Trinidad and the state of Paraíba, Brazil (over 3500 km). wCtub infects individuals regardless of caste, sex or reproductive (sexual versus parthenogenetic) origin. qPCR assays reveal that Wolbachia densities are higher in the bacteriome-like structure and in the surrounding gut compared to other somatic tissues. High-throughput 16S rRNA gene amplicon sequencing reveals that Wolbachia represents over 97% of bacterial reads present in the bacteriome structure. BLAST analyses of 16S rRNA, bioA (a gene of the biosynthetic pathway of B vitamins) and five multilocus sequence typing genes indicated that wCtub shares 99% identity with wCle, an obligate nutritional mutualist of the bedbug Cimex lectularius.

Secondary queens in the parthenogenetic termite Cavitermes tuberosus develop through a transitional helper stage.

In termite species with asexual queen succession (AQS), parthenogenetically produced immatures mostly differentiate into secondary queens, called "neotenics." In order to elucidate the ontogenetic origin of neotenics in Cavitermes tuberosus (Termitidae: Termitinae), a neotropical termite with AQS, we investigated developmental pathways of offspring according to their sex and genetic origin using both morphometric and genetic tools. The caste system of C. tuberosus follows the classical pathway of Termitidae. After the first larval instar, there is a bifurcation between two developmental lines. The apterous line is composed of a second larval instar, several worker instars, presoldiers, and soldiers. Workers display a consistent male bias and soldiers are female-only. The nymphal line is composed of five nymphal instars and the imago stage. We highlight that neotenic queens derive from third and fourth instar nymphs displaying peculiar morphological traits, here termed "aspirants," most of which are produced by parthenogenesis. Aspirants are present in all nests and perform worker tasks while waiting for the queen's death to differentiate into neotenic queens. Aspirants can successfully be used to demonstrate the occurrence of parthenogenesis in termite species whose reproductive cores are difficult to access.

An ultra performance liquid chromatography-tandem mass spectrometry method for the therapeutic drug monitoring of isavuconazole and seven other antifungal compounds in plasma samples.

A new analytical method was developed for the routine Therapeutic Drug Monitoring of 8 antifungals compounds in 50µL of plasma: isavuconazole (ISZ), voriconazole (VRZ), posaconazole (PSZ), fluconazole (FCZ), caspofungin (CSF), flucytosine (5FC), itraconazole (ITZ) and its metabolite OH-itraconazole (OH-ITZ). After adding 50µL of the internal standard, which consisted in a mixture of the deuterated isotopes of the quantified compounds, the sample treatment consisted in a simple protein precipitation with 400µL of acetonitrile. Five microliters of the supernatant were directly injected into the chromatographic system. The chromatographic separation was performed with a Waters C18-BEH column and a mobile phase consisting in a mixture of water and acetonitrile, both containing 0.1% of formic acid. The total run time was 3min and the detection of the analytes was performed by electrospray ionization in a positive mode using selected reaction monitoring. Intra and inter-day precision and inaccuracy were <15% over the calibration ranges that were determined according to their clinical relevance: 0.20-20.0mg/L for ISZ, VRZ, PSZ, ITZ, and OH-ITZ; 0.50-50.0mg/L for FCZ and CSF; 2.00-200mg/L for 5FC. This simple and fast method was found suitable for routine therapeutic drug monitoring.

Facultative asexual reproduction and genetic diversity of populations in the humivorous termite Cavitermes tuberosus.

Termite colonies are typically founded by a pair of sexually reproducing dispersers, which can sometimes be replaced by some of their offspring. Some Reticulitermes and Embiratermes species routinely practice asexual queen succession (AQS): the queen is replaced by neotenic daughters produced by parthenogenesis, which mate with the primary king. Here, to cast light on the evolution of AQS, we investigated another candidate species, Cavitermes tuberosus (Termitinae). Of 95 nests, 39 contained a primary queen and 28 contained neotenic females (2-667 individuals), usually with the primary king. Microsatellite analyses confirmed that colonies were initiated by single pairs after large dispersal flights. More than 80% of the neotenic females were of exclusively maternal origin and completely homozygous, suggesting automictic parthenogenesis with gamete duplication. Conversely, workers, soldiers, and most alates and primary reproductives were produced sexually. AQS often occurs late, after colonies have reached maturity, whereas early AQS in other species may boost the young colony's growth rate. We suggest additional benefits of AQS in C. tuberosus, related with a smaller size, lesser stability and higher mobility of colonies. Our data add to the phylogenetical dispersion and diversity of modalities of AQS in termites, supporting a multiple evolutionary origin of this process.

Social Structure and Genetic Distance Mediate Nestmate Recognition and Aggressiveness in the Facultative Polygynous Ant Pheidole pallidula.

In social insects, the evolutionary stability of cooperation depends on the privileged relationships between individuals of the social group, which is facilitated by the recognition of relatives. Nestmate recognition is based on genetically determined cues and/or environmentally derived chemical components present on the cuticle of individuals. Here, we studied nestmate recognition in the ant Pheidole pallidula, a species where both single-queen (monogyne) and multiple-queen (polygyne) colonies co-occur in the same population. We combined geographical, genetic and chemical analyses to disentangle the factors influencing the level of intraspecific aggressiveness. We show that encounters between workers from neighbouring colonies (i.e., nests less than 5 m away) are on average less aggressive than those between workers from more distant colonies. Aggressive behaviour is associated with the level of genetic difference: workers from monogyne colonies are more aggressive than workers from polygyne colonies, and the intensity of aggressiveness is positively associated with the genetic distance between colonies. Since the genetic distance is correlated with the spatial distance between pairs of colonies, the lower level of aggression toward neighbours may result from their higher relatedness. In contrast, the analysis of overall cuticular hydrocarbon profiles shows that aggressive behaviour is associated neither with the chemical diversity of colonies, nor with the chemical distances between them. When considering methyl-branched alkanes only, however, chemical distances differed between monogyne and polygyne colonies and were significantly associated with aggressiveness. Altogether, these results show that the social structure of colonies and the genetic distances between colonies are two major factors influencing the intensity of agonistic behaviours in the ant P. pallidula.

Growth Rate of Bumblebee Larvae is Related to Pollen Amino Acids.

The use of Bombus terrestris L. commercial colonies for outdoor and greenhouse crop pollination is currently widespread. Colony breeding includes bumblebee feeding, mostly by using the honeybee pollen loads of diverse palynological composition. Because the chemical content of pollen is highly variable, the choice of commercial blend should not be random but has to be carefully selected to ensure the optimal development of workers and then pollination efficacy. In this work, we compared the impact of three common commercial blends on the development of bumblebee microcolonies, namely, Actinidia deliciosa L., Cistus sp., and Salix sp. We focus on amino acids (i.e., composition and amount), as they are currently used as an indicator of diet performance. Five parameters were used to determine microcolonies growth rate: 1) number of eggs, 2) number of alive larvae, 3) number of ejected larvae, 4) number of pupae, and 5) total number of offspring. Syrup collection was also monitored to estimate energetic requirement for colony growth. Results revealed that the three commercial blends chemically differed in their amino acid contents, with those displaying higher concentrations (i.e., Salix sp. and A. deliciosa) accelerating microcolony development along with an increase of syrup collection. The advantages of rearing bumblebee commercial colonies using a pollen diet with an optimal amino acid content are discussed.

Genetic structure, nestmate recognition and behaviour of two cryptic species of the invasive big-headed ant Pheidole megacephala.

BACKGROUND: Biological invasions are recognized as a major cause of biodiversity decline and have considerable impact on the economy and human health. The African big-headed ant Pheidole megacephala is considered one of the world's most harmful invasive species. METHODOLOGY/PRINCIPAL FINDINGS: To better understand its ecological and demographic features, we combined behavioural (aggression tests), chemical (quantitative and qualitative analyses of cuticular lipids) and genetic (mitochondrial divergence and polymorphism of DNA microsatellite markers) data obtained for eight populations in Cameroon. Molecular data revealed two cryptic species of P. megacephala, one inhabiting urban areas and the other rainforests. Urban populations belong to the same phylogenetic group than those introduced in Australia and in other parts of the world. Behavioural analyses show that the eight populations sampled make up four mutually aggressive supercolonies. The maximum distance between nests from the same supercolony was 49 km and the closest distance between two nests belonging to two different supercolonies was 46 m. The genetic data and chemical analyses confirmed the behavioural tests as all of the nests were correctly assigned to their supercolony. Genetic diversity appears significantly greater in Africa than in introduced populations in Australia; by contrast, urban and Australian populations are characterized by a higher chemical diversity than rainforest ones. CONCLUSIONS/SIGNIFICANCE: Overall, our study shows that populations of P. megacephala in Cameroon adopt a unicolonial social structure, like invasive populations in Australia. However, the size of the supercolonies appears several orders of magnitude smaller in Africa. This implies competition between African supercolonies and explains why they persist over evolutionary time scales.

Reproductive system, social organization, human disturbance and ecological dominance in native populations of the little fire ant, Wasmannia auropunctata.

The invasive ant species Wasmannia auropunctata displays both ecologically dominant and non-dominant populations within its native range. Three factors could theoretically explain the ecological dominance of some native populations of W. auropunctata: (i) its clonal reproductive system, through demographic and/or adaptive advantages; (ii) its unicolonial social organization, through lower intraspecific and efficient interspecific competition; (iii) the human disturbance of its native range, through the modification of biotic and abiotic environmental conditions. We used microsatellite markers and behavioural tests to uncover the reproductive modes and social organization of dominant and non-dominant native populations in natural and human-modified habitats. Microsatellite and mtDNA data indicated that dominant and non-dominant native populations (supercolonies as determined by aggression tests) of W. auropunctata did not belong to different evolutionary units. We found that the reproductive system and the social organization are neither necessary nor sufficient to explain W. auropunctata ecological dominance. Dominance rather seems to be set off by unknown ecological factors altered by human activities, as all dominant populations were recorded in human-modified habitats. The clonal reproductive system found in some populations of W. auropunctata may however indirectly contribute to its ecological dominance by allowing the species to expand its environmental niche, through the fixation over time of specific combinations of divergent male and female genotypes. Unicoloniality may rather promote the range expansion of already dominant populations than actually trigger ecological dominance. The W. auropunctata model illustrates the strong impact of human disturbance on species' ecological features and the adaptive potential of clonal reproductive systems.

Evolution: no-male's land for an Amazonian ant.

Recent work has shown that, in the Amazonian fungus-growing ant Mycocepurus smithii, queens use exclusively asexual reproduction and the male sex seems to have disappeared from the species. This finding illustrates the remarkable diversity of reproductive systems in ants.

Genetics, behaviour and chemical recognition of the invading ant Pheidole megacephala.

Introduced species often become ecologically dominant, displacing native species and posing a serious threat to ecosystem function and global biodiversity. Ants are among the most widespread and damaging alien species; introductions are often accompanied by population-level behavioural and genetic changes contributing to their success. We investigated the genetic structure, chemical profile and nestmate recognition in introduced populations of the invasive big-headed ant, Pheidole megacephala, in Australia. Behavioural analyses show that workers are not aggressive towards conspecifics from different nests, even at large geographical scales (up to 3000 km) and between populations encompassing a wide range of environmental conditions. By contrast, interactions with workers of other species invariably result in agonistic behaviours. Genetic analyses reveal that populations have low genetic diversity. No genetic differentiation occurs among nests of the same population; differentiation between populations, though significant, remains weak. Chemical analyses indicate that cuticular lipids are similar between colonies of a population, and that differentiation between populations is low. Altogether, these results indicate that the big-headed ant P. megacephala forms a large unicolonial population across northern/eastern Australia.

Isolation and characterization of microsatellite loci from the invasive ant Pheidole megacephala.

We report the characterization of eight microsatellite markers in the big-headed ant Pheidole megacephala, a pest ant registered in the list of '100 of the world's worst invasive alien species'. An enrichment protocol was used to isolate microsatellite loci, and polymorphism was explored with 36 individuals collected in an invasive population from Australia and 20 individuals collected in a population from the native mainland location in South Africa. These primers showed a number of alleles per locus ranging from two to 10, and expected heterozygosities ranging from 0.083 to 0.826. Moreover, results of cross-species amplification are reported in five other Pheidole species and in seven other ants of the subfamily Myrmicinae.

Sex and clonality in the little fire ant.

Reproduction systems are controlling the creation of new genetic variants as well as how natural selection can operate on these variants. Therefore, they had historically been one of the main foci of evolutionary biology studies. The little fire ant, Wasmannia auropunctata, has been found to display an extraordinary reproduction system, in which both males and female queens are produced clonally. So far, native sexual populations of W. auropunctata have not been identified. Our goals were to identify such sexual populations and investigate the origins of female parthenogenesis and male clonality. Using mitochondrial DNA and microsatellite markers in 17 native populations, we found that traditional sexual populations occurred in W. auropunctata and are likely the recent source of neighboring clonal populations. Queen parthenogenesis has probably evolved several times through mutational events. Male clonality is tightly linked to queen parthenogenesis and thus appears to be female controlled. Its origin could be accounted for by 2 mutually exclusive hypotheses: either by the expected coevolution of the 2 sexes (i.e., a variant of the maternal genome elimination hypothesis) or by a shared mechanistic origin (i.e., by the production of anucleate ovules by parthenogenetic queens). Our results also show that W. auropunctata males and females do not form separate evolutionary units and are unlikely to be engaged in an all-out battle of sexes. This work opens up new perspectives for studies on the adaptive significance and evolutionary stability of mixed sexual and clonal reproduction systems in living organisms.

Clonal reproduction by males and females in the little fire ant.

Sexual reproduction can lead to major conflicts between sexes and within genomes. Here we report an extreme case of such conflicts in the little fire ant Wasmannia auropunctata. We found that sterile workers are produced by normal sexual reproduction, whereas daughter queens are invariably clonally produced. Because males usually develop from unfertilized maternal eggs in ants and other haplodiploid species, they normally achieve direct fitness only through diploid female offspring. Hence, although the clonal production of queens increases the queen's relatedness to reproductive daughters, it potentially reduces male reproductive success to zero. In an apparent response to this conflict between sexes, genetic analyses reveal that males reproduce clonally, most likely by eliminating the maternal half of the genome in diploid eggs. As a result, all sons have nuclear genomes identical to those of their father. The obligate clonal production of males and queens from individuals of the same sex effectively results in a complete separation of the male and female gene pools. These findings show that the haplodiploid sex-determination system provides grounds for the evolution of extraordinary genetic systems and new types of sexual conflict.

Colony sex ratios in the facultatively polygynous ant Pheidole pallidula: a reanalysis with new data.

A recent study by Fournier et al. (2003) provides important new information on sex allocation in the ant Pheidole pallidula, and proposes a new scenario for sex-ratio evolution in P. pallidula and similar species. However, Helms proposed to the authors that two important conclusions of the study were questionable because of potential problems with the analyses. Here we provide new data and a reanalysis that strengthens the conclusion that colony sex ratio is associated with breeding system (i.e., polygyny or monogyny). However, the proposal that colonies shift from monogyny to polygyny when they become larger and more productive is weakened because there is substantial overlap in productivity between monogynous and polygynous colonies.

Colony sex ratios vary with breeding system but not relatedness asymmetry in the facultatively polygynous ant Pheidole pallidula.

We investigated sex allocation in a Mediterranean population of the facultatively polygynous (multiple queen per colony) ant Pheidole pallidula. This species shows a strong split sex ratio, with most colonies producing almost exclusively a single-sex brood. Our genetic (microsatellite) analyses reveal that P. pallidula has an unusual breeding system, with colonies being headed by a single or a few unrelated queens. As expected in such a breeding system, our results show no variation in relatedness asymmetry between monogynous (single queen per colony) and polygynous colonies. Nevertheless, sex allocation was tightly associated with the breeding structure, with monogynous colonies producing a male-biased brood and polygynous colonies almost only females. In addition, sex allocation was closely correlated with colony total sexual productivity. Overall, our data show that when colonies become more productive (and presumably larger) they shift from monogyny to polygyny and from male production to female production, a pattern that has never been reported in social insects.

Investigation of the population genetic structure and mating system in the ant Pheidole pallidula.

The origin of eusociality in haplo-diploid organisms such as Hymenoptera has been mostly explained by kin selection. However, several studies have uncovered decreased relatedness values within colonies, resulting primarily from multiple queen matings (polyandry) and/or from the presence of more than one functional queen (polygyny). Here, we report on the use of microsatellite data for the investigation of sociogenetic parameters, such as relatedness, and levels of polygyny and polyandry, in the ant Pheidole pallidula. We demonstrate, through analysis of mother-offspring combinations and the use of direct sperm typing, that each queen is inseminated by a single male. The inbreeding coefficient within colonies and the levels of relatedness between the queens and their mate are not significantly different from zero, indicating that matings occur between unrelated individuals. Analyses of worker genotypes demonstrate that 38% of the colonies are polygynous with 2-4 functional queens, and suggest the existence of reproductive skew, i.e. unequal respective contribution of queens to reproduction. Finally, our analyses indicate that colonies are genetically differentiated and form a population exhibiting significant isolation-by-distance, suggesting that some colonies originate through budding.