Extensive human-mediated jump dispersal within and across the native and introduced ranges of the invasive termite Reticulitermes flavipes.

As native ranges are often geographically structured, invasive species originating from a single source population only carry a fraction of the genetic diversity present in their native range. The invasion process is thus often associated with a drastic loss of genetic diversity resulting from a founder event. However, the fraction of diversity brought to the invasive range may vary under different invasion histories, increasing with the size of the propagule, the number of reintroduction events, and/or the total genetic diversity represented by the various source populations in a multiple-introduction scenario. In this study, we generated a SNP data set for the invasive termite Reticulitermes flavipes from 23 native populations in the eastern United States and six introduced populations throughout the world. Using population genetic analyses and approximate Bayesian computation random forest, we investigated its worldwide invasion history. We found a complex invasion pathway with multiple events out of the native range and bridgehead introductions from the introduced population in France. Our data suggest that extensive long-distance jump dispersal appears common in both the native and introduced ranges of this species, probably through human transportation. Overall, our results show that similar to multiple introduction events into the invasive range, admixture in the native range prior to invasion can potentially favour invasion success by increasing the genetic diversity that is later transferred to the introduced range.

Alteration of gut microbiota with a broad-spectrum antibiotic does not impair maternal care in the European earwig.

The microbes residing within the gut of an animal host often increase their own fitness by modifying their host's physiological, reproductive and behavioural functions. Whereas recent studies suggest that they may also shape host sociality and therefore have critical effects on animal social evolution, the impact of the gut microbiota on maternal care remains unexplored. This is surprising, as this behaviour is widespread among animals, often determines the fitness of both juveniles and parents, and is essential in the evolution of complex animal societies. Here, we tested whether life-long alterations of the gut microbiota with rifampicin-a broad-spectrum antibiotic-impair pre- and post-hatching maternal care in the European earwig. Our results first confirm that rifampicin altered the mothers' gut microbial communities and indicate that the composition of the gut microbiota differs before and after egg care. Contrary to our predictions, however, the rifampicin-induced alterations of the gut microbiota did not modify pre- or post-hatching care. Independent of maternal care, rifampicin increased the females' faeces production and resulted in lighter eggs and juveniles. By contrast, rifampicin altered none of the other 21 physiological, reproductive and longevity traits measured over the 300 days of a female's lifetime. Overall, these findings reveal that altering the gut microbiota with a large spectrum antibiotic such as rifampicin does not necessarily affect host sociality. They also emphasize that not all animals have evolved a co-dependence with their microbiota and call for caution when generalizing the central role of gut microbes in host biology.

Efficient but occasionally imperfect vertical transmission of gut mutualistic protists in a wood-feeding termite.

Although mutualistic associations between animals and microbial symbionts are widespread in nature, the mechanisms that have promoted their evolutionary persistence remain poorly understood. A vertical mode of symbiont transmission (from parents to offspring) is thought to ensure partner fidelity and stabilization, although the efficiency of vertical transmission has rarely been investigated, especially in cases where hosts harbour a diverse microbial community. Here we evaluated vertical transmission rates of cellulolytic gut oxymonad and parabasalid protists in the wood-feeding termite Reticulitermes grassei. We sequenced amplicons of the 18S rRNA gene of protists from 24 colonies of R. grassei collected in two populations. For each colony, the protist community was characterized from the gut of 14 swarming reproductives and from a pool of 10 worker guts. A total of 98 operational taxonomic units belonging to 13 species-level taxa were found. The vertical transmission rate was estimated for each protist present in a colony based on its frequency among the reproductives. The results revealed that transmission rates were high, with an average of 0.897 (±0.164) per protist species. Overall, the protist community did not differ between reproductive sexes, suggesting that both the queen and the king could contribute to the gut microbiota of the offspring. A positive relationship between the transmission rate of protists and their prevalence within populations was also detected. However, transmission rates alone do not explain the prevalence of protists. In conclusion, these findings reveal key forces behind a conserved, multispecies mutualism, raising further questions on the roles of horizontal transfer and negative selection in shaping symbiont prevalence.

An American termite in Paris: temporal colony dynamics.

Termites of the genus Reticulitermes are widespread invaders, particularly in urban habitats. Their cryptic and subterranean lifestyle makes them difficult to detect, and we know little about their colony dynamics over time. In this study we examined the persistence of Reticulitermes flavipes (Kollar) colonies in the city of Paris over a period of 15 years. The aim was (1) to define the boundaries of colonies sampled within the same four areas over two sampling periods, (2) to determine whether the colonies identified during the first sampling period persisted to the second sampling period, and (3) to compare the results obtained when colonies were delineated using a standard population genetic approach versus a Bayesian clustering method that combined both spatial and genetic information. Herein, colony delineations were inferred from genetic differences at nine microsatellite loci and one mitochondrial locus. Four of the 18 identified colonies did not show significant differences in their genotype distributions between the two sampling periods. While allelic richness was low, making it hard to reliably distinguish colony family type, most colonies appeared to retain the same breeding structure over time. These large and expansive colonies showed an important ability to fuse (39% were mixed-family colonies), contained hundreds of reproductives and displayed evidence of isolation-by-distance, suggesting budding dispersal. These traits, which favor colony persistence over time, present a challenge for pest control efforts, which apply treatment locally. The other colonies showed significant differences, but we cannot exclude the possibility that their genotype distributions simply changed over time.

Historical biogeography of Reticulitermes termites (Isoptera: Rhinotermitidae) inferred from analyses of mitochondrial and nuclear loci.

Termites of the genus Reticulitermes are ecologically and economically important wood-feeding social insects that are widespread in the Holarctic region. Despite their importance, no study has yet attempted to reconstruct a global time-scaled phylogeny of Reticulitermes termites. In this study, we sequenced mitochondrial (2096bp) and nuclear (829bp) loci from 61 Reticulitermes specimens, collected across the genus' entire range, and one specimen of Coptotermes formosanus, which served as an outgroup. Bayesian and Maximum likelihood analyses conducted on the mitochondrial and nuclear sequences support the existence of four main lineages that span four global geographical regions: North America (NA lineage), western Europe (WE lineage), a region including eastern Europe and western Asia (EA+WA lineage), and eastern Asia (EA lineage). The mitochondrial data allowed us to clarify the phylogenetic relationships among these lineages. They were also used to infer a chronogram that was time scaled based on age estimates for termite fossils (including the oldest Reticulitermes fossils, which date back to the late Eocene-early Oligocene). Our results support the hypothesis that the extant Reticulitermes lineage first differentiated in North America. The first divergence event in the ancestral lineage of Reticulitermes occurred in the early Miocene and separated the Nearctic lineages (i.e., the NA lineages) from the Palearctic lineages (i.e., WE, EE+WA, and EA lineages). Our analyses revealed that the main lineages of Reticulitermes diversified because of vicariance and migration events, which were probably induced by major paleogeographic and paleoclimatic changes that occurred during the Cenozoic era. This is the first global and comprehensive phylogenetic study of Reticulitermes termites, and it provides a crucial foundation for studying the evolution of phenotypic and life-history traits in Reticulitermes. For instance, the phylogeny we obtained suggested that 'asexual queen succession', a unique reproductive system, independently evolved at least three times during the diversification of the genus.

SnakeMAGs: a simple, efficient, flexible and scalable workflow to reconstruct prokaryotic genomes from metagenomes.

Background: Over the last decade, we have observed in microbial ecology a transition from gene-centric to genome-centric analyses. Indeed, the advent of metagenomics combined with binning methods, single-cell genome sequencing as well as high-throughput cultivation methods have contributed to the continuing and exponential increase of available prokaryotic genomes, which in turn has favored the exploration of microbial metabolisms. In the case of metagenomics, data processing, from raw reads to genome reconstruction, involves various steps and software which can represent a major technical obstacle. Methods: To overcome this challenge, we developed SnakeMAGs, a simple workflow that can process Illumina data, from raw reads to metagenome-assembled genomes (MAGs) classification and relative abundance estimate. It integrates state-of-the-art bioinformatic tools to sequentially perform: quality control of the reads (illumina-utils, Trimmomatic), host sequence removal (optional step, using Bowtie2), assembly (MEGAHIT), binning (MetaBAT2), quality filtering of the bins (CheckM), classification of the MAGs (GTDB-Tk) and estimate of their relative abundance (CoverM). Developed with the popular Snakemake workflow management system, it can be deployed on various architectures, from single to multicore and from workstation to computer clusters and grids. It is also flexible since users can easily change parameters and/or add new rules. Results: Using termite gut metagenomic datasets, we showed that SnakeMAGs is slower but allowed the recovery of more MAGs encompassing more diverse phyla compared to another similar workflow named ATLAS. Conclusions: Overall, it should make the reconstruction of MAGs more accessible to microbiologists. SnakeMAGs as well as test files and an extended tutorial are available at https://github.com/Nachida08/SnakeMAGs.

Ontogenic potentialities of the worker caste in two sympatric subterranean termites in France.

In termites, the capacity of workers to differentiate into neotenic reproductives is an important characteristic that deserves particular attention. To gain insight into the differentiation pathway, the potentialities of workers and the endocrinal changes during the formation of neotenics were compared in two sympatric termites, Reticulitermes flavipes and Reticulitermes grassei. After 1 year of development, 100% of R. flavipes worker groups produced neotenics against only 63% of R. grassei groups. The average production of female neotenics was significantly higher in R. flavipes worker groups compared with R. grassei groups and R. flavipes produced a greater proportion of female neotenics. Moreover, R. flavipes produced more offspring, not only because there were more females, but also because R. flavipes females were more productive. Moreover, the offspring produced by R. flavipes grew faster than the offspring of R. grassei. Both ecdysteroid and juvenile hormone (JH) titers varied significantly during the development of neotenics. The two species showed similar ecdysteroid titer variation patterns. However, the JH titer variation patterns strongly differed: in R. grassei, the concentration of JH increased in maturing neotenics then dropped in mature neotenics, whereas in R. flavipes, the level of JH was significantly higher than in R. grassei and remained constantly high in mature neotenics. Overall, these results suggest that these two species differ strongly in many life-history traits as well as in the physiological control of their caste differentiation system. Possible origins and mechanisms of such interspecific variations are discussed, as well as their evolutionary and ecological consequences.

Variations in worker cuticular hydrocarbons and soldier isoprenoid defensive secretions within and among introduced and native populations of the subterranean termite, Reticulitermes flavipes.

In social insects, cuticular hydrocarbons (CHCs) play a central role in nestmate recognition. CHCs have proved to be useful for identifying species and differentiating populations. In combination with CHCs, isoprenoid soldier defensive secretions (SDSs) have been previously used in some termite species for chemotaxonomic analyses. This study compared the levels of chemical variation within and among introduced (French) and native (U.S.) populations of the subterranean termite, Reticulitermes flavipes. Worker CHCs and soldier SDSs from termites collected from colonies in nine populations in Florida, Louisiana, and France were analyzed. Discriminant analyses revealed that both localities and populations can be distinguished by using the variation in CHC profiles. Principal component analyses of CHC profiles as well as the calculation of two distance parameters (Nei and Euclidean) revealed remarkable chemical homogeneity within and among French populations. These analyses also showed that the CHC profiles of French populations were closer to termite populations from Louisiana than to those from Florida. Of the six distinct SDS chemotypes, one was common to populations in France and Louisiana. The possibility that populations in France originated from Louisiana, and the potential causes and consequences of chemical homogeneity within introduced populations are discussed.

Physiological and biochemical responses to thermal stress vary among genotypes in the parasitic wasp Nasonia vitripennis.

In ectotherm species such as insects, thermal fluctuations represent a major environmental factor driving development, survival and reproduction of individuals. Reproductive traits are particularly sensitive to heat stress that can induce a permanent sterility, or at least hypofertility, of adult males. This study aims to compare physiological and biochemical responses associated to male performances to an exposure of 24 h to moderately high temperature (36 °C) among three inbred lines of N. vitripennis (AsymC, Cor, Oul). Cor males showed very specific metabolic adjustments compared to the two other lines. By contrast, Oul males showed stronger phenotypic adjustment of its life cycle, and produced metabolic water to compensate water loss by heat stress. Finally, AsymC males had probably more difficulties to acclimate at 36 °C, even for a short period, as their adult longevity was significantly reduced. Thus, the ability of developmental plasticity in N. vitripennis males exposed to heat stress appears to be dependent of their genotypes.

Ecological specificity of the metagenome in a set of lower termite species supports contribution of the microbiome to adaptation of the host.

BACKGROUND: Elucidating the interplay between hosts and their microbiomes in ecological adaptation has become a central theme in evolutionary biology. A textbook example of microbiome-mediated adaptation is the adaptation of lower termites to a wood-based diet, as they depend on their gut microbiome to digest wood. Lower termites have further adapted to different life types. Termites of the wood-dwelling life type never leave their nests and feed on a uniform diet. Termites of the foraging life type forage for food outside the nest and have access to other nutrients. Here we sought to investigate whether the microbiome that is involved in food substrate breakdown and nutrient acquisition might contribute to adaptation to these dietary differences. We reasoned that this should leave ecological imprints on the microbiome. RESULTS: We investigated the protist and bacterial microbiomes of a total of 29 replicate colonies from five termite species, covering both life types, using metagenomic shotgun sequencing. The microbiome of wood-dwelling species with a uniform wood diet was enriched for genes involved in lignocellulose degradation. Furthermore, metagenomic patterns suggest that the microbiome of wood-dwelling species relied primarily on direct fixation of atmospheric nitrogen, while the microbiome of foraging species entailed the necessary pathways to utilize nitrogen in the form of nitrate for example from soil. CONCLUSION: Our findings are consistent with the notion that the microbiome of wood-dwelling species bears an imprint of its specialization on degrading a uniform wood diet, while the microbiome of the foraging species might reflect its adaption to access growth limiting nutrients from more diverse sources. This supports the idea that specific subsets of functions encoded by the microbiome can contribute to host adaptation.

Invasion Dynamics of A Termite, Reticulitermes flavipes, at Different Spatial Scales in France.

Termites are social insects that can also be major pests. A well-known problem species is the subterranean termite, Reticulitermes flavipes. It is invasive in France and is thought to have arrived from Louisiana during the 18th century. While the putative source of French populations has been identified, little is known about how the termite spread following its establishment. Here, we examined expansion patterns at different spatial scales in urban areas to clarify how R. flavipes spread in France. Based on our analyses of phylogeography and population genetics, results suggest a scenario of successive introductions into the Charente-Maritime region, on the Atlantic Coast. Two major expansion fronts formed: one that spread toward the northeast and the other toward the southeast. At the regional scale, different spatial and genetic distribution patterns were observed: there was heterogeneity in Île-de-France and aggregation in Centre-Val de Loire. At the local scale, we found that our three focal urban sites each formed a single large colony that contained several secondary reproductives. Our findings represent a second step in efforts to reconstruct termite's invasion dynamics. They also highlight the role that may have been played by the French railway network in transporting termites over long distances.

Differential Ecological Specificity of Protist and Bacterial Microbiomes across a Set of Termite Species.

The gut microbiome of lower termites comprises protists and bacteria that help these insects to digest cellulose and to thrive on wood. The composition of the termite gut microbiome correlates with phylogenetic distance of the animal host and host ecology (diet) in termites collected from their natural environment. However, carryover of transient microbes from host collection sites are an experimental concern and might contribute to the ecological imprints on the termite gut microbiome. Here, we set out to test whether an ecological imprint on the termite gut microbiome remains, when focusing on the persistent microbiome. Therefore, we kept five termite species under strictly controlled dietary conditions and subsequently profiled their protist and bacterial gut microbial communities using 18S and 16S rRNA gene amplicon sequencing. The species differed in their ecology; while three of the investigated species were wood-dwellers that feed on the piece of wood they live in and never leave except for the mating flight, the other two species were foragers that regularly leave their nests to forage for food. Despite these prominent ecological differences, protist microbiome structure aligned with phylogenetic relatedness of termite host species. Conversely, bacterial communities seemed more flexible, suggesting that microbiome structure aligned more strongly with the foraging and wood-dwelling ecologies. Interestingly, protist and bacterial community alpha-diversity correlated, suggesting either putative interactions between protists and bacteria, or that both types of microbes in the termite gut follow shared structuring principles. Taken together, our results add to the notion that bacterial communities are more variable over evolutionary time than protist communities and might react more flexibly to changes in host ecology.

Virulence, multiple infections and regulation of symbiotic population in the Wolbachia-Asobara tabida symbiosis.

The density and regulation of microbial populations are important factors in the success of symbiotic associations. High bacterial density may improve transmission to the next generation, but excessive replication could turn out to be costly to the host and result in higher virulence. Moreover, differences in virulence may also depend on the diversity of symbionts. Using the maternally transmitted symbiont Wolbachia, we investigated how bacterial density and diversity are regulated and influence virulence in host insects subject to multiple infection. The model we used was the wasp Asobara tabida that naturally harbors three different Wolbachia strains, of which two are facultative and induce cytoplasmic incompatibility, whereas the third is necessary for the host to achieve oogenesis. Using insect lines infected with different subsets of Wolbachia strains, we show that: (i) some traits of A. tabida are negatively affected by Wolbachia; (ii) the physiological cost increases with the number of co-infecting strains, which also corresponds to an increase in the total bacterial density; and (iii) the densities of the two facultative Wolbachia strains are independent of one another, whereas the obligatory strain is less abundant when it is alone, suggesting that there is some positive interaction with the other strains.

Correlation between the green-island phenotype and Wolbachia infections during the evolutionary diversification of Gracillariidae leaf-mining moths.

Internally feeding herbivorous insects such as leaf miners have developed the ability to manipulate the physiology of their host plants in a way to best meet their metabolic needs and compensate for variation in food nutritional composition. For instance, some leaf miners can induce green-islands on yellow leaves in autumn, which are characterized by photosynthetically active green patches in otherwise senescing leaves. It has been shown that endosymbionts, and most likely bacteria of the genus Wolbachia, play an important role in green-island induction in the apple leaf-mining moth Phyllonorycter blancardella. However, it is currently not known how widespread is this moth-Wolbachia-plant interaction. Here, we studied the co-occurrence between Wolbachia and the green-island phenotype in 133 moth specimens belonging to 74 species of Lepidoptera including 60 Gracillariidae leaf miners. Using a combination of molecular phylogenies and ecological data (occurrence of green-islands), we show that the acquisitions of the green-island phenotype and Wolbachia infections have been associated through the evolutionary diversification of Gracillariidae. We also found intraspecific variability in both green-island formation and Wolbachia infection, with some species being able to form green-islands without being infected by Wolbachia. In addition, Wolbachia variants belonging to both A and B supergroups were found to be associated with green-island phenotype suggesting several independent origins of green-island induction. This study opens new prospects and raises new questions about the ecology and evolution of the tripartite association between Wolbachia, leaf miners, and their host plants.

Comparative Analysis of Transcriptomes from Secondary Reproductives of Three Reticulitermes Termite Species.

Termites are eusocial insects related to cockroaches that feed on lignocellulose. These insects are key species in ecosystems since they recycle a large amount of nutrients but also are pests, exerting major economic impacts. Knowledge on the molecular pathways underlying reproduction, caste differentiation or lignocellulose digestion would largely benefit from additional transcriptomic data. This study focused on transcriptomes of secondary reproductive females (nymphoid neotenics). Thirteen transcriptomes were used: 10 of Reticulitermes flavipes and R. grassei sequenced from a previous study, and two transcriptomes of R. lucifugus sequenced for the present study. After transcriptome assembly and read mapping, we examined interspecific variations of genes expressed by termites or gut microorganisms. A total of 18,323 orthologous gene clusters were detected. Functional annotation and taxonomic assignment were performed on a total of 41,287 predicted contigs in the three termite species. Between the termite species studied, functional categories of genes were comparable. Gene ontology (GO) terms analysis allowed the discovery of 9 cellulases and a total of 79 contigs potentially involved in 11 enzymatic activities used in wood metabolism. Altogether, results of this study illustrate the strong potential for the use of comparative interspecific transcriptomes, representing a complete resource for future studies including differentially expressed genes between castes or SNP analysis for population genetics.

Intra-individual coexistence of a Wolbachia strain required for host oogenesis with two strains inducing cytoplasmic incompatibility in the wasp Asobara tabida.

Cytoplasmically inherited symbiotic Wolbachia bacteria are known to induce a diversity of phenotypes on their numerous arthropod hosts including cytoplasmic incompatibility, male-killing, thelytokous parthenogenesis, and feminization. In the wasp Asobara tabida (Braconidae), in which all individuals harbor three genotypic Wolbachia strains (wAtab1, wAtab2 and wAtab3), the presence of Wolbachia is required for insect oogenesis. To elucidate the phenotype of each Wolbachia strain on host reproduction, especially on oogenesis, we established lines of A. tabida harboring different combinations of these three bacterial strains. We found that wAtab3 is essential for wasp oogenesis, whereas the two other strains, wAtabl and wAtab2, seem incapable to act on this function. Furthermore, interline crosses showed that strains wAtab1 and wAtab2 induce partial (about 78%) cytoplasmic incompatibility of the female mortality type. These results support the idea that bacterial genotype is a major factor determining the phenotype induced by Wolbachia on A. tabida hosts. We discuss the implications of these findings for current hypotheses regarding the evolutionary mechanisms by which females of A. tabida have become dependent on Wolbachia for oogenesis.

Do variable compensatory mechanisms explain the polymorphism of the dependence phenotype in the Asobara tabida-wolbachia association?

Wolbachia are symbiotic intracellular bacteria, which are classified as reproductive parasites. Although generally facultative, Wolbachia is necessary for Asobara tabida (Hymenoptera), because aposymbiotic females do not produce any offspring. Interestingly, the ovarian phenotype of aposymbiotic females is variable: some females do not produce any eggs, whereas others do produce some eggs, but these are aborted. Here, we show that the ovarian phenotype of aposymbiotic females is highly polymorphic within populations, although dependence remains complete in both cases. We also identified some lines in which aposymbiotic females were able to produce a very few viable offspring, further extending the range of variation observed. These results suggest that various factors actively maintain polymorphism. We demonstrated that Wolbachia is necessary to trigger oogenetic processes, but that the ovarian phenotype was determined by the host only. Phenotypic variation was also correlated with the differential expression of genes controlling iron homeostasis and oxidative stress, which are potentially involved in the evolution of dependence. This suggests that variation in the ovarian phenotype could reflect selection for different levels of compensatory mechanisms in response to Wolbachia infection, and that polymorphism is maintained through selection on different antagonist traits influenced by oxidative stress.

Social parasitism in fire ants (Solenopsis spp.): a potential mechanism for interspecies transfer of Wolbachia.

One possible mechanism for interspecific transfer of Wolbachia is through the intimate contact between parasites and their hosts. We surveyed 10 species of fly parasitoids (Pseudacteon spp.) and one inquiline social parasite, Solenopsis daguerrei, for the presence and sequence identity (wsp gene) of Wolbachia. Two Wolbachia variants infecting S. daguerrei were identical to known variants infecting the two common ant host species, Solenopsis invicta and Solenopsis richteri, suggesting possible transfers of Wolbachia between this parasite and their hosts have occurred. Our data also revealed an unexpectedly high diversity of Wolbachia variants within S. daguerrei: up to eight variants were found within each individual, which, to our knowledge, is the highest reported number of Wolbachia variants infecting a single individual of any host species.