Bacterial supergroup-specific "cost" of Wolbachia infections in Nasonia vitripennis.

The maternally inherited endosymbiont, Wolbachia, is known to alter the reproductive biology of its arthropod hosts for its own benefit and can induce both positive and negative fitness effects in many hosts. Here, we describe the effects of the maintenance of two distinct Wolbachia infections, one each from supergroups A and B, on the parasitoid host Nasonia vitripennis. We compare the effect of Wolbachia infections on various traits between the uninfected, single A-infected, single B-infected, and double-infected lines with their cured versions. Contrary to some previous reports, our results suggest that there is a significant cost associated with the maintenance of Wolbachia infections where traits such as family size, fecundity, longevity, and rates of male copulation are compromised in Wolbachia-infected lines. The double Wolbachia infection has the most detrimental impact on the host as compared to single infections. Moreover, there is a supergroup-specific negative impact on these wasps as the supergroup B infection elicits the most pronounced negative effects. These negative effects can be attributed to a higher Wolbachia titer seen in the double and the single supergroup B infection lines when compared to supergroup A. Our findings raise important questions on the mechanism of survival and maintenance of these reproductive parasites in arthropod hosts.

Coping with the 'Indian summer': unique nesting cycle and nest architecture of the paper wasp, Polistes wattii.

Polistes is one of the most widely distributed and extensively studied primitively eusocial wasps. Based on where they are found, there are two established nesting cycles in this genus. The temperate wasps follow an annual cycle with diapause in winter while the tropical wasps of South America can initiate nests any time of the year and do not hibernate. Additionally, some subtropical Polistes are known to form nest free aggregations during the cold, dry, unfavorable season. Although several species of Polistes wasps are found in India, our knowledge about their biology is pitifully small and is restricted to taxonomic reports. Here, we report the unique nesting cycle of Polistes wattii, a wasp abundantly found in north India and other Asian countries. P. wattii hibernates in winter as well as forms nest-free aggregations in the dry summer season and thus has a nesting cycle with two inactive periods, which no other Polistes is known to follow. The study site in North India experiences short, cold, snow-free winters, spring, a very dry early summer, and humid late summer with intermittent rain. We found that P. wattii here shows several unique adaptations to survive the long Indian summer where it shows two rounds of nest-founding in the same year, once as overwintered, solitary foundress in spring and once with multiple foundresses during summer. To meet the demands of expanding colony in late summer, P. wattii often adds multiple combs to their nest, which are architecturally different from the multiple comb nests reported from neotropical regions and strikingly different from all temperate Polistes who make only single comb nests. This study investigates the nesting biology and natural history of P. wattii to understand how they maximize survival and fitness.

In Vitro Studies Reveal that Pseudomonas, from Odontotermes obesus Colonies, can Function as a Defensive Mutualist as it Prevents the Weedy Fungus While Keeping the Crop Fungus Unaffected.

Insects that farm monocultures of fungi are canonical examples of nutritional symbiosis as well as independent evolution of agriculture in non-human animals. But just like in human agriculture, these fungal crops face constant threat of invasion by weeds which, if unchecked, take over the crop fungus. In fungus-growing termites, the crop fungus (Termitomyces) faces such challenges from the weedy fungus Pseudoxylaria. The mechanism by which Pseudoxylaria is suppressed is not known. However, evidence suggests that some bacterial secondary symbionts can serve as defensive mutualists by preventing the growth of Pseudoxylaria. However, such secondary symbionts must possess the dual, yet contrasting, capabilities of suppressing the weedy fungus while keeping the growth of the crop fungus unaffected. This study describes the isolation, identification, and culture-dependent estimation of the roles of several such putative defensive mutualists from the colonies of the wide-spread fungus-growing termite from India, Odontotermes obesus. From the 38 bacterial cultures tested, a strain of Pseudomonas showed significantly greater suppression of the weedy fungus than the crop fungus. Moreover, a 16S rRNA pan-microbiome survey, using the Nanopore platform, revealed Pseudomonas to be a part of the core microbiota of O. obesus. A meta-analysis of microbiota composition across different species of Odontotermes also confirms the widespread prevalence of Pseudomonas within this termite. These lines of evidence indicate that Pseudomonas could be playing the role of defensive mutualist within Odontotermes.

A meta-analysis testing eusocial co-option theories in termite gut physiology and symbiosis.

The termite gut accomplishes key physiologic functions that underlie termite symbiosis and sociality. However, potential candidate functions of the host-symbiont holobiome have not yet been explored across seemingly divergent processes such as digestion, immunity, caste differentiation, and xenobiotic tolerance. This study took a meta-analysis approach for concurrently studying host and symbiont gut metatranscriptome responses of the lower termite Reticulitermes flavipes, which has ancestral characteristics and hosts a diverse mix of eukaryotic and bacterial symbionts. Thirteen treatments were compared from 5 categories (dietary, social, hormonal, immunological, and xenobiotic), revealing 3 main insights. First, each of the 5 tested colonies had distinct magnitudes of transcriptome response, likely as a result of unique symbiont profiles, which highlights the uniqueness of individual termite colonies. Second, after normalization to standardize colony response magnitudes, unique treatment-linked metatranscriptome topologies became apparent. Third, despite colony and topology differences, 4 co-opted master genes emerged that were universally responsive across diverse treatments. These master genes encode host functions related to protein translation and symbiont functions related to protein degradation and pore formation in microbial cell walls. Three of the 4 master genes were from co-evolved protist symbionts, highlighting potentially co-evolved roles for gut symbiota in coordinating functional responses of the collective host-symbiont holobiome. Lastly, for host genes identified, these results provide annotations of recent termite genome sequences. By revealing conserved domain genes, as well as apparent roles for gut symbiota in holobiome regulation, this study provides new insights into co-opted eusocial genes and symbiont roles in termite sociobiology.

Molecular signatures of nicotinoid-pathogen synergy in the termite gut.

Previous studies in lower termites revealed unexpected synergies between nicotinoid insecticides and fungal entomopathogens. The present study investigated molecular mechanisms of nicotinoid-pathogen synergy in the lower termite Reticulitermes flavipes, using the nicotinoid, imidacloprid, in combination with fungal and bacterial entomopathogens. Particular focus was placed on metatranscriptome composition and microbial dynamics in the symbiont-rich termite gut, which houses diverse mixes of protists and bacteria. cDNA microarrays containing a mix of host and protist symbiont oligonucleotides were used to simultaneously assess termite and protist gene expression. Five treatments were compared that included single challenges with sublethal doses of fungi (Metharizium anisopliae), bacteria (Serratia marcescens) or imidacloprid, and dual challenges with fungi + imidacloprid or bacteria + imidacloprid. Our findings point towards protist dysbiosis and compromised social behavior, rather than suppression of stereotypical immune defense mechanisms, as the dominant factors underlying nicotinoid-pathogen synergy in termites. Also, greater impacts observed for the fungal pathogen than for the bacterial pathogen suggest that the rich bacterial symbiont community in the R. flavipes gut (>5000 species-level phylotypes) exists in an ecological balance that effectively excludes exogenous bacterial pathogens. These findings significantly advance our understanding of antimicrobial defenses in this important eusocial insect group, as well as provide novel insights into how nicotinoids can exert deleterious effects on social insect colonies.

Differential impacts of juvenile hormone, soldier head extract and alternate caste phenotypes on host and symbiont transcriptome composition in the gut of the termite Reticulitermes flavipes.

BACKGROUND: Termites are highly eusocial insects and show a division of labor whereby morphologically distinct individuals specialize in distinct tasks. In the lower termite Reticulitermes flavipes (Rhinotermitidae), non-reproducing individuals form the worker and soldier castes, which specialize in helping (e.g., brood care, cleaning, foraging) and defense behaviors, respectively. Workers are totipotent juveniles that can either undergo status quo molts or develop into soldiers or neotenic reproductives. This caste differentiation can be regulated by juvenile hormone (JH) and primer pheromones contained in soldier head extracts (SHE). Here we offered worker termites a cellulose diet treated with JH or SHE for 24-hr, or held them with live soldiers (LS) or live neotenic reproductives (LR). We then determined gene expression profiles of the host termite gut and protozoan symbionts concurrently using custom cDNA oligo-microarrays containing 10,990 individual ESTs. RESULTS: JH was the most influential treatment (501 total ESTs affected), followed by LS (24 ESTs), LR (12 ESTs) and SHE treatments (6 ESTs). The majority of JH up- and downregulated ESTs were of host and symbiont origin, respectively; in contrast, SHE, LR and LS treatments had more uniform impacts on host and symbiont gene expression. Repeat "follow-up" bioassays investigating combined JH + SHE impacts in relation to individual JH and SHE treatments on a subset of array-positive genes revealed (i) JH and SHE treatments had opposite impacts on gene expression and (ii) JH + SHE impacts on gene expression were generally intermediate between JH and SHE. CONCLUSIONS: Our results show that JH impacts hundreds of termite and symbiont genes within 24-hr, strongly suggesting a role for the termite gut in JH-dependent caste determination. Additionally, differential impacts of SHE and LS treatments were observed that are in strong agreement with previous studies that specifically investigated soldier caste regulation. However, it is likely that gene expression outside the gut may be of equal or greater importance than gut gene expression.

The hindgut lumen prokaryotic microbiota of the termite Reticulitermes flavipes and its responses to dietary lignocellulose composition.

Reticulitermes flavipes (Isoptera: Rhinotermitidae) is a highly eusocial insect that thrives on recalcitrant lignocellulosic diets through nutritional symbioses with gut-dwelling prokaryotes and eukaryotes. In the R. flavipes hindgut, there are up to 12 eukaryotic protozoan symbionts; the number of prokaryotic symbionts has been estimated in the hundreds. Despite its biological relevance, this diverse community, to date, has been investigated only by culture- and cloning-dependent methods. Moreover, it is unclear how termite gut microbiomes respond to diet changes and what roles they play in lignocellulose digestion. This study utilized high-throughput 454 pyrosequencing of 16S V5-V6 amplicons to sample the hindgut lumen prokaryotic microbiota of R. flavipes and to examine compositional changes in response to lignin-rich and lignin-poor cellulose diets after a 7-day feeding period. Of the ~475,000 high-quality reads that were obtained, 99.9% were annotated as bacteria and 0.11% as archaea. Major bacterial phyla included Spirochaetes (24.9%), Elusimicrobia (19.8%), Firmicutes (17.8%), Bacteroidetes (14.1%), Proteobacteria (11.4%), Fibrobacteres (5.8%), Verrucomicrobia (2.0%), Actinobacteria (1.4%) and Tenericutes (1.3%). The R. flavipes hindgut lumen prokaryotic microbiota was found to contain over 4761 species-level phylotypes. However, diet-dependent shifts were not statistically significant or uniform across colonies, suggesting significant environmental and/or host genetic impacts on colony-level microbiome composition. These results provide insights into termite gut microbiome diversity and suggest that (i) the prokaryotic gut microbiota is much more complex than previously estimated, and (ii) environment, founding reproductive pair effects and/or host genetics influence microbiome composition.

Evolution of cold-tolerant fungal symbionts permits winter fungiculture by leafcutter ants at the northern frontier of a tropical ant-fungus symbiosis.

The obligate mutualism between leafcutter ants and their Attamyces fungi originated 8 to 12 million years ago in the tropics, but extends today also into temperate regions in South and North America. The northernmost leafcutter ant Atta texana sustains fungiculture during winter temperatures that would harm the cold-sensitive Attamyces cultivars of tropical leafcutter ants. Cold-tolerance of Attamyces cultivars increases with winter harshness along a south-to-north temperature gradient across the range of A. texana, indicating selection for cold-tolerant Attamyces variants along the temperature cline. Ecological niche modeling corroborates winter temperature as a key range-limiting factor impeding northward expansion of A. texana. The northernmost A. texana populations are able to sustain fungiculture throughout winter because of their cold-adapted fungi and because of seasonal, vertical garden relocation (maintaining gardens deep in the ground in winter to protect them from extreme cold, then moving gardens to warmer, shallow depths in spring). Although the origin of leafcutter fungiculture was an evolutionary breakthrough that revolutionized the food niche of tropical fungus-growing ants, the original adaptations of this host-microbe symbiosis to tropical temperatures and the dependence on cold-sensitive fungal symbionts eventually constrained expansion into temperate habitats. Evolution of cold-tolerant fungi within the symbiosis relaxed constraints on winter fungiculture at the northern frontier of the leafcutter ant distribution, thereby expanding the ecological niche of an obligate host-microbe symbiosis.

Bacterial diversity in Solenopsis invicta and Solenopsis geminata ant colonies characterized by 16S amplicon 454 pyrosequencing.

Social insects harbor diverse assemblages of bacterial microbes, which may play a crucial role in the success or failure of biological invasions. The invasive fire ant Solenopsis invicta (Formicidae, Hymenoptera) is a model system for understanding the dynamics of invasive social insects and their biological control. However, little is known about microbes as biotic factors influencing the success or failure of ant invasions. This pilot study is the first attempt to characterize and compare microbial communities associated with the introduced S. invicta and the native Solenopsis geminata in the USA. Using 16S amplicon 454 pyrosequencing, bacterial communities of workers, brood, and soil from nest walls were compared between neighboring S. invicta and S. geminata colonies at Brackenridge Field Laboratory, Austin, Texas, with the aim of identifying potential pathogenic, commensal, or mutualistic microbial associates. Two samples of S. geminata workers showed high counts of Spiroplasma bacteria, a known pathogen or mutualist of other insects. A subsequent analysis using PCR and sequencing confirmed the presence of Spiroplasma in additional colonies of both Solenopsis species. Wolbachia was found in one alate sample of S. geminata, while one brood sample of S. invicta had a high count of Lactococcus. As expected, ant samples from both species showed much lower microbial diversity than the surrounding soil. Both ant species had similar overall bacterial diversities, although little overlap in specific microbes. To properly characterize a single bacterial community associated with a Solenopsis ant sample, rarefaction analyses indicate that it is necessary to obtain 5,000-10,000 sequences. Overall, 16S amplicon 454 pyrosequencing appears to be a cost-effective approach to screen whole microbial diversity associated with invasive ant species.

Microbiomes of ant castes implicate new microbial roles in the fungus-growing ant Trachymyrmex septentrionalis.

Fungus-growing ants employ several defenses against diseases, including disease-suppressing microbial biofilms on their integument and in fungal gardens. Here, we compare the phenology of microbiomes in natural nests of the temperate fungus-growing ant Trachymyrmex septentrionalis using culture-dependent isolations and culture-independent 16S-amplicon 454-sequencing. 454-sequencing revealed diverse actinobacteria associated with ants, including most prominently Solirubrobacter (12.2-30.9% of sequence reads), Pseudonocardia (3.5-42.0%), and Microlunatus (0.4-10.8%). Bacterial abundances remained relatively constant in monthly surveys throughout the annual active period (late winter to late summer), except Pseudonocardia abundance declined in females during the reproductive phase. Pseudonocardia species found on ants are phylogenetically different from those in gardens and soil, indicating ecological separation of these Pseudonocardia types. Because the pathogen Escovopsis is not known to infect gardens of T. septentrionalis, the ant-associated microbes do not seem to function in Escovopsis suppression, but could protect against ant diseases, help in nest sanitation, or serve unknown functions.

Defining host-symbiont collaboration in termite lignocellulose digestion: "The view from the tip of the iceberg".

Termites have the unique ability to exploit lignocellulose as a primary nutrition source. Traditionally, termite lignocellulose digestion has been considered as a gut-symbiont-mediated process; however, in recent years the importance of host digestive capabilities have become apparent. Despite this growing understanding, how digestive enzymes from different origins specifically collaborate (i.e., additively or synergistically) has remained largely unknown. In a recent study, we undertook translational-genomic studies to address these questions in the lower termite Reticulitermes flavipes (Isoptera: Rhinotermitidae) and its symbiotic gut fauna. We used a combination of native gut tissue preparations and recombinant enzymes derived from the host gut transcriptome to identify synergistic collaborations between host and symbiont, and also among enzymes produced exclusively by the host termite. These findings provided important new evidence of synergistic collaboration among enzymes in the release of fermentable monosaccharides from wood lignocellulose, and laid a foundation for future integrative studies into termite digestion, symbiosis and eusociality.

Nestmateship and body size do not influence mate choice in males and females: a laboratory study of a primitively eusocial wasp Ropalidia marginata.

We investigated the effect of nestmateship and body size on mate selection through a choice based assay in the primitively eusocial wasp Ropalidia marginata. A recent study has shown that male and female R. marginata mate with their nestmates and non-nestmates with equal probability if no choice is available. That study could also not detect any influence of body size on mating probability in the absence of choice. To confirm that the same results can be obtained even when the wasps have a choice, we offered a choice of two virgin partners either to a virgin test male or to a virgin test female and measured the probability that the test individual would mate with any particular partner based on nestmateship or body size. We show here that even when a choice is available, neither male nor female test wasps base their mate choice on the nestmateship or body size of the partner. We therefore suggest that the natural mating habit of these wasps is sufficiently promiscuous and not constrained by such factors as nestmateship and body size.

Placement of attine ant-associated Pseudonocardia in a global Pseudonocardia phylogeny (Pseudonocardiaceae, Actinomycetales): a test of two symbiont-association models.

We reconstruct the phylogenetic relationships within the bacterial genus Pseudonocardia to evaluate two models explaining how and why Pseudonocardia bacteria colonize the microbial communities on the integument of fungus-gardening ant species (Attini, Formicidae). The traditional Coevolution-Codivergence model views the integument-colonizing Pseudonocardia as mutualistic microbes that are largely vertically transmitted between ant generations and that supply antibiotics that specifically suppress the garden pathogen Escovopsis. The more recent Acquisition model views Pseudonocardia as part of a larger integumental microbe community that frequently colonizes the ant integument from environmental sources (e.g., soil, plant material). Under this latter model, ant-associated Pseudonocardia may have diverse ecological roles on the ant integument (possibly ranging from pathogenic, to commensal, to mutualistic) and are not necessarily related to Escovopsis suppression. We test distinct predictions of these two models regarding the phylogenetic proximity of ant-associated and environmental Pseudonocardia. We amassed 16S-rRNA gene sequence information for 87 attine-associated and 238 environmental Pseudonocardia, aligned the sequences with the help of RNA secondary structure modeling, and reconstructed phylogenetic relationships using a maximum-likelihood approach. We present 16S-rRNA secondary structure models of representative Pseudonocardia species to improve sequence alignments and identify sequencing errors. Our phylogenetic analyses reveal close affinities and even identical sequence matches between environmental Pseudonocardia and ant-associated Pseudonocardia, as well as nesting of environmental Pseudonocardia in subgroups that were previously thought to be specialized to associate only with attine ants. The great majority of ant-associated Pseudonocardia are closely related to autotrophic Pseudonocardia and are placed in a large subgroup of Pseudonocardia that is known essentially only from cultured isolates (rather than cloned 16S sequences). The preponderance of the known ant-associated Pseudonocardia in this latter clade of culturable lineages may not necessarily reflect abundance of these Pseudonocardia types on the ants, but isolation biases when screening for Pseudonocardia (e.g., preferential isolation of autotrophic Pseudonocardia with minimum-nutrient media). The accumulated phylogenetic patterns and the possibility of isolation biases in previous work further erode support for the traditional Coevolution-Codivergence model and calls for continued revision of our understanding how and why Pseudonocardia colonize the microbial communities on the integument of fungus-gardening ant species.

Generalized antifungal activity and 454-screening of Pseudonocardia and Amycolatopsis bacteria in nests of fungus-growing ants.

In many host-microbe mutualisms, hosts use beneficial metabolites supplied by microbial symbionts. Fungus-growing (attine) ants are thought to form such a mutualism with Pseudonocardia bacteria to derive antibiotics that specifically suppress the coevolving pathogen Escovopsis, which infects the ants' fungal gardens and reduces growth. Here we test 4 key assumptions of this Pseudonocardia-Escovopsis coevolution model. Culture-dependent and culture-independent (tag-encoded 454-pyrosequencing) surveys reveal that several Pseudonocardia species and occasionally Amycolatopsis (a close relative of Pseudonocardia) co-occur on workers from a single nest, contradicting the assumption of a single pseudonocardiaceous strain per nest. Pseudonocardia can occur on males, suggesting that Pseudonocardia could also be horizontally transmitted during mating. Pseudonocardia and Amycolatopsis secretions kill or strongly suppress ant-cultivated fungi, contradicting the previous finding of a growth-enhancing effect of Pseudonocardia on the cultivars. Attine ants therefore may harm their own cultivar if they apply pseudonocardiaceous secretions to actively growing gardens. Pseudonocardia and Amycolatopsis isolates also show nonspecific antifungal activities against saprotrophic, endophytic, entomopathogenic, and garden-pathogenic fungi, contrary to the original report of specific antibiosis against Escovopsis alone. We conclude that attine-associated pseudonocardiaceous bacteria do not exhibit derived antibiotic properties to specifically suppress Escovopsis. We evaluate hypotheses on nonadaptive and adaptive functions of attine integumental bacteria, and develop an alternate conceptual framework to replace the prevailing Pseudonocardia-Escovopsis coevolution model. If association with Pseudonocardia is adaptive to attine ants, alternate roles of such microbes could include the protection of ants or sanitation of the nest.