Fabio Manfredini

Sociogenomics of Cooperation and Conflict during Colony Founding in the Fire Ant Solenopsis invicta

One of the fundamental questions in biology is how cooperative and altruistic behaviors evolved. The majority of studies seeking to identify the genes regulating these behaviors have been performed in systems where behavioral and physiological differences are relatively fixed, such as in the honey bee. During colony founding in the monogyne (one queen per colony) social form of the fire ant Solenopsis invicta, newly-mated queens may start new colonies either individually (haplometrosis) or in groups (pleometrosis). However, only one queen (the “winner”) in pleometrotic associations survives and takes the lead of the young colony while the others (the “losers”) are executed. Thus, colony founding in fire ants provides an excellent system in which to examine the genes underpinning cooperative behavior and how the social environment shapes the expression of these genes. We developed a new whole genome microarray platform for S. invicta to characterize the gene expression patterns associated with colony founding behavior. First, we compared haplometrotic queens, pleometrotic winners and pleometrotic losers. Second, we manipulated pleometrotic couples in order to switch or maintain the social ranks of the two cofoundresses. Haplometrotic and pleometrotic queens differed in the expression of genes involved in stress response, aging, immunity, reproduction and lipid biosynthesis. Smaller sets of genes were differentially expressed between winners and losers. In the second experiment, switching social rank had a much greater impact on gene expression patterns than the initial/final rank. Expression differences for several candidate genes involved in key biological processes were confirmed using qRT-PCR. Our findings indicate that, in S. invicta, social environment plays a major role in the determination of the patterns of gene expression, while the queens physiological state is secondary. These results highlight the powerful influence of social environment on regulation of the genomic state, physiology and ultimately, social behavior of animals.

Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens

BackgroundOrganisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses.ResultsWe identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses.ConclusionsOur meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.

Molecular and social regulation of worker division of labour in fire ants

Reproductive and worker division of labour (DOL) is a hallmark of social insect societies. Despite a long‐standing interest in worker DOL, the molecular mechanisms regulating this process have only been investigated in detail in honey bees, and little is known about the regulatory mechanisms operating in other social insects. In the fire ant Solenopsis invicta, one of the most studied ant species, workers are permanently sterile and the tasks performed are modulated by the workers internal state (age and size) and the outside environment (social environment), which potentially includes the effect of the queen presence through chemical communication via pheromones. However, the molecular mechanisms underpinning these processes are unknown. Using a whole‐genome microarray platform, we characterized the molecular basis for worker DOL and we explored how a drastic change in the social environment (i.e. the sudden loss of the queen) affects global gene expression patterns of worker ants. We identified numerous genes differentially expressed between foraging and nonforaging workers in queenright colonies. With a few exceptions, these genes appear to be distinct from those involved in DOL in bees and wasps. Interestingly, after the queen was removed, foraging workers were no longer distinct from nonforaging workers at the transcriptomic level. Furthermore, few expression differences were detected between queenright and queenless workers when we did not consider the task performed. Thus, the social condition of the colony (queenless vs. queenright) appears to impact the molecular pathways underlying worker task performance, providing strong evidence for social regulation of DOL in S. invicta.

Parasitic infection protects wasp larvae against a bacterial challenge

Host antibacterial defense after Strepsiptera parasitization is a complex and rather unexplored topic. The way how these parasites interact with bacteria invading into the host insect during an infection is completely unknown. In the present study we demonstrate that larvae of the paper wasp Polistes dominulus are more efficient at eliminating bacteria when they are parasitized by the strepsipteran insect Xenos vesparum. We looked at the expression levels of the antimicrobial peptide defensin and we screened for the activity of other hemolymph components by using a zone of inhibition assay. Transcription of defensin is triggered by parasitization, but also by mechanical injury (aseptic injection). Inhibitory activity in vitro against the Gram positive bacterium Staphylococcus aureus is not influenced by the presence of the parasite in the wasp or by a previous immune challenge, suggesting a constitutive power of killing this bacterium by wasp hemolymph. Our results suggest either direct involvement of the parasite or that defensin and further immune components not investigated in this paper, for example other antimicrobial peptides, could play a role in fighting off bacterial infections in Polistes.

RNA-sequencing elucidates the regulation of behavioural transitions associated with the mating process in honey bee queens

BackgroundMating is a complex process, which is frequently associated with behavioural and physiological changes. However, understanding of the genetic underpinnings of these changes is limited. Honey bees are both a model system in behavioural genomics, and the dominant managed pollinator of human crops; consequently understanding the mating process has both pure and applied value. We used next-generation transcriptomics to probe changes in gene expression in the brains of honey bee queens, as they transition from virgin to mated reproductive status. In addition, we used CO2-narcosis, which induces oviposition without mating, to isolate the process of reproductive maturation.ResultsThe mating process produced significant changes in the expression of vision, chemo-reception, metabolic, and immune-related genes. Differential expression of these genes maps clearly onto known behavioural and physiological changes that occur during the transition from being a virgin queen to a newly-mated queen. A subset of these changes in gene expression were also detected in CO2-treated queens, as predicted from previous physiological studies. In addition, we compared our results to previous studies that used microarray techniques across a range of experimental time-points. Changes in expression of immune- and vision-related genes were common to all studies, supporting an involvement of these groups of genes in the mating process.ConclusionsOur study is an important step in understanding the molecular mechanisms regulating post-mating behavioural transitions in a natural system. The weak overlap in patterns of gene expression with previous studies demonstrates the high sensitivity of genome-wide approaches. Thus, while we build on previous microarray studies that explored post-mating changes in honey bees, the broader experimental design, use of RNA-sequencing, and focus on Australian honey bees, which remain free from the devastating parasite Varroa destructor, in the current study, provide unique insights into the biology of the mating process in honey bees.

The strepsipteran endoparasite Xenos vesparum alters the immunocompetence of its host, the paper wasp Polistes dominulus

It is unexplained how strepsipteran insects manipulate the physiology of their hosts in order to undergo endoparasitic development without being entrapped by the innate immune defences of the host. Here we present pioneering work that aimed to explore for the first time several components of the cellular and humoral immune response among immature stages of the paper wasp Polistes dominulus, in both unparasitized insects and after infection by the strepsipteran endoparasite Xenos vesparum. We carried out hemocyte counts, phagocytosis assays in vitro and antibacterial response in vivo. On the whole, hemocyte load does not seem to be drastically affected by parasitization: a non-significant increase in hemocyte numbers was observed in parasitized wasps as respect to control, while the two dominant hemocyte types were present with similar proportions in both groups. On the other hand, phagocytosis was significantly reduced in hemocytes from parasitized wasps while the antibacterial response seemed to be less effective in control. These somewhat unexpected results are discussed, along with the implications of a multiple approach in immune response studies.

Examining the “evolution of increased competitive ability” hypothesis in response to parasites and pathogens in the invasive paper wasp Polistes dominula

Successful invaders often become established in new ranges by outcompeting native species. The “evolution of increased competitive ability” hypothesis predicts that invasive species are subjected to less predation and parasitization than sympatric native species, and thus can allocate resources from defence and immunity to growth and fecundity, thereby achieving higher fitness. In this study, we examined whether American invasive Polistes dominula paper wasps have reduced immunocompetence. To explore this scenario, we tested their susceptibility towards parasites and pathogens at both the individual (immune defence) and colony levels, i.e. hygienic behaviour (removal of diseased individuals by nestmates). First, we examined the response to the specific coevolved parasite Xenos vesparum (lost after invasion) in terms of individual host susceptibility and hygienic behaviour. Second, we explored the response against general pathogens by quantifying the bacterial clearance in individual wasps after a challenge with Escherichia coli and hygienic behaviour after a challenge with the fungus Beauveria bassiana. Our results show that American invasive P. dominula have a higher response against X. vesparum at the colony level, but at the individual level their susceptibility is not significantly different from conspecifics of the native range. On the other hand, invasive P. dominula display lower response after a challenge with general pathogens at both the individual and colony levels. While supporting the hypothesis of a reduction of immunocompetence towards general pathogens in invasive species, these findings also suggest that the response against coevolved parasites might follow different evolutionary pathways which are not always easily predictable.

A difficult choice for tiny pests: host-seeking behaviour in Xenos vesparum triungulins

The first-instar larvae of strepsipteran parasites, commonly referred to as “triungulins”, are the host-seeking stage: they must locate, invade and successfully develop in the new host, in order to start their parasitic cycle. Little information is available about the behaviour of Xenos vesparum triungulins. They emerge in batches from the endoparasitic female infecting Polistes dominulus, the primary host, and reach the nest through a vector (a foraging wasp or the parasitised wasp itself). Once there, they have the possibility to penetrate into wasp immatures at different developmental stages. In this study, we performed preliminary analyses aimed to investigate which cues are important to direct triungulin movements during their brief stay in wasp nests. In laboratory conditions we selectively presented different stimuli to Xenos larvae: apparently, the host larva itself is attractive in an open arena, but not inside a confined space, nor are epicuticular compounds of wasp larvae able to control triungulin movements. These are more likely oriented by their gregarious behaviour, whereas light (positive phototaxy) may at a previous stage enhance their emergence via the brood canal opening in the female cephalothorax.

Parasitic castration by Xenos vesparum depends on host gender.

Host castration represents a mechanism used by parasites to exploit energy resources from their hosts by interfering with their reproductive development or to extend host lifespan by removing risks associated with reproductive activity. One of the most intriguing groups of parasitic castrators is represented by the insects belonging to the order Strepsiptera. The macroparasite Xenos vesparum can produce dramatic phenotypic alterations in its host, the paper wasp Polistes dominula. Parasitized female wasps have undeveloped ovaries and desert the colony without performing any social task. However, very little attention has been given to the parasitic impact of X. vesparum on the male phenotype. Here, we investigated the effects of this parasite on the sexual behaviour and the morpho-physiology of P. dominula males. We found that, differently from female wasps, parasitized males are not heavily affected by Xenos: they maintain their sexual behaviour and ability to discriminate between female castes. Furthermore, the structure of their reproductive apparatus is not compromised by the parasite. We think that our results, demonstrating that the definition of X. vesparum as a parasitoid does not apply to infected males of P. dominula, provide a new perspective to discuss and maybe reconsider the traditional view of strepsipteran parasites.

Transcriptomics of an extended phenotype: Parasite manipulation of wasp social behaviour shifts expression of caste-related genes

Parasites can manipulate host behaviour to increase their own transmission and fitness, but the genomic mechanisms by which parasites manipulate hosts are not well understood. We investigated the relationship between the social paper wasp, Polistes dominula, and its parasite, Xenos vesparum (Insecta: Strepsiptera), to understand the effects of an obligate endoparasitoid on its hosts brain transcriptome. Previous research suggests that X. vesparum shifts aspects of host social caste-related behaviour and physiology in ways that benefit the parasitoid. We hypothesized that X. vesparum-infested (stylopized) females would show a shift in caste-related brain gene expression. Specifically, we predicted that stylopized females, who would normally be workers, would show gene expression patterns resembling pre-overwintering queens (gynes), reflecting gyne-like changes in behaviour. We used RNA-sequencing data to characterize patterns of brain gene expression in stylopized females and compared these with those of unstylopized workers and gynes. In support of our hypothesis, we found that stylopized females, despite sharing numerous physiological and life-history characteristics with members of the worker caste, show gyne-shifted brain expression patterns. These data suggest that the parasitoid affects its host by exploiting phenotypic plasticity related to social caste, thus shifting naturally occurring social behaviour in a way that is beneficial to the parasitoid.