Influential Insider: Wolbachia, an Intracellular Symbiont, Manipulates Bacterial Diversity in Its Insect Host.

Facultative intracellular symbionts like the α-proteobacteria Wolbachia influence their insect host phenotype but little is known about how much they affect their host microbiota. Here, we quantified the impact of Wolbachia infection on the bacterial community of the cabbage root fly Delia radicum by comparing the microbiota of Wolbachia-free and infected adult flies of both sexes. We used high-throughput DNA sequencing (Illumina MiSeq, 16S rRNA, V5-V7 region) and performed a community and a network analysis. In both sexes, Wolbachia infection significantly decreased the diversity of D. radicum bacterial communities and modified their structure and composition by reducing abundance in some taxa but increasing it in others. Infection by Wolbachia was negatively correlated to 8 bacteria genera (Erwinia was the most impacted), and positively correlated to Providencia and Serratia. We suggest that Wolbachia might antagonize Erwinia for being entomopathogenic (and potentially intracellular), but would favor Providencia and Serratia because they might protect the host against chemical plant defenses. Although they might seem prisoners in a cell, endocellular symbionts can impact the whole microbiota of their host, hence its extended phenotype, which provides them with a way to interact with the outside world.

Long-lasting effects of antibiotics on bacterial communities of adult flies.

Insect symbionts benefit their host and their study requires large spectrum antibiotic use like tetracycline to weaken or suppress symbiotic communities. While antibiotics have a negative impact on insect fitness, little is known about antibiotic effects on insect microbial communities and how long they last. We characterized the bacterial communities of adult cabbage root fly Delia radicum in a Wolbachia-free population and evaluated the effect of tetracycline treatment on these communities over several generations. Three D. radicum generations were used: the first- and second-generation flies either ingested tetracycline or not, while the third-generation flies were untreated but differed with their parents and/or grandparents that had or had not been treated. Fly bacterial communities were sequenced using a 16S rRNA gene. Tetracycline decreased fly bacterial diversity and induced modifications in both bacterial abundance and relative frequencies, still visible on untreated offspring whose parents and/or grandparents had been treated, therefore demonstrating long-lasting transgenerational effects on animal microbiomes after antibiotic treatment. Flies with an antibiotic history shared bacterial genera, potentially tetracycline resistant and heritable. Next, the transmission should be investigated by comparing several insect development stages and plant compartments to assess vertical and horizontal transmissions of D. radicum bacterial communities.

Oviposition Preference of the Cabbage Root Fly towards Some Chinese Cabbage Cultivars: A Search for Future Trap Crop Candidates.

The development of integrated pest management strategies becomes more and more pressing in view of potential harmful effects of synthetic pesticides on the environment and human health. A promising alternative strategy against Delia radicum is the use of trap crops. Chinese cabbage (Brassica rapa subsp. pekinensis and subsp. chinensis) is a highly sensitive Brassicaceae species previously identified as a good candidate to attract the cabbage root fly away from other crops. Here, we carried out multi-choice experiments both in the laboratory and in field conditions to measure the oviposition susceptibilities of different subspecies and cultivars of Chinese cabbages as compared to a broccoli reference. We found large differences among subspecies and cultivars of the Chinese cabbage, which received three to eleven times more eggs than the broccoli reference in field conditions. In laboratory conditions, the chinensis subspecies did not receive more eggs than the broccoli reference. We conclude that D. radicum largely prefers to lay eggs on the pekinensis subspecies of Chinese cabbage compared to the chinensis subspecies or broccoli. Some pekinensis cultivars, which received over ten times more eggs than broccoli in the field, appear especially promising candidates to further develop trap crop strategies against the cabbage root fly.

The Toxin-Antidote Model of Cytoplasmic Incompatibility: Genetics and Evolutionary Implications.

Wolbachia bacteria inhabit the cells of about half of all arthropod species, an unparalleled success stemming in large part from selfish invasive strategies. Cytoplasmic incompatibility (CI), whereby the symbiont makes itself essential to embryo viability, is the most common of these and constitutes a promising weapon against vector-borne diseases. After decades of theoretical and experimental struggle, major recent advances have been made toward a molecular understanding of this phenomenon. As pieces of the puzzle come together, from yeast and Drosophila fly transgenesis to CI diversity patterns in natural mosquito populations, it becomes clearer than ever that the CI induction and rescue stem from a toxin-antidote (TA) system. Further, the tight association of the CI genes with prophages provides clues to the possible evolutionary origin of this phenomenon and the levels of selection at play.

Influence of the symbiont Wolbachia on life history traits of the cabbage root fly (Delia radicum).

Wolbachia is an endocellular bacteria infecting arthropods and nematodes and is only transmitted vertically by females via the cytoplasm of the egg. It is often a manipulator of host reproduction, causing cytoplasmic incompatibility, thelytokous parthenogenesis, feminization or male killing, which all increase the proportion of infected females in the population. However, Wolbachia can modify life history traits of the host without causing the above phenotypes and each species illustrates the variability of relationships between this remarkably versatile symbiont and its many hosts. We have measured maternal transmission and the impact of a natural Wolbachia infection in the cabbage root fly Delia radicum, a major agricultural pest. We used a population that is polymorphic for the infection to ensure similar genetic and microbiome backgrounds between groups. Maternal transmission of the infection was 100% in our sample. We found no evidence of cytoplasmic incompatibility, thelytokous parthenogenesis, feminization nor male killing. Wolbachia infection significantly reduced hatch rate in infected eggs (by 10%) but improved larvo-nymphal viability sufficiently so that infected eggs nevertheless yielded as many adults as uninfected ones, albeit with a 1.5% longer total development time. Starved and infected ovipositing females suffered significantly reduced viability (20% higher mortality during a 3-day oviposition period) than uninfected females, but mortality was not higher in starved virgin females nor in starved males, suggesting that the energetic cost of the infection is only revealed in extreme conditions. Wolbachia had no effect on egg hatch time or offspring size. The apparently 100% vertical transmission and the significant but mutually compensating effects found suggest that infection might be nearly benign in this host and might only drift slowly, which would explain why the infection rate has been stable in our laboratory (approximately 50% individuals infected) for at least 30 generations.

One more step toward a push-pull strategy combining both a trap crop and plant volatile organic compounds against the cabbage root fly Delia radicum.

The "push-pull" strategy aims at manipulating insect pest behavior using a combination of attractive and repulsive stimuli using either plants derived volatile organic compounds or insect host plant preferences. In a field experiment using broccoli as a crop, we combined in a "push-pull" context the oviposition deterrent effect of dimethyl disulfide and the attractive effect of a Chinese cabbage strip enhanced with Z-3-hexenyl-acetate. The push component dimethyl disulfide reduced Delia radicum L. (Diptera: Anthomyiidae) oviposition on broccoli by nearly 30%, and applying Z-3-hexenyl-acetate in the pull component of Chinese cabbage increased it by 40%. Moreover, pest infestation was 40% higher in Chinese cabbage compared to broccoli and parasitism by Trybliographa rapae Westwood (Hymenoptera: Figitidae) was four times higher on this trap plant. In addition, lab experiments confirmed that Chinese cabbage is a more suitable host plant than broccoli for the cabbage root fly. Taken together, our results demonstrate the technical possibility of using a push-pull strategy to manipulate the egg-laying behavior of D. radicum in the field.

Can soil microbial diversity influence plant metabolites and life history traits of a rhizophagous insect? A demonstration in oilseed rape.

Interactions between plants and phytophagous insects play an important part in shaping the biochemical composition of plants. Reciprocally plant metabolites can influence major life history traits in these insects and largely contribute to their fitness. Plant rhizospheric microorganisms are an important biotic factor modulating plant metabolites and adaptation to stress. While plant-insects or plant-microorganisms interactions and their consequences on the plant metabolite signature are well-documented, the impact of soil microbial communities on plant defenses against phytophagous insects remains poorly known. In this study, we used oilseed rape (Brassica napus) and the cabbage root fly (Delia radicum) as biological models to tackle this question. Even though D. radicum is a belowground herbivore as a larva, its adult life history traits depend on aboveground signals. We therefore tested whether soil microbial diversity influenced emergence rate and fitness but also fly oviposition behavior, and tried to link possible effects to modifications in leaf and root metabolites. Through a removal-recolonization experiment, 3 soil microbial modalities ("high," "medium," "low") were established and assessed through amplicon sequencing of 16S and 18S ribosomal RNA genes. The "medium" modality in the rhizosphere significantly improved insect development traits. Plant-microorganism interactions were marginally associated to modulations of root metabolites profiles, which could partly explain these results. We highlighted the potential role of plant-microbial interaction in plant defenses against Delia radicum. Rhizospheric microbial communities must be taken into account when analyzing plant defenses against herbivores, being either below or aboveground.

Bacterial Community Diversity Harboured by Interacting Species.

All animals are infected by microbial partners that can be passengers or residents and influence many biological traits of their hosts. Even if important factors that structure the composition and abundance of microbial communities within and among host individuals have been recently described, such as diet, developmental stage or phylogeny, few studies have conducted cross-taxonomic comparisons, especially on host species related by trophic relationships. Here, we describe and compare the microbial communities associated with the cabbage root fly Delia radicum and its three major parasitoids: the two staphylinid beetles Aleochara bilineata and A. bipustulata and the hymenopteran parasitoid Trybliographa rapae. For each species, two populations from Western France were sampled and microbial communities were described through culture independent methods (454 pyrosequencing). Each sample harbored at least 59 to 261 different bacterial phylotypes but was strongly dominated by one or two. Microbial communities differed markedly in terms of composition and abundance, being mainly influenced by phylogenetic proximity but also geography to a minor extent. Surprisingly, despite their strong trophic interaction, parasitoids shared a very low proportion of microbial partners with their insect host. Three vertically transmitted symbionts from the genus Wolbachia, Rickettsia, and Spiroplasma were found in this study. Among them, Wolbachia and Spiroplasma were found in both the cabbage fly and at least one of its parasitoids, which could result from horizontal transfers through trophic interactions. Phylogenetic analysis showed that this hypothesis may explain some but not all cases. More work is needed to understand the dynamics of symbiotic associations within trophic network and the effect of these bacterial communities on the fitness of their hosts.

Broccoli and turnip plants display contrasting responses to belowground induction by Delia radicum infestation and phytohormone applications.

Induced responses to insect herbivory are a common phenomenon in the plant kingdom. So far, induced responses have mostly investigated in aerial plant parts. Recently it was found that root herbivore may also elicit both local and systemic responses affecting aboveground herbivores and their natural enemies. Using broccoli (Brassica oleracea subsp. italica L.) and turnip (Brassica rapa subsp. rapa L.), two cultivated brassicaceaous plants differing in their chemistry and morphology, we analysed the local and systemic induced responses triggered by Delia radicum L. damage, JA and SA application. We also assessed whether the root induction treatments affected D. radicum larval performance. Both D. radicum damage and JA induced changes in glucosinolate and sugar content as well as affected D. radicum performance, while SA application did not. Despite the uniform chemical responses, the effect on larval performance on broccoli and turnip plants was very different. On broccoli, JA root treatment reduced herbivore performance, whereas in turnips the same treatment enhanced it. JA- and D. radicum-induced responses followed similar patterns, which suggests that the JA signalling pathway is involved in root-induced responses to larval feeding. Glucosinolate induction cannot fully explain the differences found in the performance of D. radicum on the different species. Changes in other resistance factors might significantly contribute to the induced resistance in these brassicaceaeous species as well.

Kin recognition loss following anesthesia in beetle larvae (Aleochara bilineata, Coleoptera, Staphylinidae).

Kin recognition is a complex cognitive process, where an individual should detect a phenotypic cue and compare it to an internal template, which might be genetically determined (i.e., innate or acquired). Kin recognition mechanisms will depend on whether previous encounters with kin are possible or not to form the individual internal template. When relatives have never met before, kin recognition is supposed to rely on recognition alleles (which allows the innate recognition of relatives bearing them), or on self-referent phenotype matching (the individual has formed a template using its own phenotype and recognizes as kin individuals which match it closely enough). Although self-referent phenotype matching is in theory the more likely explanation, it has not been possible so far to exclude experimentally the recognition alleles' hypothesis. Here, we report that kin recognition in the solitary parasitoid larvae of Aleochara bilineata (Coleoptera; Staphylinidae) is suppressed following carbon dioxide anesthesia or chill-coma, both treatments known to cause a temporary amnesia. Treated larvae superparasitize indifferently hosts parasitized either by siblings or by non-kin larvae, while untreated larvae avoid hosts occupied by siblings. The two types of anesthesia thus suppress kin recognition, but their global effect on larvae is different. Chill-coma suppresses the ability to distinguish parasitized from unparasitized hosts and reduces parasitism rate, suggesting an aspecific impairment of sensory receptors or cognition. However, carbon dioxide narcosis only impairs kin recognition, strongly suggesting that an intact memory is necessary for kin recognition to take place. Although this study does not address the recognition alleles' hypothesis per se, our results strongly support a self-referent phenotype matching mechanism. On the whole, kin recognition in A. bilineata larvae is effective through short-term memory, because it is affected by amnesic treatments.

Infection by Wolbachia: from passengers to residents.

Wolbachia are endosymbiotic alpha-proteobacteria harboured by terrestrial arthropods and filarial nematodes, where they are maternally transmitted through egg cytoplasm. According to the host group, Wolbachia have developed two contrasting symbiotic strategies. In arthropods, symbiosis is secondary (i.e. facultative), and Wolbachia insure their transmission as reproduction parasites. However, despite of the efficiency of the manipulation mechanisms used, Wolbachia are limited to the state of passenger because some factors can prevent the association between Wolbachia and their hosts to become permanent. On the contrary, symbiosis is primary (i.e. obligatory) in filarial nematodes where Wolbachia insure their transmission via a mutualistic relationship, leading them to become permanent residents of their hosts. However, a few examples show that in arthropods too some Wolbachia have started to present the first stages of a mutualistic behaviour, or are even truly indispensable to their host. Whatever its strategy, Wolbachia infection is a spectacular evolutionary success, this symbiotic bacterium representing one of the most important biomass of its kind.

Identification of a widespread monomolecular odor differentially attractive to several Delia radicum ground-dwelling predators in the field.

Dimethyl disulfide (DMDS) was identified as a major volatile constituent of Brassica napus roots heavily infested by Delia radicum, the cabbage root fly. Attractiveness of this widespread compound was tested in the field in a naturally complex odorous environment. By using an original setup especially designed for ground dwelling beetles, different concentrations of the pure molecule as well as attractiveness of the natural blend emitted by the rotten part of infested roots were tested simultaneously. The use of general linear model (GLM) statistics permitted us to finely discriminate the responses among the different treatments. The main predators of D. radicum (i.e., two staphylinids Aleochara bilineata and Aleochara bipustulata and carabid beetles of the genus Bembidion) were significantly attracted by DMDS, but responded in different ways to the natural blend and to the different concentrations tested. The dose-response curves were similar for the two staphylinids. However, whereas A. bilineata was more attracted by the natural volatile blend than by its preferred DMDS concentration, A. bipustulata was attracted as much by the natural blend as by its preferred DMDS concentration. Carabid beetles exhibited a different response. They were not attracted by the natural blend, but responded to a wider range of DMDS concentrations that included low concentrations that did not attract the staphylinid beetles. These results are discussed according to the potential resources searched by each taxon studied and their specificity for the resources. The possible use of DMDS for enhancing biological control of D. radicum is mentioned.

Kin recognition in Aleochara bilineata could support the kinship theory of genomic imprinting.

Genomic imprinting corresponds to the differential expression of a gene according to its paternal or maternal origin. The kinship theory of genomic imprinting proposes that maternally or paternally inherited genes may be in conflict over their effects on kin differently related along the paternal or maternal line. Most examples supporting the kinship theory of imprinting deal with competition between offspring for maternal resources. However, genomic imprinting may also explain differential behavioral expression toward kin whenever sibs are more related to each other via one parental sex than the other. Unfortunately, nothing is currently known about imprinting associated with a behavioral phenotype in insects. Here we report the first evidence of such a maternally imprinted behavior. We show that the solitary parasitoid larvae of Aleochara bilineata Gyll (Coleoptera; Staphylinidae), which avoid superparasitizing their full sibs, also avoid their cousins when they are related to them through their father, but not when they are related to them through their mother. A genetic kin recognition mechanism is proposed to explain this result and we conclude that genomic imprinting could control the avoidance of kin superparasitism in this species and have a profound influence on decision-making processes.

Kin discrimination and altruism in the larvae of a solitary insect.

Kin selection theory predicts altruism between related individuals, which requires the ability to recognize kin from non-kin. In insects, kin discrimination associated with altruistic behaviour is well-known in clonal and social species but in very few solitary insects. Here, we report that the solitary larvae of a non-social insect Aleochara bilineata Gyll. (Coleoptera; Staphylinidae) show kin discrimination and sibling-directed altruistic behaviour. Larvae superparasitize more frequently the hosts parasitized by non-kin individuals than those hosts parasitized by siblings. Kin discrimination probably occurs by self-referent phenotype matching, where an individual compares its own phenotype with that of a non-familiar related individual, a mechanism rarely demonstrated in animals. The label used to recognize kin from non-kin corresponds to substances contained in the plug placed on the hosts by the resident larvae during the parasitization process. Kin competition induced by a limited larval dispersion may have favoured the evolution of kin recognition in this solitary species.

Incipient evolution of Wolbachia compatibility types.

Cytoplasmic incompatibility (CI) is induced in arthropods by the maternally inherited bacterium Wolbachia. When infected males mate with uninfected females or with females bearing a different Wolbachia variant, paternal chromosomes behave abnormally and embryos die. This pattern can be interpreted as resulting from two bacterial effects: One (usually termed mod, for modification) would affect sperm and induce embryo death, unless Wolbachia is also present in the egg, which implies the existence of a second effect, usually termed resc, for rescue. The fact that CI can occur in crosses between males and females infected by different Wolbachia shows that mod and resc interact in a specific manner. In other words, different compatibility types, or mod/resc pairs seem to have diverged from one (or a few) common ancestor(s). We are interested in the process allowing the evolution of mod/resc pairs. Here this question is addressed experimentally after cytoplasmic injection into a single host species (Drosophila simulans) by investigating compatibility relationships between closely related Wolbachia variants naturally evolving in different dipteran hosts: D. simulans, Drosophila melanogaster, and Rhagoletis cerasi. Our results suggest that closely related bacteria can be totally or partially incompatible. The compatibility relationships observed can be explained using a formal description of the mod and resc functions, implying both qualitative and quantitative variations.

On the mechanism of Wolbachia-induced cytoplasmic incompatibility: confronting the models with the facts.

The endocellular bacterium Wolbachia manipulates the reproduction of its arthropod hosts for its own benefit by various means, the most widespread being cytoplasmic incompatibility (CI). To date, the molecular mechanism involved in CI has not been elucidated. We examine here three different CI models described in previous literature, namely, the "lock-and-key", "titration-restitution" and "slow-motion" models. We confront them with the full range of CI patterns discovered so far, including the most complex ones such as multiple infections, asymmetrical and partial compatibility relationships and the existence of Wolbachia variants that can rescue the host from CI but not induce it. We conclude that the lock-and-key model is the most parsimonious of the models and fits the observations best. The two other models cannot be categorically invalidated, but they encounter some difficulties that make additional hypotheses necessary.

WOLBACHIA INFECTION IN DROSOPHILA SIMULANS: DOES THE FEMALE HOST BEAR A PHYSIOLOGICAL COST?

Fitness traits of three Drosophila simulans strains infected by endocellular bacteria belonging to the genus Wolbachia have been compared with those of replicate stocks previously cured from the infection by an antibiotic treatment. The traits measured were development time, egg-to-adult viability, egg hatch, productivity, fecundity, and the number of functional ovarioles. Individuals of the first strain were bi-infected by two Wolbachia variants, wHa and wNo. The second strain was infected by wHa, the third one by wNo. The Wolbachia studied here cause cytoplasmic incompatibility (CI), a high embryonic mortality (70% to > 90%) when an infected male is crossed with an uninfected female. Three generations after antibiotic treatment, we observed in all strains a significant drop in productivity in the cured stocks. This drop was not due to antibiotic toxicity and was associated with the loss of the Wolbachia. However the effect had disappeared in two of the three strains five generations after treatment, and could not be found in the third strain in a third measurement carried out 14 generations after treatment. The temporary nature of the productivity difference indicates that Wolbachia do not enhance productivity in infected strains. On the other hand, in all traits measured, our results show the absence of any negative effects of the Wolbachia on their host. This could be explained when considering Wolbachia evolution, as maternally transmitted parasites bear a strong selective pressure not to harm their female host. However, CI would allow the bacteria to be maintained even when harming the female. The apparent absence of deleterious effects caused by these Wolbachia might result from a trade-off, where a relatively low bacteria density would advantage the Wolbachia by suppressing any deleterious effects on the female host, at the cost of a weaker maternal transmission rate of the infection.