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.     

Composition of Bacterial Communities Associated with Natural and Laboratory Populations of Asobara tabida Infected with Wolbachia

Asobara tabida wasps are fly endoparasitoids that naturally harbor three Wolbachia strains, which induce cytoplasmic incompatibility and control oogenesis. To investigate whether other bacteria play a role in wasp biology, we surveyed the bacterial communities of wild A. tabida populations originating from different regions of France and of laboratory colonies using PCR-denaturing gradient gel electrophoresis and culture methods. Proteobacteria and Firmicutes were found to be the main phyla represented in these populations. Among these were several cultured and uncultured representatives of the genera Acetobacter, Acidomonas, Bacillus, Brevibacillus, Duganella, Herbaspirillum, Pseudomonas, Staphylococcus, and Streptococcus. In addition to Wolbachia, wild individuals harbored Rickettsia, which tended to be lost when insects were reared in the laboratory. The antibiotic treatment used to generate wasp sublines singly infected with Wolbachia also affected the overall bacterial composition, with most fingerprint sequences being characteristic of the family Enterobacteriaceae. We also screened for potentially heritable endosymbionts by PCR and fluorescence in situ hybridization in stable laboratory lines, with only Wolbachia being consistently found in wasp ovaries.Bacteria associated with insects play a crucial role in host development, survival, and reproduction (13). Many insects harbor bacterial endosymbionts, which establish close relationships, like the mutualistic interaction between aphids and their primary endosymbiont of the genus Buchnera; the bacterium uses the host as a habitat to which it supplies essential amino acids, facilitating insect growth when the diet of plant phloem sap is insufficient (5, 23). Aphids host many other nonessential bacteria as secondary or facultative symbionts. However, aphids with secondary symbionts can gain a fitness advantage in terms of diet, regimen plant host range, heat tolerance, or resistance to pathogens and parasitoids (reviewed in references 45 and 48). Multiple infections are costly to hosts and are perhaps maintained because of the benefits they confer. Recently, Wolbachia has been shown to protect the host Drosophila melanogaster from viral damage (37). However, investigating the evolutionary significance of interspecific symbioses in bacterial communities in invertebrates is challenging in that the majority of bacteria are not yet cultivable outside host cells.Here we analyzed the main bacterial populations of Asobara tabida (Hymenoptera: Braconidae), endoparasitoids of Drosophila species and related genera (14). Usually, members of A. tabida are naturally multiply infected with bacteria of the genus Wolbachia, obligate intracellular Alphaproteobacteria of the order Rickettsiales (2, 25, 51), found in association with numerous arthropods, mainly insects, and certain nematodes, where they are mostly vertically transmitted from mother to progeny (74). The interaction between Wolbachia spp. and their hosts is very complex and ranges from parasitism to mutualism. In filarial nematodes, Wolbachia organisms are required in the host''s biology (4), but Wolbachia spp. are mostly parasites that affect arthropod reproduction, such as by inducing parthenogenesis in some parasitoid wasps (63), feminizing genetic males in isopods (9), and inducing male killing and cytoplasmic incompatibility in many insects (39, 64). The wasp A. tabida harbors three Wolbachia strains; strains wAtab1 and wAtab2 induce cytoplasmic incompatibility, whereas wAtab3 is necessary for the completion of oogenesis (19, 20, 73). The involvement of Wolbachia strains in wasp reproduction was discovered when wasps were treated with antibiotics to generate lines harboring subsets of Wolbachia or aposymbiotic lines (19, 21). Only oogenesis was affected by curing Wolbachia wAtab3; other traits, such as insect size, weight, locomotion, and behavior, were unchanged (19). While antibiotic treatment has been used to determine biological roles of symbionts in this way, their effect on the overall composition of bacterial populations has not been investigated.To investigate the potential role of bacterial endosymbionts in the biology of A. tabida, we studied the bacterial communities in insect populations originating from different regions of France using culture and nonculture methods and fluorescence in situ hybridization (FISH). We also examined whether antibiotherapy to generate lines with a subset of Wolbachia strains altered the composition or density of the bacterial communities.     

Bacteria Endosymbiont,Wolbachia, Promotes Parasitism of Parasitoid Wasp Asobara japonica

Wolbachia is the most widespread endosymbiotic bacterium that manipulates reproduction of its arthropod hosts to enhance its own spread throughout host populations. Infection with Wolbachia causes complete parthenogenetic reproduction in many Hymenoptera, producing only female offspring. The mechanism of such reproductive manipulation by Wolbachia has been extensively studied. However, the effects of Wolbachia symbiosis on behavioral traits of the hosts are scarcely investigated. The parasitoid wasp Asobara japonica is an ideal insect to investigate this because symbiotic and aposymbiotic strains are available: Wolbachia-infected Tokyo (TK) and noninfected Iriomote (IR) strains originally collected on the main island and southwest islands of Japan, respectively. We compared the oviposition behaviors of the two strains and found that TK strain females parasitized Drosophila melanogaster larvae more actively than the IR strain, especially during the first two days after eclosion. Removing Wolbachia from the TK strain wasps by treatment with tetracycline or rifampicin decreased their parasitism activity to the level of the IR strain. Morphological and behavioral analyses of both strain wasps showed that Wolbachia endosymbionts do not affect development of the host female reproductive tract and eggs, but do enhance host-searching ability of female wasps. These results suggest the possibility that Wolbachia endosymbionts may promote their diffusion and persistence in the host A. japonica population not only at least partly by parthenogenesis but also by enhancement of oviposition frequency of the host females.     

Wolbachia sp. (Rickettsiales: Rickettsiaceae) a symbiont of the almond moth,Ephestia cautella: Ultrastructure and influence on host fertility

Wolbachia sp. is a maternally inherited symbiont of the almond moth, Ephestia cautella. It is transmitted through the cytoplasm of the egg and occurs normally in the gonads of all stages of the moth. The symbiont is responsible for reproductive cytoplasmic incompatibility between crosses of experimental laboratory strains of aposymbiotic female moths and symbiotic (normal) males. Although female moths were inseminated in laboratory tests, their eggs failed to hatch and exhibited no signs of embryonic development. The reciprocal cross, i.e., symbiotic female months × aposymbiotic males, produced normal progeny.The ultrastructure of Wolbachia was studied in sections of E. cautella larval testes. Symbionts, minute rod-shaped structures, were abundant in the cytoplasm of hypertrophied spermatids. There was no indication of deleterious influence of symbionts on sperm production or activity. Strains of Wolbachia occur in allopatric populations of insects where they may function as a genetic isolation mechanism. Microorganismal reproductive incompatibility has been suggested as a possible approach for insect control.     

Condition-dependent alteration of cellular immunity by secondary symbionts in the pea aphid,Acyrthosiphon pisum

Endosymbionts can fundamentally alter host physiology. Whether such changes are beneficial or detrimental to one or both partners may depend on the dynamics of the symbiotic relationship. Here we investigate the relationship between facultative symbionts and host immune responses. The pea aphid, Acyrthosiphon pisum, maintains an obligate primary symbiont, but may also harbour one or more facultative, secondary symbionts. Given their more transient nature and relatively recent adoption of a symbiotic lifestyle compared to primary symbionts, secondary symbionts may present a challenge for the host immune system. We assessed the response of several key components of the cellular immune system (phenoloxidase activity, encapsulation, immune cell counts) in the presence of alternative secondary symbionts, investigating the role of host and secondary symbiont genotype in specific responses. There was no effect of secondary symbiont presence on the phenoloxidase response, but we found variation in the encapsulation response and in immune cell counts based largely on the secondary symbiont. Host genotype was less influential in determining immunity outcomes. Our results highlight the importance of secondary symbionts in shaping host immunity. Understanding the complex physiological responses that can be propagated by host-symbiont associations has important consequences for host ecology, including symbiont and pathogen transmission dynamics.     

Arsenophonus and Sodalis Symbionts in Louse Flies: an Analogy to the Wigglesworthia and Sodalis System in Tsetse Flies

Symbiosis between insects and bacteria result in a variety of arrangements, genomic modifications, and metabolic interconnections. Here, we present genomic, phylogenetic, and morphological characteristics of a symbiotic system associated with Melophagus ovinus, a member of the blood-feeding family Hippoboscidae. The system comprises four unrelated bacteria representing different stages in symbiosis evolution, from typical obligate mutualists inhabiting bacteriomes to freely associated commensals and parasites. Interestingly, the whole system provides a remarkable analogy to the association between Glossina and its symbiotic bacteria. In both, the symbiotic systems are composed of an obligate symbiont and two facultative intracellular associates, Sodalis and Wolbachia. In addition, extracellular Bartonella resides in the gut of Melophagus. However, the phylogenetic origins of the two obligate mutualist symbionts differ. In Glossina, the mutualistic Wigglesworthia appears to be a relatively isolated symbiotic lineage, whereas in Melophagus, the obligate symbiont originated within the widely distributed Arsenophonus cluster. Although phylogenetically distant, the two obligate symbionts display several remarkably similar traits (e.g., transmission via the host''s “milk glands” or similar pattern of genome reduction). To obtain better insight into the biology and possible role of the M. ovinus obligate symbiont, “Candidatus Arsenophonus melophagi,” we performed several comparisons of its gene content based on assignments of the Cluster of Orthologous Genes (COG). Using this criterion, we show that within a set of 44 primary and secondary symbionts, “Ca. Arsenophonus melophagi” is most similar to Wigglesworthia. On the other hand, these two bacteria also display interesting differences, such as absence of flagellar genes in Arsenophonus and their presence in Wigglesworthia. This finding implies that a flagellum is not essential for bacterial transmission via milk glands.     

Obligate gut symbiotic association in the sloe bug <Emphasis Type="Italic">Dolycoris baccarum</Emphasis> (Hemiptera: Pentatomidae)

A number of phytophagous stinkbugs are associated with specific bacterial symbionts in their alimentary tracts. The sloe bug Dolycoris baccarum (Linnaeus), a notorious pest of diverse crops, possesses a number of sac-like tissues, called crypts, in a posterior section of the midgut, wherein a specific bacterial symbiont colonizes. Here we characterized the symbiotic bacterium of D. baccarum by histological analysis, molecular phylogeny, and diagnostic PCR with a specific primer set. The cloning and sequencing analyses of bacterial 16S rRNA genes and fluorescent in situ hybridization demonstrated that the sloe bug is associated with a single species of Gammaproteobacteria in the midgut crypts. Molecular phylogenetic analysis strongly suggested that the symbiont should be placed in the genus Pantoea of the Enterobacteriaceae. Diagnostic PCR and egg surface sterilization with formalin indicated the stinkbug vertically transmits the Pantoea symbiont via egg-smearing. The sterilization-produced aposymbiotic nymphs showed high mortality and no insects reached adulthood. In addition, the Pantoea symbiont was uncultivable outside the insect host, indicating an obligate and intimate host-symbiont association.     

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. To cite this article: H. Merçot, D. Poinsot, C. R. Biologies 332 (2009).     

Specific Midgut Region Controlling the Symbiont Population in an Insect-Microbe Gut Symbiotic Association

Many insects possess symbiotic bacteria that affect the biology of the host. The level of the symbiont population in the host is a pivotal factor that modulates the biological outcome of the symbiotic association. Hence, the symbiont population should be maintained at a proper level by the host''s control mechanisms. Several mechanisms for controlling intracellular symbionts of insects have been reported, while mechanisms for controlling extracellular gut symbionts of insects are poorly understood. The bean bug Riptortus pedestris harbors a betaproteobacterial extracellular symbiont of the genus Burkholderia in the midgut symbiotic organ designated the M4 region. We found that the M4B region, which is directly connected to the M4 region, also harbors Burkholderia symbiont cells, but the symbionts therein are mostly dead. A series of experiments demonstrated that the M4B region exhibits antimicrobial activity, and the antimicrobial activity is specifically potent against the Burkholderia symbiont but not the cultured Burkholderia and other bacteria. The antimicrobial activity of the M4B region was detected in symbiotic host insects, reaching its highest point at the fifth instar, but not in aposymbiotic host insects, which suggests the possibility of symbiont-mediated induction of the antimicrobial activity. This antimicrobial activity was not associated with upregulation of antimicrobial peptides of the host. Based on these results, we propose that the M4B region is a specialized gut region of R. pedestris that plays a critical role in controlling the population of the Burkholderia gut symbiont. The molecular basis of the antimicrobial activity is of great interest and deserves future study.     

Endosymbiont costs and benefits in a parasitoid infected with both Wolbachia and Cardinium

Theory suggests that maternally inherited endosymbionts can promote their spread and persistence in host populations by enhancing the production of daughters by infected hosts, either by improving overall host fitness, or through reproductive manipulation. In the doubly infected parasitoid wasp Encarsia inaron, Wolbachia manipulates host reproduction through cytoplasmic incompatibility (CI), but Cardinium does not. We investigated the fitness costs and/or benefits of infection by each bacterium in differentially cured E. inaron as a potential explanation for persistence of Cardinium in this population. We introgressed lines infected with Wolbachia, Cardinium or both with the cured line to create a similar genetic background, and evaluated several parasitoid fitness parameters. We found that symbiont infection resulted in both fitness costs and benefits for E. inaron. The cost was lower initial egg load for all infected wasps. The benefit was increased survivorship, which in turn increased male production for wasps infected with only Cardinium. Female production was unaffected by symbiont infection; we therefore have not yet identified a causal fitness effect that can explain the persistence of Cardinium in the population. Interestingly, the Cardinium survivorship benefit was not evident when Wolbachia was also present in the host, and the reproduction of doubly infected individuals did not differ significantly from uninfected wasps. Therefore, the results of our study show that even when multiple infections seem to have no effect on a host, there may be a complex interaction of costs and benefits among symbionts.     

The evolution of obligate mutualism: if you can't beat 'em, join 'em

Wolbachia is best known as a facultative endosymbiotic parasite, manipulating host reproduction. However, it has also evolved as an obligate mutualist at least twice. In a recent paper, Pannebakker et al. identify a possible mechanism for such a transition from facultative parasitism to obligate mutualism in a parasitic wasp in which Wolbachia are required for producing eggs (oogenesis). Their proposed mechanism suggests that compensatory evolution in the host to counter the harmful effects of Wolbachia is the basis of this evolutionary transition.     

Role of Host Nutrition in Symbiont Regulation: Impact of Dietary Nitrogen on Proliferation of Obligate and Facultative Bacterial Endosymbionts of the Pea Aphid Acyrthosiphon pisum

The impact of host nutrition on symbiont regulation in the pea aphid Acyrthosiphon pisum was investigated. The population density of the obligate symbiont Buchnera aphidicola positively correlated with dietary nitrogen levels. In contrast, the population density of the facultative symbiont Serratia symbiotica increased in aphids reared on low-nitrogen diets, indicating distinct regulatory mechanisms in the same insect host.     

Molecular Characterization of Wolbachia Strains Associated with the Invasive Asian Citrus Psyllid Diaphorina citri in Brazil

Wolbachia is a symbiont intensively studied due to its ability to interfere with their host’s reproduction, and it has been recently proposed as an alternative tool to control insect pests or vectors of diseases. The Asian citrus psyllid Diaphorina citri is an important pest of citrus since it vectors the bacterium that causes the "Huanglongbing" disease in citrus. The frequency and diversity of Wolbachia associated with D. citri is unknown, limiting the utilization of Wolbachia as an alternative strategy for insect management. Thus, we aimed to determine the natural rate of infection, to characterize the Wolbachia strains associated with this psyllid by "multilocus sequencing typing” (MLST) and wsp analysis, and to verify the association of the symbiont to particular genotypes of the host. Analysis indicated Wolbachia infects 100 % of all specimens tested from all 15 sampled populations. MLST revealed the occurrence of five new sequence types (STs) of Wolbachia, while analysis based on the wsp sequences indicated only four different types of Wolbachia. ST-173 was predominant, while the remaining STs were population specific. Analysis of the host–symbiont relationship did not reveal any particular association of Wolbachia and haplotypes or a decrease in nucleotide diversity of D. citri in populations in which more than one ST was recorded. The consequences of the diversity of STs reported are still unknown, but the fact that Wolbachia infection is fixed and that there is one ST with a broad distribution highlights the use of this symbiont as an alternative strategy to control D. citri.     

Loss and resiliency of social amoeba symbiosis under simulated warming

Anthropogenic global change is increasingly raising concerns about collapses of symbiotic interactions worldwide. Therefore, understanding how climate change affects symbioses remains a challenge and demands more study. Here, we look at how simulated warming affects the social ameba Dictyostelium discoideum and its relationship with its facultative bacterial symbionts, Paraburkholderia hayleyella and Paraburkholderia agricolaris. We cured and cross‐infected ameba hosts with different symbionts. We found that warming significantly decreased D. discoideum''s fitness, and we found no sign of local adaptation in two wild populations. Experimental warming had complex effects on these symbioses with responses determined by both symbiont and host. Neither of these facultative symbionts increases its hosts’ thermal tolerance. The nearly obligate symbiont with a reduced genome, P. hayleyella, actually decreases D. discoideum''s thermal tolerance and even causes symbiosis breakdown. Our study shows how facultative symbioses may have complex responses to global change.     

Micronucleus‐Specific Bacterium Holospora elegans Irreversibly Enhances Stress Gene Expression of the Host Paramecium caudatum

ABSTRACT. The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear‐specific symbiont Holospora obtusa survived better than symbiont‐free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25 °C, a usual growth temperature. We report herein that paramecia bearing the micronuclear‐specific symbiont Holospora elegans also acquire the heat‐shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock‐resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25 °C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat‐shock resistance.     

Comparative Genomics Suggests an Independent Origin of Cytoplasmic Incompatibility in Cardinium hertigii

Terrestrial arthropods are commonly infected with maternally inherited bacterial symbionts that cause cytoplasmic incompatibility (CI). In CI, the outcome of crosses between symbiont-infected males and uninfected females is reproductive failure, increasing the relative fitness of infected females and leading to spread of the symbiont in the host population. CI symbionts have profound impacts on host genetic structure and ecology and may lead to speciation and the rapid evolution of sex determination systems. Cardinium hertigii, a member of the Bacteroidetes and symbiont of the parasitic wasp Encarsia pergandiella, is the only known bacterium other than the Alphaproteobacteria Wolbachia to cause CI. Here we report the genome sequence of Cardinium hertigii cEper1. Comparison with the genomes of CI–inducing Wolbachia pipientis strains wMel, wRi, and wPip provides a unique opportunity to pinpoint shared proteins mediating host cell interaction, including some candidate proteins for CI that have not previously been investigated. The genome of Cardinium lacks all major biosynthetic pathways but harbors a complete biotin biosynthesis pathway, suggesting a potential role for Cardinium in host nutrition. Cardinium lacks known protein secretion systems but encodes a putative phage-derived secretion system distantly related to the antifeeding prophage of the entomopathogen Serratia entomophila. Lastly, while Cardinium and Wolbachia genomes show only a functional overlap of proteins, they show no evidence of laterally transferred elements that would suggest common ancestry of CI in both lineages. Instead, comparative genomics suggests an independent evolution of CI in Cardinium and Wolbachia and provides a novel context for understanding the mechanistic basis of CI.     

Influence of oxidative homeostasis on bacterial density and cost of infection in Drosophila–Wolbachia symbioses

The evolution of symbioses along the continuum between parasitism and mutualism can be influenced by the oxidative homeostasis, that is the balance between reactive oxygen species (ROS) and antioxidant molecules. Indeed, ROS can contribute to the host immune defence to regulate symbiont populations, but are also toxic. This interplay between ROS and symbiosis is notably exemplified by recent results in arthropod–Wolbachia interactions. Wolbachia are symbiotic bacteria involved in a wide range of interactions with their arthropods hosts, from facultative, parasitic associations to obligatory, mutualistic ones. In this study, we used Drosophila–Wolbachia associations to determine whether the oxidative homeostasis plays a role in explaining the differences between phenotypically distinct arthropod–Wolbachia symbioses. We used Drosophila lines with different Wolbachia infections and measured the effects of pro‐oxidant (paraquat) and antioxidant (glutathione) treatments on the Wolbachia density and the host survival. We show that experimental manipulations of the oxidative homeostasis can reduce the cost of the infection through its effect on Wolbachia density. We discuss the implication of this result from an evolutionary perspective and argue that the oxidative homeostasis could underlie the evolution of tolerance and dependence on Wolbachia.