Phylogeny and host-symbiont interactions of thelytoky inducing Wolbachia in Hymenoptera

Summary and conclusionsBacteria of the genus Wolbachia (α-Proteobacteria, Rickettsia) are widespread in arthropods and can induce thelytoky (T) in parasitoids (Hymenoptera). Infection with thelytoky inducing Wolbachia (T- Wolbachia ) enables infected females to produce daughters from unfertilised eggs. Thelytokous strains can be maintained without the involvement of males. T- Wolbachia may represent a tool to improve biological control because only female parasitoids attack target pest species. Advantages of thelytokous reproduction of parasitic wasps in biological control programs may include: lower costs of mass rearing; faster population growth after release and easier establishment of thelytokous wasps in pest populations.Therefore, it was suggested to render sexual reproducing parasitoids thelytokous by infecting them with T- Wolbachia . To determine whether this approach was feasible, several experiments were done to improve our understanding of this specific host-symbiont relationship.PhylogenyThe phylogeny of Wolbachia has been studied using 16S ribosomal DNA (rDNA) and the cell cycle gene ftsZ but sequence variation of those genes is limited. The spacer 2 region (SR2), the region between 23S rDNA and 5S rDNA, was amplified to determine if this region would improve phylogenetic resolution. The SR2 of Wolbachia is 66 basepairs (bp) long and shows slightly higher sequence differences between strains than ftsZ . Due to the short length of SR2 of Wolbachia , little phylogenetic information could be retrieved. Additional phylogenetic research was done using the sequence of an outer membrane protein ( wsp ) of Wolbachia . Previous research in Yale (USA) showed that this gene evolved at a much faster rate than 16S rDNA or ftsZ and the Wolbachia clade was subdivided into twelve distinct groups based on the proposed 2.5% wsp sequence divergence grouping criterion. We extended this former Wolbachiawsp data set with fifteen T- Wolbachia strains and our results showed that: 1)Four new tested thelytokous parasitoids species were as well infected with Wolbachia ( Amitus fuscipennis , Apoanagyrus diversicornis , Coccidoxenoides peregrinus , Eretmocerus staufferi ); 2) T- Wolbachia clones are not closely related; 3) Based on identical wsp sequences of the moth Ephestia kuehniella (Lepidoptera) and its parasitoid Trichogramma , possible horizontal Wolbachia transfer between them was postulated. The exact mechanism of horizontal transfer remains to be clarified.Micro-injectionOur goal was to do a T- Wolbachia transfer experiment between hymenopteran species to test whether arrhenotokous wasps could be rendered thelytokous. Infection of a host with Wolbachia can be accomplished by microinjection of the bacteria in an insect egg as shown for Drosophila sp. However, no suitable micro-injection protocol for parasitoids was available. We developed a injection protocol for the gregarious fly pupa parasitoid Nasoniavitripennis because with this species, large number of eggs could be collected relatively easy. This facilitated the testing of the different steps of the micro-injection procedure. Different available micro-injection protocols of Drosophila spp. and Tribolium confusum were combined and optimal conditions for each of the different steps were determined. In addition, an in vitro incubation step for the N. vitripennis larvae had to be included. The final protocol enables us to do Wolbachia transfer studies in this species.Host Wolbachia interactionsDifferent fitness parameters of T- Wolbachia infected and non-infected Trichogramma species were studied. Trichogramma are minute wasps which are widely used in biological control programs against lepidopteran pests. We distinguished two different Trichogramma populations: 1) 'Fixed' populations in which the infection is fully established so that only thelytokous females are present and 2) 'Mixed' populations in which thelytokous females coexist with arrhenotokous ones. In mixed populations thelytokous females are still able to mate and to produce daughters sexually. In mixed populations, a potential cytoplasmic-nuclear conflict exists but in fixed populations, this conflict is absent. It is theorised that fixation of the infection results in a reduction of negative impact of the symbiont on its host. This hypothesis was tested with the egg parasitoid Trichogramma because both mixed and fixed populations exist within this genus.Two isofemale lines from fixed populations and four isofemale lines from mixed populations were 'cured' of Wolbachia infection using antibiotics and different lifespan fitness parameters were measured. Daughter production was significantly higher for the thelytokous fixed lines (16-131% more daughters) compared to the conspecific arrhenotokous ones. This is in contrast to the three mixed lines where the opposite was found (6-61% less daughters). Only slight fecundity effects of Wolbachia were found in the fixed lines (varying among 19% less offspring and 6% more offspring) while these effects are clearly negative in the mixed lines (34-49% less offspring).Finally, we determined whether thelytokous wasps do equally well as biological control agents as their arrhenotokous counterparts. Theoretically, thelytokous wasps may be better biological control agents than the arrhenotokous ones. However, previous studies, show that Wolbachia can have a negative fecundity impact on their host. Therefore, other fitness parameters such as host searching efficacy, dispersal etc. could also be affected. We assessed whether Wolbachia infection had an impact on the 'parasitization efficiency' of the Trichogramma species T. deion and T. cordubensis in greenhouse compartments. Laboratory studies, to assess the effect of Wolbachia on host fecundity and dispersal were also done to correlate these results with the greenhouse experiment results. Laboratory results showed: 1) The fecundity of the thelytokous wasps species was reduced compared to the arrhenotokous counterparts; 2) Experiments of Trichogramma in a laboratory chamber showed that for both species, the arrhenotokous lines dispersed more than their thelytokous counterparts, suggesting a negative effect of Wolbachia on dispersal.Greenhouse experiments showed for both species that thelytokous wasps parasitize approximately equal number of patches but parasitize fewer eggs per patch than the arrhenotokous females.These results correspond with the laboratory fecundity experiments. However, in contrast to laboratory chamber experiments, thelytokous T. deion females dispersed equally well as their arrhenotokous counterparts while thelytokous T. cordubensis females showed significant more dispersal than the arrhenotokous ones. No explanation was found for these dispersal differences in greenhouse or laboratory chamber experimental set-ups. According to calculations, it is still advantageous to use thelytokous parasitoids for biological control when the negative impact of Wolbachia on host fitness is taken into account.</p

Molecular evolution and phylogenetic utility of Wolbachia ftsZ and wsp gene sequences with special reference to the origin of male-killing

A detailed assessment of the evolution and phylogenetic utility of two genes, ftsZ and wsp, was used to investigate the origin of male-killing Wolbachia, previously isolated from the ladybird Adalia bipunctata and the butterfly Acraea encedon. The analysis included almost all available sequences of B-group Wolbachia and two outgroup taxa and showed that (1) the two gene regions differ in phylogenetic utility, (2) sequence variation is here correlated with phylogenetic information content, (3) both genes show significant rate heterogeneity between lineages, (4) increased substitution rates are associated with homoplasy in the data, (5) wsp sequences of some taxa appear to be subject to positive selection, and (6) only a limited number of clades can be inferred with confidence due to either lack of phylogenetic information or the presence of homoplasy. With respect to the evolution of male-killing, the two genes nevertheless seemed to provide unbiased information. However, they consistently produce contradictory results. Current data therefore do not permit clarification of the origin of this behavior. In addition, A. bipunctata was found to be a host to two recently diverged strains of male-killing Wolbachia that showed increased substitution rates for both genes. Moreover, the wsp gene, which codes for an outer membrane protein, was found to be subject to positive selection in these taxa. These findings were postulated to be the product of high selection pressures due to antagonistic host-symbiont interactions in this ladybird species. In conclusion, our study demonstrates that the results of a detailed phylogenetic analysis, including characterization of the limitations of such an approach, can serve as a valuable basis for an understanding of the evolution of Wolbachia bacteria. Moreover, particular features of gene evolution, such as elevated substitution rates or the presence of positive selection, may provide information about the dynamics of Wolbachia-host associations

Phylogeny of the arthropod endosymbiont Wolbachia based on the wsp gene

Bacteria of the genus Wolbachia (Rickettsiae) are widespread in arthropods and can induce cytoplasmic incompatibility (CI), thelytoky (T) or feminization (F) in their host. Recent research on the wsp gene of mainly CI inducing Wolbachia has shown that this gene evolves at a much faster rate than previously sequenced genes such as 16S or ftsZ. As a result this gene appears to be very useful in subdividing the Wolbachia and twelve groups have been distinguished to date. Here we extend the Wolbachia wsp data set with fifteen T-Wolbachia, one F-Wolbachia and three other CI-Wolbachia strains. The results showed: (i) the addition of seven groups; (ii) no relation between host phenotype and Wolbachia phylogenetic position; and (iii) possible horizontal Wolbachia transfer between the moth Ephestia kuehniella and its parasitoid Trichogramma spp

Phylogeny of the arthropod endosymbiont Wolbachia based on the wsp gene

Bacteria of the genus Wolbachia (Rickettsiae) are widespread in arthropods and can induce cytoplasmic incompatibility (CI), thelytoky (T) or feminization (F) in their host. Recent research on the wsp gene of mainly CI inducing Wolbachia has shown that this gene evolves at a much faster rate than previously sequenced genes such as 16S or ftsZ. As a result this gene appears to be very useful in subdividing the Wolbachia and twelve groups have been distinguished to date. Here we extend the Wolbachia wsp data set with fifteen T-Wolbachia, one F-Wolbachia and three other CI-Wolbachia strains. The results showed: (i) the addition of seven groups; (ii) no relation between host phenotype and Wolbachia phylogenetic position; and (iii) possible horizontal Wolbachia transfer between the moth Ephestia kuehniella and its parasitoid Trichogramma spp