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

M.M.M. van Meer

Research output: Thesisinternal PhD, WU


<H3>Summary and conclusions</H3><p>Bacteria of the genus <em>Wolbachia</em> (α-Proteobacteria, Rickettsia) are widespread in arthropods and can induce thelytoky (T) in parasitoids (Hymenoptera). Infection with thelytoky inducing <em>Wolbachia</em> (T- <em>Wolbachia</em> ) enables infected females to produce daughters from unfertilised eggs. Thelytokous strains can be maintained without the involvement of males. T- <em>Wolbachia</em> 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- <em>Wolbachia</em> . To determine whether this approach was feasible, several experiments were done to improve our understanding of this specific host-symbiont relationship.</p><H3>Phylogeny</H3><p>The phylogeny of <em>Wolbachia</em> has been studied using 16S ribosomal DNA (rDNA) and the cell cycle gene <em>ftsZ</em> 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 <em>Wolbachia</em> is 66 basepairs (bp) long and shows slightly higher sequence differences between strains than <em>ftsZ</em> . Due to the short length of SR2 of <em>Wolbachia</em> , little phylogenetic information could be retrieved. Additional phylogenetic research was done using the sequence of an outer membrane protein ( <em>wsp</em> ) of <em>Wolbachia</em> . Previous research in Yale (USA) showed that this gene evolved at a much faster rate than 16S rDNA or <em>ftsZ</em> and the <em>Wolbachia</em> clade was subdivided into twelve distinct groups based on the proposed 2.5% <em>wsp</em> sequence divergence grouping criterion. We extended this former <em>Wolbachia</em><em>wsp</em> data set with fifteen T- <em>Wolbachia</em> strains and our results showed that: 1)Four new tested thelytokous parasitoids species were as well infected with <em>Wolbachia</em> ( <em>Amitus fuscipennis</em> , <em>Apoanagyrus diversicornis</em> , <em>Coccidoxenoides peregrinus</em> , <em>Eretmocerus staufferi</em> ); 2) T- <em>Wolbachia</em> clones are not closely related; 3) Based on identical <em>wsp</em> sequences of the moth <em>Ephestia kuehniella</em> (Lepidoptera) and its parasitoid <em>Trichogramma</em> , possible horizontal <em>Wolbachia</em> transfer between them was postulated. The exact mechanism of horizontal transfer remains to be clarified.</p><H3>Micro-injection</H3><p>Our goal was to do a T- <em>Wolbachia</em> transfer experiment between hymenopteran species to test whether arrhenotokous wasps could be rendered thelytokous. Infection of a host with <em>Wolbachia</em> can be accomplished by microinjection of the bacteria in an insect egg as shown for <em>Drosophila</em> sp. However, no suitable micro-injection protocol for parasitoids was available. We developed a injection protocol for the gregarious fly pupa parasitoid <em>Nasonia</em><em>vitripennis</em> 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 <em>Drosophila</em> spp. and <em>Tribolium confusum</em> were combined and optimal conditions for each of the different steps were determined. In addition, an <em>in vitro</em> incubation step for the <em>N. vitripennis</em> larvae had to be included. The final protocol enables us to do <em>Wolbachia</em> transfer studies in this species.</p><H3><em>Host</em> Wolbachia <em>interactions</em></H3><p>Different fitness parameters of T- <em>Wolbachia</em> infected and non-infected <em>Trichogramma</em> species were studied. <em>Trichogramma</em> are minute wasps which are widely used in biological control programs against lepidopteran pests. We distinguished two different <em>Trichogramma</em> 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 <em>Trichogramma</em> because both mixed and fixed populations exist within this genus.</p><p>Two isofemale lines from fixed populations and four isofemale lines from mixed populations were 'cured' of <em>Wolbachia</em> 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 <em>Wolbachia</em> 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).</p><p>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 <em>Wolbachia</em> 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 <em>Wolbachia</em> infection had an impact on the 'parasitization efficiency' of the <em>Trichogramma</em> species <em>T. deion</em> and <em>T. cordubensis</em> in greenhouse compartments. Laboratory studies, to assess the effect of <em>Wolbachia</em> 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 <em>Trichogramma</em> in a laboratory chamber showed that for both species, the arrhenotokous lines dispersed more than their thelytokous counterparts, suggesting a negative effect of <em>Wolbachia</em> 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.</p><p>These results correspond with the laboratory fecundity experiments. However, in contrast to laboratory chamber experiments, thelytokous <em>T. deion</em> females dispersed equally well as their arrhenotokous counterparts while thelytokous <em>T. cordubensis</em> 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 <em>Wolbachia</em> on host fitness is taken into account.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • van Lenteren, Joop, Promotor
  • Stouthamer, R., Promotor
Award date7 May 1999
Place of PublicationS.l.
Print ISBNs9789058080509
Publication statusPublished - 1999


  • wolbachia
  • symbionts
  • hosts
  • hymenoptera
  • host parasite relationships
  • thelytoky
  • phylogeny
  • biological control
  • agricultural entomology
  • insect pests

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