A new case of Wolbachia dependence in the genus Asobara: evidence for parthenogenesis induction in Asobara japonica

Abstract

Wolbachia is a maternally inherited bacterium that is widely distributed among arthropods, in which it manipulates the reproduction of its hosts. Although generally facultative for its hosts, Wolbachia has recently become obligatory in Asobara tabida (Hymenoptera: Braconidae) in which it is required for the completion of oogenesis. Here, we describe a new Wolbachia strain (wAjap) that is associated with the genus Asobara and infects Asobara japonica. wAjap was detected in all female-biased populations of A. japonica found in the main islands of Japan, but not in the arrhenotokous populations from the southern islands. Using phylogenetic analyses based on multi-locus sequence typing (MLST), we show that this strain is closely related to wAtab3 (the strain required for oogenesis in A. tabida), even though they differ on Wolbachia surface protein (WSP) and WO phage sequences. Using antibiotic treatments, we show that cured thelytokous females are not dependent on Wolbachia for oogenesis. However, they produced only sons, showing that wAjap induces thelytokous parthenogenesis. Analyses of mating behavior and offspring production of individuals from Wolbachia-infected populations showed that while males were still sexually functional, females no longer attract males, making Wolbachia an obligate partner for daughter production in thelytokous populations. The fact that Wolbachia has become independently obligatory in two species of the same genus tends to show that dependence evolution can be common and swift, although no clear benefit for the parasitoid can be attributed to this dependence. Although dependence should lead to co-divergence between Wolbachia and its hosts, the very few cases of co-speciation observed in host-Wolbachia associations question the stability of these obligatory associations.

Occasional males in parthenogenetic populations of Asobara japonica (Hymenoptera: Braconidae): low Wolbachia titer or incomplete coadaptation?

“…Populations of A. japonica on the main islands of Japan exhibit highly femalebiased sex ratios (92.7-99.2% females), whereas population sex ratios on the smaller southern islands are not biased (Mitsui et al, 2007). The populations on the main islands are infected with parthenogenesis-inducing Wolbachia, while the populations on the smaller southern islands are not (Kremer et al, 2009). Also, during routine culturing in our lab, Wolbachia-infected A. japonica females regularly produce small numbers of male offspring and in rare cases even male-biased sex ratios.…”

“…An alternative possibility might be that the strain from Amami was infected with Wolbachia before, but has lost its infection. However, this seems unlikely because thelytokous females that have been cured from their Wolbachia infection with antibiotics are not capable of sexual reproduction (Kremer et al, 2009).…”

“…One-week-old females from five thelytokous strains (Sapporo, Hirosaki, Sendai, Tokyo and Kagoshima) were placed individually in a glass jar with a medium of agar covered by a layer of 2 ml baker's yeast suspension, in which they were allowed to parasitize about 100-200 2-day-old (first or second instar) larvae of D. melanogaster. As thelytokous A. japonica females are not capable of sexual reproduction (Kremer et al, 2009), we assumed that all females used in the sex ratio experiment were virgins. For each strain, half of the females were kept at 25 1C, light:dark 16:8, 65% relative humidity, while the other half were kept at 20 1C, light:dark 16:8, 65% relative humidity.…”

“…To optimize the qPCRs, we designed new primers for all three genes. Primer sequences for wsp and gatB were based on the sequences for wAjap described in Kraaijeveld et al (2011b), while the primer sequences for ITS2 were based on the sequence for A. japonica described in Kremer et al (2009). The following primers were used: wsp-wAjap-F: 5¢-GAG GCA AAA TTT ACG CCA GA-3¢ and wsp-wAjap-R: 5¢-AAC TAG CCC TGA AAT TGC TGT TA-3¢, producing a 60-bp amplicon; gatB-wAjap-F: 5¢-GAA GCA AAG AGG ATG CAA GC-3¢ and gatB-wAjap-R: 5¢-TCC TGG CTT ACC TCA ACA GG-3¢, producing a 73-bp amplicon; ITS2-Ajap-F: 5¢-GGC AAG CAC AAT CAA GGT CT-3¢ and ITS2-Ajap-R: 5¢-ACA AAA ACA AAT TTT GCG GC-3¢, producing a 93-bp amplicon.…”

“…Also, during routine culturing in our lab, Wolbachia-infected A. japonica females regularly produce small numbers of male offspring and in rare cases even male-biased sex ratios. The production of males in parthenogenetic populations of A. japonica seems to be maladaptive, because parthenogenetic females are not capable of sexual reproduction (Kremer et al, 2009).…”