Wolbachia are maternally inherited endosymbiotic bacteria of arthropods and nematodes. In arthropods, they manipulate the reproduction of their hosts to facilitate their own spread in host populations, causing cytoplasmic incompatibility, parthenogenesis induction, feminization of genetic males and male-killing. In this study, we investigated Wolbachia infection and studied wsp (Wolbachia surface protein) sequences in three wasp species associated with the unisexual galls of A. mukaigawae with the aim of determining the transmission mode and the reason for multiple infections of Wolbachia. Frequency of Wolbachia infected populations for A. mukaigawae, Synergus japonicus (inquiline), and Torymus sp. (parasitoid) was 75%, 100%, and 100%, respectively. Multiple Wolbachia infections were detected in A. mukaigawae and S. japonicus, with 5 and 8 Wolbachia strains, respectively. The two host species shared 5 Wolbachia strains and were infected by identical strains in several locations, indicating horizontal transmission of Wolbachia. The transmission potentially takes place through gall tissues, which the larvae of both wasps feed on. Furthermore, three recombination events of Wolbachia were observed: the strains W8, W2 and W6 apparently have derived from W3 and W5a, W6 and W7, W4 and W9, respectively. W8 and W2 and their respective parental strains were detected in S. japonicus. W6 was detected with only one parent (W4) in S. japonicus; W9 was detected in Torymus sp., suggesting horizontal transmission between hosts and parasitoids. In conclusion, our research supports earlier studies that horizontal transmission of Wolbachia, a symbiont of the Rickettsiales order, may be plant-mediated or take place between hosts and parasitoids. Our research provides novel molecular evidence for multiple recombination events of Wolbachia in gall wasp communities. We suggest that genomic recombination and potential plant-mediated horizontal transmission may be attributable to the high levels of multiple Wolbachia infections observed in A. mukaigawae and S. japonicus.

The Oriental chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), is a global invasive pest that causes serious damage to almost all chestnut species belonging to the Castanea genus (Fagaceae). Dryocosmus zhuili Liu et Zhu is a recently described sibling species of D. kuriphilus, which induces galls on Castanea henryi (Skan) Rehd. et Wils. There are many indigenous parasitoid species in China which play an important role in the natural regulation of their population dynamics. Wolbachia is a maternally inherited α‐proteobacterium widely found in arthropods. This study screened for the presence of Wolbachia in the two chestnut gall wasps and in six parasitoid species from 12 populations, to investigate the prevalence patterns of Wolbachia in the chestnut gall wasp‐parasitoid communities. We found that D. zhuili and four parasitoid species were infected with Wolbachia; among them, all individuals of the two populations of Megastigmus sp. had multiple Wolbachia infections. By using multilocus sequence types to characterize bacterial strains, three new sequence types were identified. The Wolbachia strains infecting D. zhuili (ST‐507), Torymus sinensis Kamijo (ST‐508), and Sycophila variegata (Curtis) (ST‐508) belonged to supergroup A, whereas the Wolbachia strain infecting Megastigmus nipponicus Kamijo (ST‐503) belonged to supergroup B. Our results also suggested that horizontal transmission of Wolbachia occurs between chestnut gall wasps and their parasitoids. Moreover, multiple Wolbachia infections of Megastigmus sp. may be due to gene recombination and horizontal transmission.

The phage WO was characterized in Wolbachia, a strictly intracellular bacterium causing several reproductive alterations in its arthropod hosts. This study aimed to screen the presence of Wolbachia and phage WO in 15 gall wasp species from six provinces of southern China to investigate their diversity and prevalence patterns. A high incidence of Wolbachia infection was determined in the gall wasp species, with an infection rate of 86.7% (13/15). Moreover, seven species had double or multiple infections. All Wolbachia-infected gall wasp species were found to harbor phage WO. The gall wasp species infected with a single Wolbachia strain were found to harbor a single phage WO type. On the contrary, almost all species with double or multiple Wolbachia infections harbored a high level of phage WO diversity (ranging from three to 27 types). Six horizontal transfer events of phage WO in Wolbachia were found to be associated with gall wasps, which shared identical orf7 sequences among their respective accomplices. The transfer potentially took place through gall inducers and associated inquilines infected with or without Wolbachia. Furthermore, 10 putative recombination events were identified from Andricus hakonensis and Andricus sp2, which harbored multiple phage WO types, suggesting that intragenic recombination was the important evolutionary force, which effectively promoted the high level of phage WO diversity associated with gall wasps.

A new gall wasp species, Dryocosmus zhuili Liu et Zhu, is herein described from the southeastern Fujian province of China. The new species induces galls on trees of Henry’s chestnut, Castanea henryi, which is also a native host for the notorious Oriental chestnut gall wasp (OCGW, Dryocosmus kuriphilus Yasumatsu). D. zhuili overlaps with OCGW in emergence time and induces galls morphologically similar to that of OCGW on similar plant parts. In a previous study, we reported considerable divergence between mtDNA CO1 (mitochondrial DNA Cytochrome c oxidase subunit I) sequences of these wasps and the true OCGW wasps and suggested the existence of a cryptic species. Herein, we confirm the identity of the new species based on morphological and biological differences and provide a formal description. Although the new species is relatively easily separated from OCGW on basis of morphology, field identification involving the two species can still be problematic because of their small body size, highly similar gall morphology, and other life history traits. We further discussed the potential of the new species to be a pest for the chestnut industry and the consequences of accidental introduction of this species into nonnative areas, especially with regard to the bisexual reproduction mode of the new species in contrast to the parthenogenetic reproduction mode of OCGW.

A new gall wasp species, Dryocosmus liui Pang, Su et Zhu n. sp., is described from Hunan Province, China. The new species forms integral galls on branches of Castanopsis tibetana Hance. The results of morphological comparison, pair-wise genetic distance of mtDNA COI, and phylogenetic analysis based on COI gene sequences did not support treating Chilaspis and Dryocosmus as separate genera. Our results also provided new evidence supporting the notion that the Nearctic Dryocosmus species may represent a lineage distinct from the Old World species of the genus.