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In Hymenoptera parasitoids, the reproductive mode is arrhenotoky, while a few species reproduce by thelytoky. The thelytoky of Hymenoptera parasitoids is generally genetically determined by the parasitoids themselves or induced by bacteria, including Wolbachia, Cardinium, and Rickettsia. Diglyphus wani (Hymenoptera: Eulophidae), a recently reported thelytokous species is a main parasitoid attacking agromyzid leafminers. To assess whether endosymbionts induce thelytoky in D. wani, we performed universal PCR detection and sequenced the V3-V4 region of 16S ribosomal RNA gene. In addition, bacteria were removed through high-temperature and antibiotic treatments, and the localized bacteria were detected using FISH. Based on general PCR detection, Wolbachia, Cardinium, Rickettsia, Arsenophonus, Spiroplasma, and Microsporidia were absent in laboratory and field individuals of thelytokous D. wani. Furthermore, 16S rRNA gene sequencing revealed that the dominant endosymbionts in thelytokous D. wani were not reproductive manipulators. High-temperature and antibiotic treatment for five consecutive generations cannot reverse the thelytokous pattern of D. wani, and no male offspring were produced. Moreover, no bacterial spots were found in the ovaries of D. wani. Thus, it is considered that the thelytoky of D. wani does not result in the presence of endosymbionts. This species is thus the second reported eulophid parasitoid whose thelytoky appears not to be associated with endosymbionts.

Multiple Data Demonstrate That Bacteria Regulating Reproduction Could Be Not the Cause for the Thelytoky of Diglyphus wani (Hymenoptera: Eulophidae)