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

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.