In the butterfly Eurema hecabe infected with two different strains of bacterial endosymbiont of the genus Wolbachia, genetic males are transformed into functional females, resulting in production of all-female broods.

In an attempt to understand how and when the Wolbachia feminizes genetically male insects, larval insects were fed with an antibiotic-containing diet from different developmental stages until pupation. When these insects emerged, strikingly, many of them exhibited sexually intermediate traits in their wings, reproductive organs and genitalia. The expression of intersexual phenotypes was strong in the insects treated from 1st instar, moderate in the insects treated from 3rd instar, and weak in the insects treated from 4th instar.

These results suggest that the antibiotic treatment suppressed the feminizing effect of Wolbachia, leading to expression of intersexual host traits, and hence, the feminizing Wolbachia continuously acts on the host insects during larval development for expression of female phenotypes under male genotype. These findings may prompt reconsideration of the notion that Wolbachia-induced reproductive manipulations have been already completed before early embryonic stage.

I consider that Wolbachia may target the downstream of the sex determining gene cascade (e.g., doublesex gene) of E. hecabe to feminize genetic males.