Effect of Spiroplasma infection on the mating behavior of Glossina fuscipes fuscipes

Tsetse flies are insects of significant public health and zoonotic importance as they are the main vectors of African trypanosomes. To date, an effective vaccine is unavailable and efforts to limit the spread of the disease primarily rely on controlling the tsetse populations. The discovery of Spiroplasma (class Mollicutes) in Glossina fuscipes fuscipes (Gff) (palpalis subgroup), offers promising insights into its potential use as a biological control agent to hinder trypanosomes infection in tsetse flies. Indeed, a negative correlation between Spiroplasma and trypanosome co-infection has been observed. Using a laboratory strain of Gff, we provide fundamental biological insights into the effects of Spiroplasma infection on the mating behavior of the fly. We found a sex-biased Spiroplasma infection, with males exhibiting a higher infection rate. Mass mating experiments revealed a higher mating propensity in Spiroplasma-infected flies. Additionally, the presence of Spiroplasma influenced premating isolation, leading to nonrandom mating patterns that favored the pairing of individuals with matching infection statuses. Moreover, we present evidence of Spiroplasma vertical paternal transmission. By analyzing female reproductive tissues at 2 and 24 h postmating, we confirmed that an infected male can transfer Spiroplasma to the female via the spermatophore, which can subsequently migrate to the spermathecae. This study provides foundational insights into the role of Spiroplasma in tsetse fly mating behavior and provides supporting evidence for vertical transmission from infected males.