Rapid tissue-specific screening of Wolbachia, Cardinium and Rickettsia in Flies (Diptera: Sepsidae; Drosophilidae)

Maternally transmitted endosymbionts can negatively influence the reproduction of their arthropod hosts (e.g. male-killing, cytoplasmic incompatibility). However, such infections are rarely assessed in insect models such as Sepsidae or Drosophilidae that are routinely used in sexual selection studies. To detect infection and bacterial localisation in the host, we developed and optimised a tissue-specific multiplex screening protocol for Wolbachia, Cardinium and Rickettsia that can be completed in a day. The robustness of the protocol was tested with the screening of multiple species and populations of flies commonly used in reproductive studies (N=147 flies; n=426 tissues). With triplex PCRs and more effective duplex PCRs, we detected both single and co-infections in most individuals from both families (Drosophilidae | Sepsidae; Single infection: 51.4% | 62.7%; Dual infection: 29.2% | 9.3%; Triple infection: 4.2% | 0%). Surprisingly, we documented the presence of all three reproductive bacteria in 32 wild-caught drosophilids from Singapore. Also, we note that most sepsid populations (19 out of 22) tested positive for Cardinium. We found that the Rickettsia infection was overall low, but it was predominantly detected in the gastrointestinal tract instead of the reproductive tract, suggesting a potential horizontal transmission. Finally, we found that amplicon sequences of equivalent sizes between the three tissues from the same individuals share at least 98.8% identity, which suggests that the same endosymbiont strain inhabits within the whole arthropod. Overall, we believe this protocol is effective in detecting co-infections and understanding the transmission of various reproductive endosymbionts. It can also be used to assess endosymbiont infections in other insects.