The facultative intracellular symbiont Lariskella is neutral for lifetime fitness and spreads through cytoplasmic incompatibility in the leaffooted bug, Leptoglossus zonatus
We set out to determine the role that symbiotic Lariskella bacteria play in the western leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae). Our results reveal Lariskella to be among a growing number of microbial symbionts that cause cytoplasmic incompatibility, a reproductive manipulation that increases the relative fitness of females harboring the symbiont. To verify that reproductive manipulators besides Lariskella (e.g. Wolbachia, Cardinium) were not present in the insects in our study, we performed universal 16S amplicon sequencing on 5 whole-body 4th instar nymphs and 8 pairs of testes and ovaries. Two DNA extraction blanks were included during the preparation of the sequencing library, from the first PCR through sequencing. These were processed identically to the samples.Sample preparation followed Illumina's two-step amplification protocol. In an initial PCR, we amplified the V3-V4 hypervariable regions of the 16S rRNA gene using primers 341F/785R. In a second PCR we added 8 bp barcodes to the forward and reverse ends of the amplicons; these uniquely identified each sample, allowing multiplexing. We sequenced an equal mass of each sample's PCR product on a 600 cycle paired-end Illumina MiSeq run at the University of Texas Arlington's Life Science Core Facility.