Rickettsia induces strong cytoplasmic incompatibility in a predatory insect

Crossing experiment Treatment Females and males of the Nesidiocoris tenuis OW1 (Rickettsia infected) and OW1tet (cured) lines were crossed in all possible combinations to compare the number of eggs laid and the hatch rates. Males and females were collected separately from newly emerged adults. A 3–6day old female was allowed to mate with a male for 7 days and then transferred to a container with a leaf of Crassula ovata for oviposition. Leaves were replaced every 2 days, and oviposition continued for 6 days. Ten days after the last day of oviposition, hatched nymphs were counted and leaves were dissected to count unhatched eggs. The food source was renewed every 2 days during the crossing experiments. Statistical analysis The total number of eggs laid was analysed using the Kruskal–Wallis test. Egg hatch rates were analysed using a generalised linear mixed model (GLMM) with binomial error and a logit-link function. Each mating pair was assigned a random effect. Based on the results of the GLMM, ANOVA was performed for each treatment to estimate the P-value using the chi-squared test with Bonferroni correction. Mating pairs with no oviposition were excluded from the analysis of egg hatch rates. The analyses were performed using R version 4.2.2. Phylogenetic analysis 16S rRNA The 16S rRNA sequences (1,494 bp) obtained through next-generation sequencing of the Rickettsia rNten-OW1 isolate were used for phylogenetic analyses. Phylogenetic trees based on the nucleotide sequences were constructed by the maximum likelihood method using MEGA11. Kimura’s two-parameter model, evaluated using the best fit method, was applied for the calculation. cif gene To infer the phylogeny of the cif gene, we obtained the nucleotide sequences of previously identified cif homologues. After manual reannotation as described by Martinez et al. (2020) (doi:10.1093/molbev/msaa209), cifA and cifB nucleotide sequences were aligned according to their amino acid translations using MUSCLE implemented in MEGA11. The aligned data were cleaned using the GBlocks tool in NGPhylogeny.fr to remove weakly conserved regions. Phylogenetic trees based on the cleaned nucleotide sequences were constructed by the maximum likelihood method using MEGA11. A general time-reversible model, evaluated by the best fit method, was applied for the calculation.