DOI: 10.5061/dryad.t1g1jwt9b
title: Rickettsia induces strong cytoplasmic incompatibility in a predatory insect

description: Rickettsia, a group of intracellular bacteria found in eukaryotes,
 exhibits diverse lifestyles, with some acting as vertebrate pathogens
 transmitted by arthropod vectors and others serving as maternally
 transmitted arthropod endosymbionts, some of which manipulate host
 reproduction for their own benefit. Two phenotypes, namely male killing
 and parthenogenesis induction are known as Rickettsia-induced host
 reproductive manipulations, but it remains unknown whether Rickettsia can
 induce other types of host manipulation. In this study, we discovered that
 Rickettsia induced strong cytoplasmic incompatibility (CI), in which
 uninfected females produce no offspring when mated with infected males, in
 the predatory insect Nesidiocoris tenuis (Hemiptera: Miridae). Molecular
 phylogenetic analysis revealed that the Rickettsia strain was related to
 Rickettsia bellii, a common insect endosymbiont. Notably, this strain
 carried plasmid-encoded homologues of the CI-inducing factors (namely
 cifA-like and cifB-like genes), typically found in Wolbachia, which are
 well-known CI-inducing endosymbionts. Protein domain prediction revealed
 that the cifB-like gene encodes PD-(D/E)XK nuclease and deubiquitinase
 domains, which are responsible for Wolbachia-induced CI, as well as
 OTU-like cysteine protease and ankyrin repeat domains. These findings
 suggest that Rickettsia and Wolbachia endosymbionts share underlying
 mechanisms of CI and that CI-inducing ability was acquired by microbes
 through horizontal plasmid transfer.
description: <strong>Crossing experiment</strong>
  
 <em>Treatment</em>
   Females and males of the
 <em>Nesidiocoris tenuis</em> OW1
 (<em>Rickettsia</em> infected) and
 OW1<sup>tet</sup> (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 <em>Crassula ovata</em> 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.  
 <em>Statistical analysis</em>
   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 <em>P</em>-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.  
 <strong>Phylogenetic analysis</strong>
   <em>16S
 rRNA</em>   The 16S
 rRNA sequences (1,494 bp) obtained through next-generation sequencing of
 the <em>Rickettsia</em> <em>r</em>Nten-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.
   <em>cif
 gene</em>   To infer
 the phylogeny of the <em>cif</em> gene, we obtained the
 nucleotide sequences of previously identified <em>cif</em>
 homologues. After manual reannotation as described by Martinez
 <em>et al.</em> (2020) (doi:10.1093/molbev/msaa209),
 <em>cifA</em> and <em>cifB</em> 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.
description: # *Rickettsia* induces strong cytoplasmic incompatibility in a predatory
 insect
 [https://doi.org/10.5061/dryad.t1g1jwt9b](https://doi.org/10.5061/dryad.t1g1jwt9b) This dataset includes the data related to the crossing experiment and phylogenetic analysis. Crossing experiment data contains raw data of eggs laid (**NTeggs.csv**), R script for eggs laid (**NTeggs.R**), raw data of egg hatch rate (**NTHatchRate.csv**), R script for egg hatch rate (**NTHatchRate.R**). Phylogenetic analysis data  contains fasta data of 16SrRNA nucleotide sequence (**16SrRNA.fas**) and *cif* gene nucleotide sequence (**cifA-B.fas**). ## **Description of the data and file structure** ### **Crossing experiment** ##### NTeggs.csv * *treatment*: Mating pair of crossing experiments RR: the pair of male of *Rickettsia* infected line (OW1) and female of *Rickettsia* infected line (OW1) RC: the pair of male of *Rickettsia* infected line (OW1) and female of cured line (OW1<sup>tet</sup>) CR: the pair of male of cured line (OW1<sup>tet</sup>) and female of *Rickettsia* infected line (OW1) CC: the pair of male of cured line (OW1<sup>tet</sup>) and female of cured line (OW1<sup>tet</sup>) * *pair*: The number identifying the mating pair * *eggs*: Numbers of eggs laid in 6days ##### NTeggs.R The R command for the Kruskal–Wallis test is shown in the file "NTeggs.R". Shapiro-Wilk normality tests were performed for each treatment and the null hypothesis was rejected, followed by a Kruskal-Wallis rank sum test was performed. Please import the "NTeggs.csv" file into R and run this R command. ##### NTHatchRate.csv * *sample*: Mating pair of crossing experiments RR: the pair of male of *Rickettsia* infected line (OW1) and female of *Rickettsia* infected line (OW1) RC: the pair of male of *Rickettsia* infected line (OW1) and female of cured line (OW1<sup>tet</sup>) CR: the pair of male of cured line (OW1<sup>tet</sup>) and female of *Rickettsia* infected line (OW1) CC: the pair of male of cured line (OW1<sup>tet</sup>) and female of cured line (OW1<sup>tet</sup>) * *pair*: The number identifying the mating pair * *hatch*: The binomial data of the egg hatch rate 0: unhatched 1: hatched ##### NTHatchRate.R The R command for the GLMM analysis is shown in the file "NTHatchRate.R". The "glmer" function in the "lme4" package was used to test each combination after the global test. Please import the "NTHatchRate.csv" file into R and run this R command. ### **Phylogenetic analysis** ##### 16SrRNA.fas 51 nucleotide sequences used for the phylogenetic tree construction of the 16S rRNA are shown in the file "16SrRNA.fas". Each OTU is labelled with the bacterial species and strain name, the common and Latin name of the host insect and the accession number of the sequence. ##### cifA-B.fas 68 nucleotide sequences used for the phylogenetic tree construction of the *cif* gene are shown in the file "cifA-B.fas". Each OTU is labelled with the strain name of the bacterium, the common and Latin names of the host insect species and the sequence accession number of the sequence. ## **Sharing/Access information** The sequence read data were deposited in the DDBJ under the accession numbers LC801390-LC801396. ## **Code/Software** The analyses were performed using R version 4.2.2, MEGA11 version 11.0.13, and GBlocks tool in NGPhylogeny.fr.