Rickettsia induces strong cytoplasmic incompatibility in a predatory insect

Owashi, Yuta1 ; Arai, Hiroshi1; Adachi-Hagimori, Tetsuya2; Kageyama, Daisuke1

Published May 18, 2024 on Dryad. https://doi.org/10.5061/dryad.t1g1jwt9b

Data files

May 18, 2024 version files 544.12 KB

16SrRNA.fas

80.45 KB
cifA-B.fas

416.10 KB
NTeggs.csv

1.84 KB
NTHatchRate.csv

42.28 KB
README.md

3.44 KB

Abstract

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.

README: Rickettsia induces strong cytoplasmic incompatibility in a predatory insect

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^tet)

CR: the pair of male of cured line (OW1^tet) and female of Rickettsia infected line (OW1)

CC: the pair of male of cured line (OW1^tet) and female of cured line (OW1^tet)
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^tet)

CR: the pair of male of cured line (OW1^tet) and female of Rickettsia infected line (OW1)

CC: the pair of male of cured line (OW1^tet) and female of cured line (OW1^tet)
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.

Methods

Crossing experiment

Treatment

Females and males of the Nesidiocoris tenuis OW1 (Rickettsia infected) and OW1^tet (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.