Highly transmissible cytoplasmic incompatibility by the extracellular insect symbiont Spiroplasma

Abstract

Cytoplasmic incompatibility (CI) is a form of reproductive manipulation caused by maternally inherited endosymbionts infecting arthropods, like Wolbachia, whereby matings between infected males and uninfected females produce few or no offspring. We report the discovery of a new CI symbiont, a strain of Spiroplasma causing CI in the parasitoid wasp Lariophagus distinguendus. Its extracellular occurrence enabled us to establish CI in uninfected adult insects by transferring Spiroplasma-infected hemolymph. We sequenced the CI-Spiroplasma genome and did not find any homologues of any of the cif genes discovered to cause CI in Wolbachia, suggesting independent evolution of CI. Instead, the genome contains other potential CI-causing candidate genes, such as homologues of high-mobility group (HMG) box proteins that are crucial in eukaryotic development but rare in bacterial genomes. Spiroplasma's extracellular nature and broad host range encompassing medically and agriculturally important arthropods make it a promising tool to study CI and its applications.

Keywords: Entomology; Microbiology; Microbiology parasite; Zoology.

Graphical abstract

Figure 1

Numbers of F1 female (red) and male (blue) offspring of crosses between endosymbiont-carrying (+) and uninfected (−) females and males of the STU strain Numbers of replicates (i.e the numbers of crosses) are given in parentheses below the paternal male of each combination. Statistical significances among numbers of female offspring, numbers of male offspring, and total offspring numbers, respectively, were tested using GLMs (family = negative binomial) followed by Tukey tests for multiple comparisons; different lower case letters (red: female offspring, blue: male offspring, black: total offspring) indicate statistical differences at p < 0.05 (see Table S1 for test statistics). Medians are represented by the middle horizontal lines; the 25% and 75% quartiles are shown as the lower and upper boundaries of the boxes, respectively. Minimum and maximum values within a range across 1.5 times the distances between the quartiles above the 75% quartile and below the 25% quartile are included in the whiskers; outliers outside of this range are shown as single points; see Data S1 for raw data.

Figure 2

FISH images depicting the presence of sDis in the ovaries of L. distinguendus females of the strain STU Blue: cell nuclei stained with Hoechst DNA staining, Red: Spiroplasma-specific probe (SPR) or anti-sense probe with Cy3 fluorochrome, nc: nurse cells, ooc: oocyte. Scale bars: 50 μm. Brightness and contrast were set to “auto” in ImageJ 1.x for all images. (A) Specific, localized signals in STU ovaries hybridized with SPR. (B) Background fluorescence in STU ovaries with anti-sense probe. (C) Fluorescence of STU ovaries with no probe.

Figure 3

Hemolymph transfer of sDis infection and CI (A) Proportion of sDis-positive F1 male offspring of injected females by day of emergence from consecutively parasitized host batches. Only males used for subsequent crossing experiments are shown. Numbers of replicates (numbers of males emerged in the respective time frame) are given in parentheses below the bars. White part of the bars: sDis-negative males; red part of the bars: sDis-positive males. (B) Numbers of F2 female (red) and male (blue) offspring of sDis-negative (−) and positive (+) F1 males and uninfected (−) STU females. Numbers of replicates (numbers of males of the given infection status used for this experiment) are given in parentheses below the bars; crosses without any offspring are excluded. Statistical significances between crosses were tested for F2 female offspring and F2 male offspring using Wilcoxon rank sum tests with continuity correction (W = 2718, p = 3.581 × 10⁻¹³ for females, W = 839, p = 0.0001971 for males) and for F2 total offspring using a Welch two sample t test (t = 1.341, p = 0.1853); different lower case letters (red: female offspring, blue: male offspring, black: total offspring) indicate statistical differences at p < 0.05. The middle horizontal line shows the median; the lower boundary of the box indicates the 25% quartile, whereas the upper boundary represents the 75% quartile. The whiskers indicate minimum and maximum values within a range stretching from 1.5 times the distances between the quartiles above the 75% quartile and below the 25% quartile. Data outside of this range are outliers and are shown as single points. See Data S2 and Data S3 for raw data and Spiroplasma titers.

Figure 4

Evolutionary relationships of the CI-inducing sDis (A) Maximum likelihood phylogram of concatenated rpoB, 16S rRNA, and 23S rRNA nucleotide sequences supports the phylogenetic position of the novel symbiont within the Ixodetis clade of Spiroplasma. Branches with 100% ML-like FastTree support are labeled with a small filled circle. Resolved phylogenomic relationships within Ixodetis clade members are shown in Figure S2. (B) Phylogenetic relationships between eukaryotic and Spiroplasma-encoded high-mobility group (HMG) box proteins. Two well-supported clades of Spiroplasma HMG box proteins are identified and distantly allied with fungal and protist HMG box proteins. One clade is interspersed with spirochete-encoded proteins. All Spiroplasma genes are indicated by gray or red (sDis) helical cartoons, and sDis genes are additionally shown in bold typeface. Branches with ML-like FastTree support values of 0.75 or greater are labeled with a small filled circle; two circles indicate support > 0.8 and three indicate support > 0.9.

Figure 5

Gene content and putative carbon utilization in sDis (A) Summary of gene content in the sDis genome assembly organized by function as predicted by matches to KEGG gene databases. Numbers in the center of the chart and beside category headings indicate numbers of genes. (B) Summary of putative carbon utilization by sDis. Complete coding regions corresponding to each named gene are present in the assembly. Dashed black outlines indicate multifunctional genes whose functions are not confidently predicted based on presence. Dashed red lines indicate genes that are absent from the assembly. Color schemes are not shared across panels (A) and (B).