Male-killing-associated bacteriophage WO identified from comparisons of Wolbachia endosymbionts of Homona magnanima

Abstract

The origin and mechanism of male-killing, an advantageous strategy employed by maternally transmitted symbionts such as Wolbachia, remain unclear. We compared genomes of four Wolbachia strains derived from Homona magnanima, a male-killing strain wHm-t (1.5 Mb), and three non-male-killing strains, wHm-a (1.1 Mb), wHm-b (1.3 Mb), and wHm-c (1.4 Mb). A wHm-t-specific 76-kbp prophage region harboured two tandemly arrayed WO-mediated killing (wmk) gene homologs (wmk-1/wmk-2 and wmk-3/wmk-4). Of these, wmk-1 or wmk-3 killed almost all Drosophila melanogaster individuals when transgenically overexpressed. Dual expression of wmk-3 and wmk-4 killed all males and rescued females. We propose a novel hypothesis wherein horizontally transmitted proto-Wolbachia with a single wmk killed both sexes, and tandem duplication of wmk allowed an evolutionary transition to a vertically transmitted symbiont, causing male-killing. Our study highlights the bacteriophage as a critical driver of the evolution of male-killing and argues for a conserved male-killing mechanism in diverse insects.

Male-killing mechanisms vary between Spiroplasma species

“…Intriguingly, MK sDa and CI sDis strains had 11 and 12 ankyrin genes, respectively. These findings suggest that S. ixodetis has similar characteristics to Wolbachia endosymbionts (Duplouy et al, 2013;Arai et al, 2022b) in terms of phenotypes (i.e., CI and MK) and genetic compositions (i.e., multiple ankyrin genes). Some ankyrin genes encoded by S. poulsonii and Wolbachia have been implicated in reproductive manipulation (LePage et al, 2017;Harumoto and Lemaitre, 2018), and the ankyrin genes found in the sHm genome may also be involved in MK mechanisms.…”

“…1 Phage WO infections were annotated using PHASTER (Arndt et al, 2016). Protein homology between different Spiroplasma strains was analyzed using S. apis B31 (CP006682.1), S. citri strain BLH-MB (CP047437.1-CP047446.1), S. syrphidicola strain EA-1 (NC_021284.1), D. melanogaster endosymbiont S. poulsonii MSRO (CM020866.1-CM020867.1) (sMel, MK strain, Masson et al, 2018) (Harumoto and Lemaitre, 2018), ankyrin genes from S. ixodetis (Yeoman et al, 2019;Martin et al, 2020;Vera-Ponce León et al, 2021), and the Wolbachia MK candidate factor responsible for WO-mediated killing (Wmk, presumed helixturn-helix transcriptional regulator, Perlmutter et al, 2019;Arai et al, 2022b) was evaluated using both BLASTn and BLASTp. Moreover, to verify whether MK microbes of H. magnanima carried conserved genes, the genes on the MK-associated prophage region WOwHm-t76 of MK Wolbachia wHm-t (Arai et al, 2022b) and those of the Partiti-like virus OGVs (Fujita et al, 2021) were compared to the sHm genes using both BLASTn and BLASTp.…”

“…Protein homology between different Spiroplasma strains was analyzed using S. apis B31 (CP006682.1), S. citri strain BLH-MB (CP047437.1-CP047446.1), S. syrphidicola strain EA-1 (NC_021284.1), D. melanogaster endosymbiont S. poulsonii MSRO (CM020866.1-CM020867.1) (sMel, MK strain, Masson et al, 2018) (Harumoto and Lemaitre, 2018), ankyrin genes from S. ixodetis (Yeoman et al, 2019;Martin et al, 2020;Vera-Ponce León et al, 2021), and the Wolbachia MK candidate factor responsible for WO-mediated killing (Wmk, presumed helixturn-helix transcriptional regulator, Perlmutter et al, 2019;Arai et al, 2022b) was evaluated using both BLASTn and BLASTp. Moreover, to verify whether MK microbes of H. magnanima carried conserved genes, the genes on the MK-associated prophage region WOwHm-t76 of MK Wolbachia wHm-t (Arai et al, 2022b) and those of the Partiti-like virus OGVs (Fujita et al, 2021) were compared to the sHm genes using both BLASTn and BLASTp. Unique genomic features of the sHm strain in the MK S+ and non-MK S+M+ H. magnanima lines 10.3389/fmicb.2022.1075199 were analyzed using GView 3 and BV-BRC variation analysis service.…”

“…However, we found that strain sHm did not harbor any gene homologous to those of MK Partiti-like virus OGVs (Fujita et al, 2021). Moreover, strain sHm did not harbor wmk or effector genes (e.g., CifB-like) that are present on the MK-associated prophage WOwHm-t76 region of strain wHm-t (Arai et al, 2020(Arai et al, , 2022b. The wmk gene, a candidate gene for Wolbachiainduced MK (Perlmutter et al, 2019(Perlmutter et al, , 2021Arai et al, 2022b), possesses a helix-turn-helix (HTH) domain containing putative transcriptional regulator.…”

Harnessing Wolbachia cytoplasmic incompatibility alleles for confined gene drive: a modeling study

“…Wolbachia are maternally inherited bacteria that are found in many arthropod species. They can manipulate the reproduction of their hosts by several mechanisms, such as by distorting sex ratios, thus increasing their frequency in insect populations [42][43][44]. The most common form of manipulation is cytoplasmic incompatibility.…”