Transgenic Testing Does Not Support a Role for Additional Candidate Genes in Wolbachia Male Killing or Cytoplasmic Incompatibility

Abstract

Endosymbiotic bacteria in the genus Wolbachia remarkably infect nearly half of all arthropod species. They spread in part because of manipulations of host sexual reproduction that enhance the maternal transmission of the bacteria, including male killing (death of infected males) and unidirectional cytoplasmic incompatibility (CI; death of offspring from infected fathers and uninfected mothers). Recent discoveries identified several genes in prophage WO of Wolbachia (wmk, cifA, and cifB) that fully or partially recapitulate male killing or CI when transgenically expressed in Drosophila melanogaster. However, it is not yet fully resolved if other gene candidates contribute to these phenotypes. Here, we transgenically tested 10 additional gene candidates for their involvement in male killing and/or CI. The results show that despite sequence and protein architecture similarities or comparative associations with reproductive parasitism, transgenic expression of the candidates does not recapitulate male killing or CI. Sequence analysis across Wmk and its closest relatives reveals amino acids that may be important to its function. In addition, evidence is presented to propose new hypotheses regarding the relationship between wmk transcript length and its ability to kill a given host, as well as copy number of wmk homologs within a bacterial strain, which may be predictive of host resistance. Together, these analyses continue to build the evidence for identification of wmk, cifA, and cifB as the major genes that have thus far been shown to cause reproductive parasitism in Wolbachia, and the transgenic resources provide a basis for further functional study of phage WO genes. IMPORTANCE Wolbachia are widespread bacterial endosymbionts that manipulate the reproduction of diverse arthropods to spread through a population and can substantially shape host evolution. Recently, reports identified three prophage WO genes (wmk, cifA, and cifB) that transgenically recapitulate many aspects of reproductive manipulation in Drosophila melanogaster. Here, we transgenically tested 10 additional gene candidates for CI and/or male killing in flies. The results yield no evidence for the involvement of these gene candidates in reproductive parasitism, bolstering the evidence for identification of the cif and wmk genes as the major factors involved in their phenotypes. In addition, evidence supports new hypotheses for prediction of male-killing phenotypes or lack thereof based on wmk transcript length and copy number. These experiments inform efforts to understand the full basis of reproductive parasitism for basic and applied purposes and lay the foundation for future work on the function of an interesting group of Wolbachia and phage WO genes.

A single synonymous nucleotide change impacts the male-killing phenotype of prophage WO genewmk

“…When wmk was transgenically expressed with the most likely upstream start codon, the phenotype was lost, and no biased sex ratio resulted. We also showed that some non-male-killing strains tended to have more of the alternative start codons, establishing a hypothesis that differences in transcript length could significantly alter phenotype 22 . To determine if w Rec and w Suzi wmk transgene phenotypes are similarly sensitive to transcript changes, we expressed them with upstream codons that are native to each of their genetic sequences.…”

“…Relevant to the work here, we previously provided evidence that transcriptional changes in wmk expression may contribute to differences in male-killing abilities between Wolbachia strains or hosts 22 . For instance, some strains contain alternative start codons upstream of the annotated start for wmk and these upstream regions are expressed in the w Mel strain.…”

“…We also tested homologs with predicted alternative start codons to determine their impact on phenotype. The homologs are derived from a set of male-killing and non-male-killing strains and are tested using the Gal4-UAS Drosophila melanogaster expression system and assays for reproductive parasitism 19,22,23 .…”

“…We hypothesized that since male killing is often suppressed by host background 14,15,24 and transgenic phenotypes can be influenced by alternative start codons 22 , adaptation between the host background and wmk homolog will be important for expression of male killing. Related, phylogenetically similar wmk sequences will cause male lethality in the same host, while distantly-related variants will not.…”

Comparative genomics reveals factors associated with phenotypic expression ofWolbachia

“…It is worth noting that none of the CI-associated genes presented here are among those that were previously shown not to cause CI when transgenically expressed in Wolbachia free D. melanogaster (Perlmutter, et al 2020). It is also important to consider that the potential CIassociated effect of these genes may be indirect rather than a direct role in mod or resc.…”