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

Abstract

Endosymbiotic bacteria in the genus <i>Wolbachia</i> 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 <i>Wolbachia</i> (<i>wmk</i>, <i>cifA</i>, and <i>cifB</i>) that fully or partially recapitulate male killing or CI when transgenically expressed in <i>Drosophila melanogaster</i> 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 <i>wmk</i> transcript length and its ability to kill a given host, as well as copy number of <i>wmk</i> homologs within a bacterial strain, which may be predictive of host resistance. Together, these analyses continue to build the evidence for identification of <i>wmk</i>, <i>cifA</i>, and <i>cifB</i> as the major genes that have thus far been shown to cause reproductive parasitism in <i>Wolbachia</i>, and the transgenic resources provide a basis for further functional study of phage WO genes.<b>IMPORTANCE</b> <i>Wolbachia</i> 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 (<i>wmk</i>, <i>cifA</i>, and <i>cifB</i>) that transgenically recapitulate many aspects of reproductive manipulation in <i>Drosophila melanogaster</i> 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 <i>cif</i> and <i>wmk</i> 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 <i>wmk</i> 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 <i>Wolbachia</i> and phage WO genes.