Wolbachia are maternally transmitted, obligate intracellular bacteria primarily residing in the cells of germline tissues in many arthropod species worldwide (Hurst and Frost, 2015; Taylor et al., 2018.) To facilitate their spread through the host matriline, these bacteria hijack sex ratios, sex determination systems, and embryonic viability to cause various reproductive parasitism phenotypes (Yen and Barr, 1971; Hurst et al., 1999; Bouchon et al., 1998; Hunter, 1999). One such phenotype is male killing, whereby sons of infected females are selectively killed (Hurst et al., 1999; Hurst and Jiggins, 2000; Charlat et al., 2005). Male killing selfishly drives the spread of the bacteria when, for instance, brothers and sisters compete for limited resources, and male lethality yields females (the transmitters) a relative fitness benefit due to reduced competition with fewer siblings (Jaenike et al., 2003; Unckless and Jaenike, 2012; Skinner, 1985; Hurst, 1997). This form of reproductive parasitism can have profound impacts on host behavior and evolution, including a sex role reversal (Jiggins et al., 2000), host nuclear genome changes to resist the phenotype (Majerus and Majerus, 2010; Hornett et al., 2006), and potentially host population extinction (Groenenboom and Hogeweg, 2002). Population modelling also suggests male killing may be deployed as a control method to crash the population size of arthropod pests and disease vectors (Berec et al., 2016).

Many lines of evidence suggest a complex relationship between male-killing genotype and phenotype. For example, male killing can be suppressed by host background (Majerus and Majerus, 2010; Hornett et al., 2006; Jaenike, 2007; Mitsuhashi et al., 2011), where male hosts exhibit resistance to the phenotype even in the presence of Wolbachia. In addition, Wolbachia that do not cause male killing in one host species can cause male killing in a second host species upon introgression or transfer to a naïve host (Jaenike, 2007; Fujii et al., 2001). Furthermore, some strains induce death at different host developmental stages (embryo vs. larvae) or across different sex determination systems (ZW lepidopterans, XY dipterans, XO arachnids), and the number of surviving males may vary widely (Riparbelli et al., 2012; Sasaki et al., 2002; Zeh et al., 2005). Such findings specify that the expression of male killing, and particularly the genotype-phenotype relationship in these symbioses, is more complex than simply the presence or absence of a male-killing gene.

We recently identified a male-killing candidate gene, wmk (WO-mediated killing), from prophage WO in the wMel Wolbachia strain of Drosophila melanogaster based on comparative genomic, transgenic, and cytological approaches (Bordenstein and Bordenstein, 2016; Perlmutter et al., 2019). It is a putative transcription factor with two predicted helix-turn-helix (HTH) DNA-binding domains, and transgenic expression in D. melanogaster embryos recapitulates many cytological and molecular aspects of male killing including accumulation of DNA damage in male embryos that overlaps with sites of dosage compensation activity (Riparbelli et al., 2012; Perlmutter et al., 2019; Harumoto et al., 2018). The wmk gene and two cytoplasmic incompatibility factor (cif) genes that underlie cytoplasmic incompatibility (a parasitism phenotype whereby offspring die in crosses between infected males and uninfected females) occur nearby specifically in the eukaryotic association module (EAM) of prophage WO (Bordenstein and Bordenstein, 2016; Perlmutter et al., 2019; LePage et al., 2017), which refers to the phage genome that is inserted into the bacterial chromosome. The EAM is common in WO phages across several Wolbachia strains (Masui et al., 2001; Wu et al., 2004) and is rich in genes that are homologous to eukaryotic genes or annotated with eukaryotic functions (Bordenstein and Bordenstein, 2016). As such, the expression of reproductive parasitism genes from the EAM and tripartite interactions between phage WO, Wolbachia, and eukaryotic hosts are central to Wolbachia’s ability to interact with and modify host reproduction.

The discovery of wmk has now enabled investigation of the impacts of genetic variation on the transgenic male-killing phenotype. Although the role of wmk in male killing has not yet been assessed in nature, evidence based on native wmk genotypes and transgenic wmk expression in flies suggest refined intricacy to the interactions between genotype and phenotype. For example, the native Wolbachia wmk homologs in three closely-related strains (wMel of D. melanogaster, wRec of D. recens, and wSuzi of D. suzukii) have nearly identical nucleotide sequences that vary by one to three SNPs (Perlmutter et al., 2019), and yet these strains induce different forms of reproductive parasitism. wMel and wRec induce cytoplasmic incompatibility, wRec also causes male killing in some strains of a sister species, and wSuzi is not known to induce any reproductive phenotype (Jaenike, 2007; Hamm et al., 2014; Hoffmann, 1988). Previous transgenic testing also demonstrated that addition of nine additional amino acids to the N-terminal region of the Wmk protein at the site of a putative alternative start codon ablates the phenotype (Perlmutter et al., 2020). Therefore, the Wmk N-terminus is particularly sensitive to genetic alterations, which is notable since small modifications such as protein tags in this location are often common additions to proteins that do not typically interfere with function. Furthermore, D. melanogaster does not harbor Wolbachia known to cause male killing, although transgenic wmk expression recapitulates the phenotype in this host (Perlmutter et al., 2019), and the wMel strain is most closely-related to the wRec male-killing strain (Metcalf et al., 2014). In addition, some D. melanogaster contain male-killing Spiroplasma symbionts whose plasmid gene Spaid can also transgenically recapitulate male killing (Harumoto and Lemaitre, 2018). In summary, the evidence for Wolbachia and wmk transgenic male killing suggests an intricate and multifaceted genotype-phenotype landscape.

Building on this background, several key questions emerge: Do closely-related or distantly-related homologs induce male killing? How sensitive is the wmk phenotype to small or large genetic changes? And how adapted are wmk homologs to their arthropod hosts? Here, we evaluate codon-optimized wmk homologs that span a spectrum of genetic divergence (Figure 1), including homologs (i) from distantly-related hosts, (ii) with putative upstream alternative start codons, (iii) with a single amino acid change, or (iv) with one or more synonymous codon changes, all in the D. melanogaster host. In this way, we aimed to investigate how a variety of genetic alterations to wmk, from a single synonymous nucleotide to the gene level, affect the phenotype. In particular, we hypothesized that divergent strains would not induce the phenotype due to co-adaptation with a distantly-related host. We also anticipated that the alternative start codons would inhibit function based on previous results (Perlmutter et al., 2020). In contrast, we hypothesized that neither a single amino acid change nor synonymous codon changes would alter the male-killing phenotype. We report that while distant homologs do not cause a male-killing phenotype in this host, single amino acid and synonymous nucleotide changes remarkably do alter the phenotype. Thus, wmk male killing is sensitive to the full spectrum of genetic alterations at a fine-scale level not previously recognized. Notably, synonymous sequence changes and post-transcriptional processes appear to play a role in controlling the genotype-phenotype relationships that underpin wmk male killing.

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Linking mutations to function is crucial in resolving the evolutionary dynamics of adaptations, especially when the functions emerge in a nested system of tripartite phage-bacteria-animal interactions. In a simple case, the direct impact of genetic divergence is functional divergence with increasing numbers of mutations leading to increased functional divergence. Here, we investigated evolutionary and molecular hypotheses related to the genotype-phenotype relationships between the prophage WO-mediated killing (wmk) gene (Perlmutter et al., 2019; Perlmutter et al., 2020) and male killing in D. melanogaster. Sequences among wmk homologs in nature can differ across divergent hosts, leading to the hypothesis that highly divergent alleles are functionally fine-tuned. Using a variety of sequences, we report three key results: (i) mutations even at the synonymous codon and single nucleotide levels can alter the male-killing phenotype, (ii) phenotype, genotype, and RNA structural variation exhibit some correlation with each other, and (iii) distantly-related homologs do not induce a male-killing phenotype in transgenic D. melanogaster, while some closely-related homologs from Drosophila species/populations with female-biased sex ratios induce all-killing phenotypes. We discuss how these findings expand and support mechanistic models of wmk male killing, emphasize important implications for transgenic assays in endosymbiont studies, and relate these findings to aspects of male killing such as phenotype switching and host resistance.

The most surprising and unanticipated result was that transgene expression of highly similar homologs and even single synonymous site changes alter phenotype, and they make the difference between life and death for some males. We tested wmk homologs with high native sequence identity compared to native wMel wmk: wRec wmk from the mushroom-feeding D. recens (native sequence has two synonymous and one non-synonymous nucleotide differences) and wSuzi wmk from the fruit crop pest D. suzukii (native sequence has one synonymous nucleotide difference). Although we anticipated similar results to wMel wmk expression, we found that transgenic expression of these genes killed all flies (Figure 2), even though the wSuzi wmk transgene produces an identical protein to transgenic wMel Wmk. Of particular note, transgenic wSuzi wmk (all killing) and the original transgenic wMel wmk sequence (male killing) produce proteins of the exact same amino acid sequence, but they shared only a 91% sequence identity in the codon-optimized transgene, largely due to an updated codon optimization algorithm. Codon optimization is the norm in transgenic symbiosis research under the common assumption that synonymous codons are functionally redundant. However, codon optimization algorithms often choose a codon based on factors including codon adaptation, mRNA folding, regulatory motifs, nucleotide bias, or codon correlations and biases (Plotkin and Kudla, 2011). With even a few different codons input into the algorithm as well as algorithm updates over the years, the tested transgenes had different nucleotide sequences.

Based on the aforementioned results, we next sought to assess the sensitivity of male killing to wmk transcriptional and post-transcriptional changes and asked if one synonymous codon or site change was sufficient to alter phenotype. We created three transgenic lines, each with different DNA sequences coding for the same Serine at position 16. With only these minor changes that encode an identical protein sequence to wMel Wmk, the transgenic male-killing phenotype was ablated (Figure 4). Thus, one single codon and even a single nucleotide, remarkably determined male viability. Importantly, this key result implies at the molecular level that endosymbiont phenotypes of reproductive parasites may not simply be governed by DNA sequence alone.

A key question related to our findings is how synonymous changes cause vastly different phenotypes. It is often assumed in endosymbiont transgenic experiments that synonymous changes do not affect function, and codons for the same amino acid are functionally redundant. However, several decades of research have uncovered mounting evidence that the functional redundancy hypothesis is not always accurate (Plotkin and Kudla, 2011). Indeed, codon bias varies across species (hence the need for codon optimization) (Sharp et al., 1997; Andersson and Kurland, 1990), and there are several mechanisms through which single or few codons can influence transcription or translation. When there is a rare codon, there are fewer tRNAs available, and the translation rate may be correspondingly lowered (Li et al., 2012). Notably, there is a bias for rare codons in N-terminal regions of bacterial genes, which is likely due to their influence on mRNA structure (Goodman et al., 2013). Lower 5’ RNA stability or weaker structure can also be correlated with ease of initiation and faster translation (Gu et al., 2010), and stem loop structures may affect translation rate in varying ways. Indeed, strong 5’ RNA secondary structure may inhibit ribosomal initiation or translational efficiency (de Smit and van Duin, 1990; Bettany et al., 1989), and there can be selection against SNPs that alter RNA secondary structure (Salari et al., 2013). There is also evidence for selection across the domains of life on codon usage in the first 30–60 nucleotides of a gene, likely due to their impact on mRNA structure near the site of initiation (Gu et al., 2010). Additionally, early codons with low frequencies can be beneficial by slowing elongation, thus preventing ribosome collisions, or potentially helping to recruit chaperones to the emerging peptide for proper folding (Tuller et al., 2010; Fredrick and Ibba, 2010). In addition, some specific codons across the sequence are favored for their lower likelihood of mistranslation (Qin et al., 2004). Indeed, synonymous codon differences in genes can result, for instance, in altered gene expression (Kudla et al., 2009), lower organism fitness (Agashe et al., 2013), or disease (Sauna and Kimchi-Sarfaty, 2011).

Beyond codon sequence, gene expression levels across representatives of all phenotypes are similar (Figure 2), supporting post-transcriptional differences as the source of functional variation and refuting the functional redundancy hypothesis in this context. Examining the three synonymous transgenes more closely, we find that codon usage indeed correlates with phenotype. According to the codon usage table for D. melanogaster on the Genscript website, the original wMel wmk codon at this position (TCG) has a frequency 16.7 per 1000 codons. In contrast, the wSuzi codon (TCC) has a frequency of 19.5/1000, and the wRec codon (AGC) has a frequency of 20.5/1000 (GenScript, 2020). Therefore, there is a pattern where the more common codons are present in the all-killing transgenes, and at similar frequencies, while the strain expressing partial male killing has a codon at a lower frequency. Thus, some of the phenomena described in studies on synonymous codons and function likely underlie the phenotypic variation observed here.

Given that transgenic wmk expression with silent site changes results in different phenotypes, we used software that estimated RNA secondary structures for the tested transgenes. Indeed, we found that the predicted structures correlate somewhat with phenotype, so synonymous codons may change transcript structure or other post-transcriptional features of the transcript or protein (Figure 2). Other small changes including adding a 9-codon N-terminal sequence ablated the phenotype and reversed it to no death, despite the additional sequence being smaller than many common gene tags that typically do not interfere with function. This small change also alters predicted RNA structure (Figure 2—figure supplement 1). We were unable to assess protein levels to determine translational differences due to lack of an antibody. However, the data demonstrates that the wmk transgene is functionally sensitive to some post-transcriptional changes, at least in the N-terminal region.

What, then, do these results illuminate about transgenic wmk function and the utility of transgenic research in light of the potential for marked influences of synonymous changes on phenotype? First, as discussed, the wmk transgenic phenotypes are likely sensitive to post-transcriptional processes. This has important implications for understanding wmk in a natural male-killing context as soon as feasible techniques are available, since heterologous expression and its reliance on codon optimization may obscure our understanding of the gene’s biology. Second, since three different transgenic phenotypes (sex ratio bias, all killing, no killing) have been found so far with only a few sequences analyzed, it is possible that further testing of new codons may increase the partial male killing to a full male-killing phenotype. Therefore, it would be fruitful and may be possible to continue to uncover a transgenic sequence that fully recapitulates the phenotype to refine this system as a study tool for Wolbachia male killing. Third and critically, although the results show that there is some relationship between synonymous codons and phenotype, several points remain for further testing. For example, we cannot conclude that the particular codon tested here is responsible for phenotype alterations in other host genetic backgrounds or species. It is possible that this codon plays a functional role only in a singular host genetic context. Here, we changed wmk sequences while holding the host genetic background fixed, but the reverse is required to conclude whether or not the particular codon plays a general role in other genotypes or natural contexts. Second, due to possible coevolution, various codons may or may not yield similar functional effects across different host backgrounds, and additional synonymous sites may contribute to the male-killing phenotype. Thus, the results here illuminate a previously unrecognized need for future research on the functional impacts of synonymous substitutions in endosymbionts. Future work may focus on determining if there is one specific synonymous codon that affects the male-killing function in all cases, if a more general feature exists where alteration of any or a subset of N-terminal or other wmk codons affects function, or if the effect of synonymous changes is specific to this background.

In addition, these findings are informative with regards to the more general study of phenotypes induced by endosymbiont transgenes. It is standard practice to codon optimize genes for maximizing host expression when testing endosymbiont gene function (Perlmutter et al., 2019; LePage et al., 2017; Harumoto and Lemaitre, 2018), with the assumption that synonymous codons will not alter function. However, if codon optimization potentially changes the interpretation of transgenic findings, then phenotypes should be corroborated in natural contexts once tools such as genetic editing are available in the relevant organism. Specifically, wmk should be knocked out in native contexts once it is more technically feasible. In addition, codon optimization algorithms are updated with new information periodically with the assumption that they yield improved results, although it is unclear in practice whether an algorithm is better optimized to produce results that reflect the true biology of a transgene. Future work is necessary to explore these concepts further. For example, comparisons of alleles may need to be performed with alleles identical in sequence except for any engineered differences, and the algorithm should remain constant across all transgenes that are compared to each other. Further, careful analysis and comparison of transgenic phenotypes produced by different algorithms may be necessary in some cases where the phenotype is known or expected. This approach could ensure the algorithm produces a transgenic phenotype that most closely resembles the natural phenotype. In addition, the reliability of certain molecular evolutionary signatures, such as dN/dS, may be compromised since synonymous mutations are assumed to be neutral in these analyses. These principles are particularly important for research on endosymbionts that increasingly relies on heterologous gene expression for functional studies. The smaller genomes of endosymbionts tend to have low GC content overall (McCutcheon et al., 2009), and GC content is the strongest driver of codon usage bias (Knight et al., 2001; Chen et al., 2004) and contributes to the strength of mRNA secondary structure (Plotkin and Kudla, 2011), so careful attention to the effects of synonymous changes may be of acute interest to the endosymbiont research community.

We also find interesting support for the hypothesis that there is co-adaptation between wmk homologs and their hosts since male-killing genes may be evolutionary matched for the host sex determination and molecular machinery that they manipulate. We first analyzed wmk homologs that are more distantly related to wMel wmk, including from male-killing strains wBol1b, wBif, wCaub, and wInn/wBor (Sasaki et al., 2002; Dyson et al., 2002; Dyer and Jaenike, 2004; Hurst et al., 2000; Sheeley and McAllister, 2009). They did not recapitulate male killing when transgenically expressed in D. melanogaster. The lack of a sex ratio bias is expected if either wmk does not underpin male killing in these systems or divergent homologs are required to be closely co-adapted to their hosts. This latter hypothesis is based on observations that resistance to male killing is common (evidence of a potential host-microbe arms race) (Charlat et al., 2005; Majerus and Majerus, 2010; Jaenike, 2007; Mitsuhashi et al., 2011; Hornett et al., 2008), and some strains can cause male killing anew when transferred to another, usually closely-related host (Jaenike, 2007; Fujii et al., 2001; Sasaki et al., 2002) while not causing male killing in all recipient host species (Veneti et al., 2012; Hughes and Rasgon, 2014). It is possible, then, that the wmk homologs tested induce a sex ratio bias in their natural hosts, but not in D. melanogaster.

If we make the assumption that wmk is involved in male killing in nature, which requires confirmation beyond transgenic recapitulation of the phenotype, then the results here give the basis for additional hypotheses that require further testing. First, the killing of both males and females by the closely-related wRec and wSuzi wmk homologs could indicate that the target is something shared in both males and females, but functions differently within each sex (assuming no off-target effects). Indeed, previous studies on wmk and Wolbachia male killing demonstrate a positive correlation with between dosage compensation complex (DCC) activity and DNA defects in male embryos (Riparbelli et al., 2012; Perlmutter et al., 2019; Harumoto et al., 2018). Notably, four of the five protein components of the DCC are expressed in both males and females, and it is only msl-2 that is male-specific and catalyzes formation of the complex (Lucchesi and Kuroda, 2015). Thus, there are several non-sex-specific wmk target candidates that the gene product may interact with to cause lethality in males and females. Second, protein divergence and resultant conformational changes may impact the specificity between host target and the Wmk toxin, and could underlie development of host resistance. Male-killing genes are expected to evolve rapidly within hosts in order to counteradapt host resistance mechanisms and keep evolutionary pace with the rapid evolution of sex-related genes that may be the target of a male-killing toxin (Marín and Baker, 1998; Rodriguez et al., 2007). As such, major or minor divergence in protein or transcript sequence of either the host target or the microbial toxin may underpin changes that lead to common host resistance, wmk-host coadaptation, and functional transgene differences in the foreign D. melanogaster host. Third, these findings leave open the possibility of a variety of functionally relevant wmk protein or transcript conformations in nature, which could contribute to the marked diversity of Wolbachia male killing in terms of host species and sex determination systems (Hurst et al., 1999; Charlat et al., 2005; Sasaki et al., 2002; Zeh et al., 2005; Dyer and Jaenike, 2004; Hurst et al., 2000; Fialho and Stevens, 2000; Van Borm et al., 2008).

Taken together, this work reports previously unrecognized relationships for wmk-induced killing and establishes new hypotheses for the impacts of RNA structure and post-transcriptional processes in wmk-induced male killing. It also highlights several critical features for the research community regarding assumptions about the broad use of transgenes and the role of synonymous mutations in gene function. If wmk is involved in natural male killing, then this work could indicate how silent sequence changes may relate to known male-killing phenomena such as frequent host resistance or male-killing function in diverse hosts.

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 3-6.

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Author details

1. Jessamyn I Perlmutter

   1. Department of Biological Sciences, Vanderbilt University, Nashville, United States
   2. Department of Molecular Biosciences, University of Kansas, Lawrence, United States
   3. Vanderbilt Microbiome Innovation Center, Vanderbilt University, Nashville, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Visualization, Writing – original draft, Writing – review and editing

   For correspondence

   jessamyn.perlmutter@ku.edu

   Competing interests

   Is listed as an author on a patent related to the use of wmk in vector control. US Patent 20210000092 16/982708

   "This ORCID iD identifies the author of this article:" 0000-0002-9789-4674

2. Jane E Meyers

   1. Department of Biological Sciences, Vanderbilt University, Nashville, United States
   2. Vanderbilt Microbiome Innovation Center, Vanderbilt University, Nashville, United States

   Contribution

   Investigation, Writing – original draft

   Competing interests

   No competing interests declared

3. Seth R Bordenstein

   1. Department of Biological Sciences, Vanderbilt University, Nashville, United States
   2. Vanderbilt Microbiome Innovation Center, Vanderbilt University, Nashville, United States
   3. Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, United States
   4. Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University, Nashville, United States

   Contribution

   Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   s.bordenstein@vanderbilt.edu

   Competing interests

   Is listed as an author on a patent related to the use of wmk in vector control. US Patent 20210000092 16/982708

   "This ORCID iD identifies the author of this article:" 0000-0001-7346-0954

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