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Wolbachia endosymbionts manipulate the self-renewal and differentiation of germline stem cells to reinforce fertility of their fruit fly host [1]

['Shelbi L. Russell', 'Department Of Biomolecular Engineering', 'University Of California Santa Cruz', 'Santa Cruz', 'California', 'United States Of America', 'Jennie Ruelas Castillo', 'Division Of Infectious Diseases', 'Department Of Medicine', 'The Johns Hopkins Hospital']

Date: 2023-11

The alphaproteobacterium Wolbachia pipientis infects arthropod and nematode species worldwide, making it a key target for host biological control. Wolbachia-driven host reproductive manipulations, such as cytoplasmic incompatibility (CI), are credited for catapulting these intracellular bacteria to high frequencies in host populations. Positive, perhaps mutualistic, reproductive manipulations also increase infection frequencies, but are not well understood. Here, we identify molecular and cellular mechanisms by which Wolbachia influences the molecularly distinct processes of germline stem cell (GSC) self-renewal and differentiation. We demonstrate that wMel infection rescues the fertility of flies lacking the translational regulator mei-P26 and is sufficient to sustain infertile homozygous mei-P26-knockdown stocks indefinitely. Cytology revealed that wMel mitigates the impact of mei-P26 loss through restoring proper pMad, Bam, Sxl, and Orb expression. In Oregon R files with wild-type fertility, wMel infection elevates lifetime egg hatch rates. Exploring these phenotypes through dual-RNAseq quantification of eukaryotic and bacterial transcripts revealed that wMel infection rescues and offsets many gene expression changes induced by mei-P26 loss at the mRNA level. Overall, we show that wMel infection beneficially reinforces host fertility at mRNA, protein, and phenotypic levels, and these mechanisms may promote the emergence of mutualism and the breakdown of host reproductive manipulations.

Funding: This work was supported by the UC Santa Cruz Chancellor’s Postdoctoral Fellowship and the NIH (R00GM135583 to SLR; R35GM139595 to WTS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Russell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We report that D. melanogaster flies infected with the wMel strain of Wolbachia partially rescue mei-P26 mutations and exhibit reinforced fertility. In flies homozygous for hypomorphic mei-P26, infection with wMel elevates fertility in both males and females to a level sufficient to maintain a stable stock, whereas the uninfected stock is unsustainable. Infection rescues GSC maintenance and cyst differentiation by mitigating the downstream effects of perturbed mei-P26 function on other protein and mRNA expression. Specifically, wMel infection restores a wild-type-like expression profile for phosphorylated Mothers against decapentaplegic (pMad), Sxl, Bam, and Oo18 RNA-binding (Orb) proteins, as well as tumorous testis (tut) and benign gonadal cell neoplasm (bgcn) mRNAs. We find evidence of wMel’s beneficial reproductive manipulator abilities in Oregon R (OreR) wild-type flies, illustrating how bacterially mediated developmental resilience may be selected for in nature. These results are essential to understanding how wMel reaches high frequencies in natural D. melanogaster populations and these beneficial functions may be able to be harnessed for biological control applications.

In a previous screen, our lab identified the essential fertility gene meiotic-P26 (mei-P26) as a host factor that influences wMel infection intensity in D. melanogaster cell culture, potentially through modulating protein ubiquitination [ 37 ]. Mei-P26 is a Trim-NHL protein that confers a wide range of functions through its multiple domains: its protein-binding NHL and B-box domains allow it to act as an adapter for multiple translational repressor complexes (e.g., Sxl, Nanos (Nos), Argonaute-1 (AGO1), see S1 to S2A Figs and S1 to S2 Tables). Its E3-ubiquitin ligase domain likely enables it to modulate proteolysis [ 38 ]. Given mei-P26’s in vitro role in infection [ 37 ] and its in vivo role in GSC maintenance [ 38 ], differentiation [ 39 , 40 ], and meiosis [ 40 , 41 ], we selected it as a candidate gene for modulating wMel–host interactions.

Host germline stem cell (GSC) maintenance and differentiation pathways are powerful targets for Wolbachia-mediated reproductive manipulation. Wolbachia strains have strong affinities for host germline tissues [ 26 , 27 ], positioning them at the right place to manipulate and enhance host fertility. In the strains that form obligate associations with Brugia filarial nematodes [ 28 ] and Asobara wasps [ 29 ], Wolbachia is required in the germline to prevent premature differentiation and achieve successful oogenesis (reviewed in [ 30 ]). In the facultative wMel-D. melanogaster association, the wMel strain can partially rescue select loss of function alleles of the essential germline maintenance and differentiation genes sex lethal (sxl) and bag-of-marbles (bam) in female flies [ 31 – 33 ]. It is known that wMel encodes its own factor, toxic manipulator of oogenesis (TomO), that partially recapitulates Sxl function in the GSC through derepression and overexpression of the translational repressor Nanos (Nos) [ 34 , 35 ]. However, Nos expression is negatively correlated with Bam expression in the early germarium [ 36 ]. Therefore, Bam’s function in cyst patterning and differentiation in wMel-infected mutant flies cannot be explained by shared mechanisms with sxl rescue or TomO’s known functions.

Currently, the wMel strain of Wolbachia and its encoded CI mechanism are successfully being used to biologically control non-native hosts [ 3 ]. In Aedes aegypti mosquitoes, CI causes nearly 100% mortality of offspring born to uninfected mothers [ 13 ]. However, in its native host, the fruit fly Drosophila melanogaster, wMel exhibits CI that rarely exceeds 50% mortality and is extremely sensitive to paternal age [ 14 – 16 ], as well as grandmother age because titer increases with age [ 17 , 18 ]. Despite weak CI in its native host, wMel is found at moderate to high infection frequencies in populations worldwide [ 19 , 20 ]. Other data suggest that these frequencies may be explained by some emergent beneficial function that increases host fitness [ 21 – 25 ]. The molecular basis for these beneficial functions could be related to the loss of CI efficacy in D. melanogaster. Given that wMel’s use in non-native hosts relies on strong and efficient CI, it is essential that we learn the basis for its beneficial functions that could ultimately undermine CI function.

Despite the success of parasitic reproductive manipulation, natural selection favors symbionts that increase the fertility of infected mothers, even if these variants reduce the efficacy of the parasitic mechanisms that initially drove the infection to high frequency [ 8 ]. In associations between arthropods and strains of the alphaproteobacterium Wolbachia pipientis, this scenario may be common. For example, measured fecundity in populations of Drosophila simulans infected with the wRi strain of Wolbachia swung from −20% to +10% across a 20-year span following wRi’s CI-mediated sweep across California in the 1980s [ 9 ]. This transition from fitness cost to benefit coincided with weakening of CI strength over the same time frame [ 10 ]. Fertility-enhancing mechanisms may be at work in other strains of Wolbachia that have reached high infection frequencies in their native hosts, yet do not currently exhibit evidence of parasitic reproductive manipulation [ 11 ]. Importantly, these fitness benefits may have also played a role in the early stages of population infection, when infected hosts are at too low of frequencies for CI to be effective [ 12 ].

Endosymbiotic bacteria have evolved diverse strategies for infecting and manipulating host populations [ 1 , 2 ], which are now being leveraged for biological control applications [ 3 ]. Many of these bacteria reside within host cells and navigate female host development to colonize offspring, thus linking their fitness to that of their hosts through vertical transmission [ 4 ]. Inherited endosymbionts with reproductive manipulator capabilities go a step further by altering host development in ways that rapidly increase the frequency of infected, reproductive females in the host population. Reproductive manipulator strategies include cytoplasmic incompatibility (CI), where infected sperm require rescue by infected eggs, male-killing, feminization, and parthenogenesis [ 5 , 6 ]. These manipulations can be highly effective at driving bacterial symbionts into host populations, regardless of costs to the host individual or population [ 7 ].

Results

Here, we explore the effects of wMel Wolbachia infection on D. melanogaster’s essential fertility gene mei-P26 through dosage knockdown with an RNAi construct, disrupted function with a hypomorphic allele, mei-P26[1], and full knockdown with a null allele, mei-P26[mfs1]. Using fly fecundity assays, immunocytochemistry, and dual-RNAseq of both host and bacterial transcripts, we show that wMel can compensate for the loss of mei-P26 to significantly rescue host fertility.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002335

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