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Forward genetics in Wolbachia: Regulation of Wolbachia proliferation by the amplification and deletion of an addictive genomic island

['Elves H. Duarte', 'Instituto Gulbenkian De Ciência', 'Oeiras', 'Faculdade De Ciências E Tecnologia', 'Universidade De Cabo Verde', 'Palmarejo', 'Cabo Verde', 'Ana Carvalho', 'Sergio López-Madrigal', 'João Costa']

Date: 2021-08

Wolbachia is one of the most prevalent bacterial endosymbionts, infecting approximately 40% of terrestrial arthropod species. Wolbachia is often a reproductive parasite but can also provide fitness benefits to its host, as, for example, protection against viral pathogens. This protective effect is currently being applied to fight arboviruses transmission by releasing Wolbachia-transinfected mosquitoes. Titre regulation is a crucial aspect of Wolbachia biology. Higher titres can lead to stronger phenotypes and fidelity of transmission but can have a higher cost to the host. Since Wolbachia is maternally transmitted, its fitness depends on host fitness, and, therefore, its cost to the host may be under selection. Understanding how Wolbachia titres are regulated and other aspects of Wolbachia biology has been hampered by the lack of genetic tools. Here we developed a forward genetic screen to identify new Wolbachia over-proliferative mutant variants. We characterized in detail two new mutants, wMelPop2 and wMelOctoless, and show that the amplification or loss of the Octomom genomic region lead to over-proliferation. These results confirm previous data and expand on the complex role of this genomic region in the control of Wolbachia proliferation. Both new mutants shorten the host lifespan and increase antiviral protection. Moreover, we show that Wolbachia proliferation rate in Drosophila melanogaster depends on the interaction between Octomom copy number, the host developmental stage, and temperature. Our analysis also suggests that the life shortening and antiviral protection phenotypes of Wolbachia are dependent on different, but related, properties of the endosymbiont; the rate of proliferation and the titres near the time of infection, respectively. We also demonstrate the feasibility of a novel and unbiased experimental approach to study Wolbachia biology, which could be further adapted to characterize other genetically intractable bacterial endosymbionts.

Insects often carry bacteria that live within their cells and are transmitted from the mother to the progeny. Wolbachia is one the most common of such bacteria and can strongly influence the insect biology. Its capacity to protect some hosts from viral infection is being used in the fight against mosquitoes-transmitted viruses by introducing Wolbachia in these insects. The amount of Wolbachia within the host can impact their interaction and must be well controlled. To understand this process we screened for new mutants of Wolbachia that proliferate too much in the fruit fly Drosophila melanogaster. We identified two mutants and characterized them in detail. One mutant has too many copies of a specific set of genes, confirming previous similar results. However, the other mutant Wolbachia lost those exact same genes, showing that they are particularly important in growth regulation. Moreover, we show that proliferation of different Wolbachia variants depends on temperature, and the developmental stage of the insect host. Finally, the data indicate that protection to viruses and cost of Wolbachia depend on related but different aspects of this control of growth. In summary, we show that we can screen for new mutants of Wolbachia and understand better how control of growth is genetically controlled by Wolbachia.

Funding: L.T. received funding for this project from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement n°773260 – WOLBAKIAN, https://erc.europa.eu ) and the Fundação para a Ciência e Tecnologia (grant IF/00839/2015, www.fct.pt ). E.H.D. was supported by the fellowship SFRH/BD/113757/2015 from Fundação para a Ciência e Tecnologia ( www.fct.pt ), in the context of the Graduate Program Science for the Development. The fly work at the Fly Facility of Instituto Gulbenkian de Ciência (Oeiras, Portugal), was partially supported by the research infrastructure Congento, co-financed by Lisboa Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and Fundação para a Ciência e Tecnologia (Portugal) under the project LISBOA-01-0145-FEDER-022170. The NGS analysis at the Genomics Unit of Instituto Gulbenkian de Ciência (Oeiras, Portugal), was partially supported by ONEIDA project (LISBOA-01-0145-FEDER-016417) co-funded by FEEI - "Fundos Europeus Estruturais e de Investimento" from "Programa Operacional Regional Lisboa 2020" and by national funds from FCT - "Fundação para a Ciência e a Tecnologia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All numerical data and analysis files are available from the figshare ( https://doi.org/10.6084/m9.figshare.14079920.v2 ). Sequencing data and assembled genomes are available from BioProject: PRJNA587443 ( https://www.ncbi.nlm.nih.gov/bioproject/ ).

Copyright: © 2021 Duarte 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.

The genetic intractability of Wolbachia, which remains unculturable so far, hampers the identification of more genetic modifications altering Wolbachia proliferation. Hence, unbiased approaches such as genetic screens could contribute to our understanding of the genetic bases of Wolbachia-host interactions. Here, we developed a screening strategy in Wolbachia to isolate novel over-proliferating variants. The strategy was based on random mutagenesis, which has been applied before to other unculturable bacteria [ 23 ]. We fed the mutagen ethyl methanesulfonate (EMS) to D. melanogaster females carrying Wolbachia and screened for over-proliferative Wolbachia in their progeny. This approach allowed us to isolate new over-proliferating Wolbachia mutants. We identified the genetic changes in Wolbachia causing over-proliferation and made a detailed phenotypical characterization in terms of proliferation, cost to the host, and antiviral protection. We identified a new mutation leading to Wolbachia over-proliferation and revealed a complex role for the Octomom region in regulating Wolbachia proliferation. Moreover, we demonstrated the feasibility of a novel and unbiased experimental approach to study Wolbachia biology.

So far, a single Wolbachia genetic factor, the Octomom region, has been shown to influence proliferation [ 16 , 17 ]. This genomic region, predicted to encode eight genes, is amplified in the highly proliferative and pathogenic wMelPop. Moreover, the degree of amplification of the Octomom region determines the proliferation rate of wMelPop and the strength of its life shortening phenotype [ 17 ].

Wolbachia titres are a critical factor regulating its biology and interaction with the host [ 3 ]. Titres correlate positively with transmission fidelity and the strength of Wolbachia-induced phenotypes, including the Wolbachia pathogen blocking phenotype [ 3 , 16 – 20 ]. In contrast, higher titres are associated with a reduction in host lifespan [ 16 , 17 , 21 , 22 ]. This may also have a cost to Wolbachia, since as a vertically transmitted bacterium, its fitness depends on the host fitness. Thus, Wolbachia titres regulation by the symbiont or the host may be under selection. Although several host and environmental factors (e.g. temperature) have been shown to affect Wolbachia titres, less is known about Wolbachia genes that regulate its titres [ 3 ].

The discovery of Wolbachia-induced protection against viruses in Drosophila melanogaster, prompted its use to control arboviruses transmission by insect vectors [ 9 ]. Aedes aegypti mosquitoes trans-infected with Wolbachia have increased resistance to viruses, including dengue, chikungunya, Zika, and yellow fever viruses, and, therefore, reduced vector competence [ 10 – 13 ]. Release of Wolbachia-carrying mosquitoes in dengue-endemic areas is likely to reduce dengue burden [ 14 , 15 ]. Despite the preliminary successful results of this strategy, we still lack knowledge on several fundamental aspects of Wolbachia biology and interaction with viral pathogens, which hinders predicting the long-term outcome of Wolbachia-based interventions to control insect-vector transmitted viruses.

Wolbachia is one of the most prevalent bacterial endosymbionts in arthropods, being found in approximately 40% of terrestrial arthropod species [ 4 ]. Wolbachia is broadly known as a host reproduction manipulator [ 5 ]. However, it can also be mutualistic, by, for example, providing vitamins [ 6 ] or protecting against viral pathogens [ 7 , 8 ].

Intracellular maternally-transmitted bacterial symbionts are widespread in insects [ 1 ]. These bacterial endosymbionts can be mutualistic by, for instance, complementing the diets of their hosts, and may expand the range of ecological niches of their insect hosts [ 1 ]. They can also be parasitic, often manipulating the reproduction of their hosts and promoting their spread in the host population [ 1 ]. Understanding the interaction of endosymbionts with their hosts is crucial to understand much of insect biology. A key aspect of this interaction is the regulation of endosymbiont titres, which influence the strength of the induced phenotypes and the cost to the hosts [ 2 , 3 ].

Results

Isolation of over-proliferative Wolbachia in an unbiased forward genetic screen We implemented a classical forward genetic screen in order to isolate new over-proliferative Wolbachia variants. We attempted to mutagenize Wolbachia by feeding the mutagen EMS to Wolbachia-carrying D. melanogaster females. EMS is extensively used in D. melanogaster [24] and has been previously used to mutagenize intracellular bacteria in cell culture [23]. We then tested Wolbachia titres, by real-time quantitative PCR (qPCR), in the progeny of treated females, since this bacterium is maternally transmitted. We used flies with wMelCS_b as our starting variant because of its potential to easily become over-proliferative, given its genetic proximity to the over-proliferative and pathogenic wMelPop variant [16,17,22,25]. Putative mutagenized Wolbachia cells within the host would be in a mixed population, which would make it harder to assess their specific phenotype. However, we hypothesized that over-proliferating Wolbachia cells could overtake the population and that the resulting higher titres could be detectable. Moreover, we decided to pre-treat some of the EMS exposed females with tetracycline to reduce the Wolbachia population in these females and their progeny. This Wolbachia titre reduction should decrease competition for any new mutated Wolbachia, increase drift during vertical transmission, and, therefore, potentially facilitate fixation of new variants. To set up the conditions for tetracycline treatment, we tested different doses of this antibiotic on females, without EMS. The progeny of treated females had from 0 to 90% of the Wolbachia titres in controls (S1 Fig, p < 0.001 for all doses compared with control, at generation 1). We then followed the subsequent progeny of these flies to test how many fly generations it takes to recover normal Wolbachia titres. Except for higher tetracycline doses which lead to infection loss, Wolbachia titres recovered to normal within four fly generations (S1 Fig, linear mixed model [lmm], p > 0.48 for all doses compared with control at generation 4). We also tested for the effect of different EMS doses on the fecundity of D. melanogaster females and Wolbachia titres. We observed that increasing doses of EMS reduce female fecundity (S2A and S2B Fig, linear model [lm], p < 0.001 for both egg number and adult progeny per female). Moreover, we found that EMS feeding strongly reduces Wolbachia titres in the next generation, in a dose-dependent manner (S2C and S2D Fig, non-linear model [nls] fit, p < 0.001). Titres were reduced by up to 90% when 8,000 mM EMS was supplied, leading to the loss of Wolbachia in the next generation in some lines (S2C and S2D Fig). Given these results and the recovery time after tetracycline treatment detailed above, we quantified Wolbachia titres at the first generation (F1), the immediate progeny of EMS-treated females, and at the fourth generation after treatment (F4), when we would expect Wolbachia titres to recover after the severe reduction due to EMS treatment. We screened approximately one thousand F1 progeny of EMS-treated females, in a range of experimental conditions, and at least one F4 female descendent per treated female. We varied EMS dose from 10 mM to 8,000 mM, and tetracycline dose from 0 μg/ml to 12.5 μg/ml, in different combinations (S1 Table). The relative Wolbachia titre was determined when females were ten days old, after they laid eggs, so that any putatively interesting progeny could be followed up. In three independent batches of EMS-treated flies, we detected females with 3 to 14-fold more Wolbachia than controls, suggesting the presence of over-proliferative variants (Fig 1 and S3 Fig). In two batches, over-proliferating Wolbachia were identified in the F1 and in the other batch in the F4. We assessed Wolbachia titres in the next generation and found that the over-proliferative phenotypes were inherited. Subsequent selection allowed us to establish D. melanogaster lines carrying new potentially over-proliferative Wolbachia variants. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Isolation of over-proliferative Wolbachia variants by a forward genetic screen. (A and B) Relative Wolbachia titres in a control (wMelCS_b) and EMS-treated flies (Lines 1A and 2A). 5–10 virgin females were randomly collected each generation for egg-laying and Wolbachia titre measurement using qPCR. Bacterial titres are normalized to that of control flies. The female used in the first generation to start the next generation is coloured. At the other generations the progeny of the female with the higher Wolbachia titre was used to set up the next generation. The selection of the other putative over-proliferating Wolbachia line in panel B is shown in S3A Fig. (C) Relative titres of over-proliferating Wolbachia variants in a host isogenic genetic background. Both lines kept the over-proliferative phenotype (p < 0.001). Each dot represents the Wolbachia titre of a single female. https://doi.org/10.1371/journal.pgen.1009612.g001 We designed the screen to find new mutants of Wolbachia that lead to the endosymbiont over-proliferation. However, EMS will most likely also induce mutations in the host, in the nuclear or mitochondrial genomes, that can be transmitted. To minimize the influence of host nuclear mutations on our screen, we backcrossed the EMS-treated females and their progeny, at every generation, with males from the control isogenic line. To verify that new mutations in the host nuclear genome were not the cause of Wolbachia over-proliferation, we replaced the first, second and third chromosomes of D. melanogaster females carrying the over-proliferating Wolbachia variants in lines 1A, 2A, and 3A, with the chromosomes of the control line, through the use of balancer chromosomes (S4 Fig). We then repeated Wolbachia titres quantification and found that the over-proliferative phenotypes were maintained (Figs 1C and S5, lmm, p < 0.001 for all compared with wMelCS_b). Since mitochondria are maternally transmitted and could have been also mutated by EMS, the experiments described above cannot exclude the possibility that Wolbachia over-proliferation is mitochondria-determined. Thus, the mitogenome of the lines 1A and 2A, showing higher Wolbachia titres, were sequenced with Illumina short-reads and aligned to the mitochondrial reference genome release 6 (GenBank: KJ947872.2:1–14,000, S2 Table). We did not find SNPs or indels unique to the mitochondria of these flies, which shows that flies with over-proliferative Wolbachia did not inherit mutated mitochondria (S3 Table). Therefore, we concluded that the observed Wolbachia over-proliferative phenotypes did not result from mutations in neither the nuclear or mitochondrial host genome.

Deletion and amplification of the Octomom region differently impact titres and growth of Wolbachia In order to characterize better the phenotypes of the new Wolbachia variants wMelOctoless and wMelPop2, we analysed their proliferation, together with wMelCS_b and wMelPop, in adult males kept at 18°C, 25°C, and 29°C (Fig 3 and S10 Fig). The flies were reared at 25°C and placed at the different temperatures when 0–1 day-old adults. At this initial point, at adult eclosion, there are differences in titres between lines carrying different Wolbachia variants (S11 Fig, p < 0.028 for all comparisons). Flies carrying wMelCS_b have the lowest relative titre of Wolbachia. Flies carrying variants with low amplification of the Octomom region have approximately twice the titres of Wolbachia, while flies carrying variants with high copy number of this region have three times more Wolbachia than wMelCS_b. Finally, flies carrying wMelOctoless have the highest titres, approximately four-fold higher than flies carrying wMelCS_b. Therefore, the deletion or amplification of the Octomom region impact Wolbachia titres at adult eclosion. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. The amplification or deletion of Octomom increase Wolbachia proliferation rate in adults. Time-course of relative Wolbachia titres in adults at 18°C (A), 25°C (B) and 29°C (C) with different Wolbachia variants. D. melanogaster males used in these experiments developed at 25°C, were collected on the day of adult eclosion and aged at the given temperatures (18°C, 25°C or 29°C). Ten males were collected at each time-point for Wolbachia titre measurement using qPCR. Wolbachia titres were normalized to that of 0–1 day-old wMelCS_b-infected males. A replicate of the experiment is shown in S10 Fig. Exponential models were used to estimate Wolbachia doubling time, using both full replicates, and a summary of the results is given in Table 1. Each dot represents the relative Wolbachia titre of a single male. https://doi.org/10.1371/journal.pgen.1009612.g003 To analyse proliferation during adult life, we fitted an exponential model to the titres over adult age and estimated doubling time of the Wolbachia variants, at different temperatures (Table 1). Doubling time varies widely with Wolbachia variant and temperature, from approximately one day to seventeen days. A model with all the data shows a complex interaction between proliferation, Wolbachia variant and temperature (lmm, p < 0.001). We analysed this dataset by comparing specific set of variants to test differences between wMelOctoless and wMelCS_b, differences between wMelPop2 and wMelCS_b, and differences between levels of Octomom amplification in wMelPop and wMelPop2. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Doubling time of Wolbachia variants in larvae and adults at different temperatures. https://doi.org/10.1371/journal.pgen.1009612.t001 A direct comparison between wMelOctoless with wMelCS_b shows that this new variant replicates faster than wMelCS_b (lmm, p < 0.001), although it is a relatively small difference at all temperatures (in the full model with all variants, however, the proliferation of wMelOctoless and wMelCS_b is only statistically different at 18°C, Table 1). Both strains interact equally with temperature. Their growth rate does not significantly change between 18°C and 25°C (p = 0.94), but increases at 29°C (p < 0.001). A comparison of wMelPop2 having high and low Octomom copy number with wMelCS_b and wMelOctoless shows that these variants with Octomom amplification have the highest growth rates at 25°C and 29°C (p < 0.001 for all comparisons of wMelPop2 (3 or 8–9 copies) compared to wMelCS_b and wMelOctoless). At 18°C wMelPop2 with 3 copies of Octomom has a growth rate similar to wMelCS_b (p = 0.79) and lower than wMelOctoless (p < 0.001). While at this temperature the growth rate of wMelPop2 with 8–9 copies of Octomom is not significantly different from either wMelCS_b or wMelOctoless (p > 0.088 in both comparisons), and the estimated value is in-between the two (Table 1). The analysis also shows a strong interaction between wMelPop2 growth and temperature. Both low and high Octomom copy number wMelPop2 growth rates increase from 18°C to 25°C, and from 25°C to 29°C (p < 0.001 for these comparisons). To test the effect of the degree of Octomom amplification on growth rate and differences between wMelPop and wMelPop2, we compared these variants with low or high copy number of the Octomom region. The variants with high copy number have a higher growth rate than the variants with low copy number at all temperatures (p < 0.025 at all temperatures). These results confirm that the degree of amplification of the Octomom region controls the intensity of the over-proliferation of these variants, as shown before [17]. Both low and high Octomom copy number wMelPop and wMelPop2 increase growth rate with temperature (p < 0.001 for low and high copy number variants compared between 18°C and 25°C, and between 25°C and 29°C), confirming the analysis above. The statistical model comparing wMelPop and wMelPop2, which differ in two SNPs (see above), indicated a significant difference in growth between them at 25°C (p < 0.001). This could mean that these two SNPs also influence growth of Wolbachia. However, this could also be due to the fact that the copy number of the Octomom region was not equally controlled in wMelPop and wMelPop2 lines during these experiments. wMelPop low copy number line had 2–3 copies of Octomom, while the wMelPop2 line had 3 copies. To test if wMelPop and wMelPop2 indeed vary in proliferation rate, we repeated this experiment with a more tightly controlled Octomom copy number in these two lines, at 25°C (S12A and S12B Fig). Both wMelPop and wMelPop2 carrying 3 copies of Octomom grow faster than wMelCS_b (lmm, p < 0.001 for both) and there is no difference in growth between them (p = 0.39). This indicates that the genetic differences between these lines do not affect their growth and that they are equally influenced by Octomom copy number. Overall, the data and analysis show a complex interaction between Wolbachia variants, temperature and growth rate. There is a strong interaction between temperature and the increased proliferation of variants with amplification of the Octomom region, wMelPop and wMelPop2, when compared with wMelCS_b. The effect of the amplification is not significant at 18°C and becomes increasingly stronger at higher temperatures. On the other hand, loss of Octomom leads to a smaller effect in growth, but similar at all temperatures, when compared with wMelCS_b. Therefore, although both genomic mutations lead to an increase in Wolbachia titres they have different impacts in the growth rates and interaction with temperature.

Wolbachia variants with a deletion or amplification of the Octomom region induce different life-shortening phenotypes The over-proliferation of wMelPop has been associated with a shortening of the host lifespan [16,22]. We, therefore, tested if these new over-proliferative variants also shorten the lifespan of their host, at different temperatures, in males (Figs 5A–5D and S14A–S14C). We also performed this assay in females at 25°C, with similar results to males at 25°C (S14D and S14E Fig). There was a significant interaction between Wolbachia variant and temperature (Cox proportional hazard model with mixed effects (CHR), p < 0.001). All lines, including the Wolbachia-free line have a shorter lifespan at 25°C than at 18°C, and even shorter at 29°C (p < 0.001 for all these comparisons). wMelCS_b did not affect the host lifespan at any temperature (p > 0.16 for all comparisons with the Wolbachia-free line). wMelOctoless strongly reduces host lifespan at all tested temperatures (p < 0.001, each comparison with wMelCS_b) (Figs 5A–5D and S14). This deleterious effect is stronger at 18°C, where wMelOctoless is the tested variant with the highest impact on lifespan, although very similar and not statistically different from wMelPop2 with high Octomom copy number (p < 0.001, for all comparisons with other lines, p = 1 when compared with wMelPop2 with 8–9 Octomom copies). The effect of wMelOctoless on host lifespan is weaker at 25°C than at 18°C (p = 0.001), and similar at 25°C and 29°C (p = 0.95). These results demonstrate that the new over-proliferative wMelOctoless also has a cost to the host in terms of lifespan and this effect interacts with temperature, being stronger at lower temperature. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. wMelOctoless and wMelPop2 are pathogenic. Lifespan of males with different Wolbachia variants at 18°C (A), 25°C (B), and 29°C (C). For survival analyses, fifty males were collected on the day of eclosion and kept in groups of 10 per vial until all flies died. Flies were transferred to new vials every five days. A full replicate of these experiments is shown in S14A–S14C Fig. (D) Coefficients of a Cox mixed model, which represent the effect of Wolbachia on the lifespan of flies relative to the lifespan of Wolbachia-free flies. Both experimental replicates were analysed together. Bars represent the standard error of the coefficient, and letters statistically significant groups after p-value correction. (E-G) Correlation between the strength of life-shortening phenotype and Wolbachia doubling time at 18°C (E), 25°C (F), and 29°C (G). The y-axis represents the strength of Wolbachia life-shortening phenotype (estimated using Cox mixed models) and the x-axis the Wolbachia doubling time (in days). The Pearson correlation coefficient (r) and its significance (p) are given in each panel. https://doi.org/10.1371/journal.pgen.1009612.g005 wMelPop2, similarly to wMelPop, also shortens host lifespan (Figs 5 and S14). The variants containing high copy number of Octomom (8–9 copies) shorten lifespan at all temperatures (p < 0.001, for each comparison with wMelCS_b). This effect is much stronger at 25°C than at 18°C (p < 0.001 for contrasts between both lines and wMelCS_b), and similar at 25°C and 29°C (p > 0.21 for these contrasts). At these two higher temperatures the lines carrying the variants with high copy number of Octomom have the shortest lifespan of all tested lines (p < 0.001 for all comparisons). wMelPop2 and wMelPop with low copy number of Octomom (2–3 copies) always have a weaker effect on host lifespan shortening than high copy number variants (p < 0.001 for all these comparisons). As observed with the high copy number variants, their effect increases with temperature, being stronger at 25°C than at 18°C, and even stronger at 29°C (p < 0.05 for these comparisons). In fact, wMelPop2 and wMelPop with low copy number are only pathogenic at 25°C and 29°C, not at 18°C. These data confirm the association of Octomom region amplification with host lifespan shortening, and the increase in the severity of this phenotype with an increase in Octomom copy number, and an increase in temperature. In some comparisons wMelPop2 and wMelPop differ significantly in their pathogenic phenotype (Fig 5D). This could indicate that there were differences in this phenotype between these two lines. Therefore, and as done above in the analysis of proliferation, we repeated this experiment comparing the lifespan phenotype in wMelPop2 and wMelPop lines with a tightly controlled Octomom copy number (S12 Fig). At 25°C lines both wMelPop and wMelPop2 with 3 copies of the Octomom region had a shorter lifespan than the line with wMelCS_b (p < 0.001), and no difference between them (p = 0.29). These results show that wMelPop2 and wMelPop have the same phenotype. To further demonstrate that the life shortening phenotypes were due to the new Wolbachia variants, and not to EMS-induced mutations in the host nuclear genome, we performed reciprocal crosses between flies carrying wMelCS_b and flies carrying either wMelOctoless or wMelPop2 (with 3 or 8–9 copies of Octomom) and followed the survival of their female progeny at 29°C. The female progeny from reciprocal crosses should be identical in the nuclear genome but differ in the Wolbachia variant, which is maternally transmitted. The life-shortening phenotype segregated maternally, thus demonstrating that the Wolbachia variants carried by the lines are the cause of the phenotypes (S15 Fig). The relative strength of the life-shortening phenotype of the progeny of the reciprocal crosses matches the strength of the phenotypes in the maternal lines, observed in Figs 5 and S14. Moreover, all the tested lines that inherited wMelCS_b had a similar lifespan (p > 0.78 for all comparisons), indicating, as expected, no contribution of the host genotype in this set of experiments. The life shortening phenotype of wMelPop has been associated with its over-proliferation and higher titres since its discovery [22]. We tested if these phenotypes were correlated by taking advantage of the data on titres, proliferation and lifespan shortening that we collected from this set of variants at different temperatures. We found a negative correlation between the strength of the life-shortening phenotype and Wolbachia doubling time, at all temperatures (Fig 5E–5G, |r| > 0.86, p < 0.027, for all correlations). However, we found no significant correlations between the strength of the life-shortening phenotype and Wolbachia titres in 0–1 day-old adults (S16 Fig, p > 0.05 for all correlations). These results show that over-proliferative variants shorten the host lifespan and the strength of this phenotype correlates with their proliferation rates.

[END]

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