The cellular phenotype of cytoplasmic incompatibility in Culex pipiens in the light of cidB diversity

Wolbachia are maternally inherited endosymbiotic bacteria, widespread among arthropods thanks to host reproductive manipulations that increase their prevalence into host populations. The most commonly observed manipulation is cytoplasmic incompatibility (CI). CI leads to embryonic death in crosses between i) infected males and uninfected females and ii) individuals infected with incompatible Wolbachia strains. CI can be conceptualized as a toxin-antidote system where a toxin deposited by Wolbachia in the sperm would induce embryonic death unless countered by an antidote produced by Wolbachia present in the eggs. In Drosophila melanogaster, transgenic expression of Wolbachia effector cidB revealed its function of CI-inducing toxin. Moreover in Culex pipiens, the diversity of cidB variants present in wPip strains accounts for the diversity in crossing-types. We conducted cytological analyses to determine the CI mechanisms that lead to embryonic death in C. pipiens, and assess whether diversity in crossing-types could be based on variations in these mechanisms. We revealed that paternal chromatin condensation and segregation defects during the first embryonic division are always responsible for embryonic death. The strongest observed defects lead to an exclusion of the paternal chromatin from the first zygotic division, resulting in haploid embryos unable to hatch. The proportion of unhatched haploid embryos, developing with only maternal chromatin, which reflects the frequency of strong defects can be considered as a proxy of CI intensity at the cellular level. We thus studied the putative effect of variations in crossing types and cidB diversification on CI defects intensity. Incompatible crosses involving distinct wPip strains revealed that CI defects intensity depends on the Wolbachia strains hosted by the males and is linked to the diversity of cidB genes harbored in their genomes. These results support that, additionally to its implication in C. pipiens crossing type variability, cidB diversification also influences the strength of CI embryonic defects.

Introduction during mitosis, reflecting incomplete replication of paternal DNA [22]. It has been envisioned that these DNA replication defects might be responsible for the paternal chromatin bridges and segregation failure during the first mitotic division, which result in early embryonic arrest [22,24,25]. Nevertheless, some embryos reaching late development stages have been reported in CI crosses [21,25,26]. Late development in CI embryos was interpreted as resulting from a complete paternal chromatin exclusion during the first division, which allows successful maternal chromatin segregation and the formation of two haploid nuclei [25]. These haploid nuclei which further divide, lead to gynogenetic development (i.e. haploid development with only maternal genetic material) until late embryonic stages [27]. These haploid embryos are never viable in diploid species such as D. simulans [25]. However, in the haplodiploid parasitoid wasps Leptopilina heterotoma and N. vitripenis, CI-induced paternal chromosome defects can lead either i) to the death of the embryos or ii) to the production of healthy males [19,21,28,29]. It has been proposed that these two CI developmental outcomes could result from different degree of paternal chromatin defect (improper condensation) before the first division [29][30][31]. Severe defects would lead to complete elimination of male chromosomes from the first zygotic division resulting in haploidization and male development, whereas less severe defects would lead in partial exclusion of the paternal chromatin resulting in incomplete elimination of male chromosomes and early arrest of the aneuploid development [29][30][31]. In diploid species such as C. pipiens and D. melanogaster, the proportion of unhatched developed haploid embryos observed in fully incompatible CI crosses would be a proxy of the frequency of total paternal chromatin exclusion during embryogenesis due to strong CI intensity at the cellular level. However, this hypothesis is counter intuitive as one could expect that strong CI defects would prevent any development to occur while soft CI defect would allow development.
The molecular mechanism underlying CI can be conceptualized as a toxin-antidote system in which i) a toxin produced by Wolbachia in the testes, more generally called a "mod factor", and introduced in the sperm during spermatogenesis would interfere ("modify") with the paternal chromatin and induce embryonic perturbations, and ii) an antidote released by Wolbachia in the egg, more generally called "resc factor", would "rescue" these paternal chromatin defects to allow normal embryogenesis to occur [32,33]. The recent discoveries of i) Wolbachia genes cidA and cidB ability to recapitulate the CI phenotypes when expressed in transgenic Drosophila [34,35], and ii) the link between specific allelic cidA wPip /cidB wPip variations in worldwide natural C. pipiens populations and the capacity of males to sterilize females [36], open new paths into understanding CI mechanisms. CidA and cidB genes are syntenic genes within the WO phage region (S6 Table) [34,35,[37][38][39]. CidB encodes a deubiquitylating enzyme (DUB) and when a cidB wPip construct bearing this catalytically inactivated DUB domain was expressed in D. melanogaster males, CI was no longer observed, showing the implication of the deubiquitylating activity in the mod function [34]. The role of cidA in the CI mechanism is more debated because i) both cidA wMel and cidB wMel are required to induce CI in transgenic Drosophila [35] and ii) in natural populations of C. pipiens, specific cidA wPip allelic variations were found to be linked to mod variations [36]. However, the implication of cidA in the resc function is supported by the capacity of cidA to prevent cidB toxicity in yeast [34] and the capability of transgenic uninfected females expressing cidA wMel throughout oogenesis to rescue the effect of cidB wMel [40].
In C. pipiens, all individuals are infected with different Wolbachia strains belonging to the monophyletic wPip group, but divided in five subgroups wPipI to wPipV. MLST (Multi Locus Sequence Typing) genes from Baldo et al. (2006) [41] were not polymorphic between wPip strains, thus a wPip specific MLST with more polymorphic genes MutL, ank2, pk1, pk2, GP12, GP15, and RepA was used to resolve wPip phylogeny (S6 Table) [12]. Mosquitoes hosting wPip from the same group are likely to be compatible with each other but incompatible with mosquitoes infected with Wolbachia from other wPip groups [42]. This diversity of wPip strains distributed all around the world is responsible for the unique complexity of CI crossing types described in this host species [12,43]. Unlike wVitA and wMel, which harbors only one cidA/cidB copy, and wRi, which harbors two identical copies of cidA/cidB, high intra and inter-genomic diversities of cidA wPip /cidB wPip genes were uncovered between and within all wPip strains studied [36]. This diversity certainly explains the unrivaled diversity of crossing types described in C. pipiens [36]. This cidA wPip /cidB wPip genes amplification and diversification within the same Wolbachia genome may also account for the impressive CI penetrance described in C. pipiens. Indeed, expression of multiple cidA wPip and cidB wPip variants in males could i) be responsible for differences in CI cellular phenotype(s) and ii) influence the penetrance of CI. Here, we investigated the putative impact of crossing type variations and cidA wPip /cidB wPip diversification on CI cellular phenotypes and CI intensity during C. pipiens embryogenesis. To this end, we monitored the development of embryos derived from various incompatible crosses involving males from C. pipiens lines infected with Wolbachia strains from distinct wPip groups and exhibiting different crossing types.

A single cellular phenotype of CI in C. pipiens
Three different types of crosses were performed using different laboratory mosquito lines: i) fertile crosses between individuals from the same line, representing our control to monitor normal embryonic early development, ii) sterile crosses between mosquito lines harboring different wPip strains, and iii) sterile crosses between infected males and uninfected females (TC lines), to test the effect of Wolbachia absence on embryo development and CI cellular mechanism (S1 and S2 Tables).
The cellular phenotype during embryogenesis in fertile intra-line crosses is illustrated in Fig  1. To differentially visualize the paternal from the maternal chromatin, we used propidium iodide to mark both maternal and paternal chromatin and an anti-acetylated histone H4 labelling that preferentially marks the de novo assembled paternal chromatin after protamine removal [22]. Paternal chromatin appears in green/yellow (acetylated histone H4 labelling is dominant) and maternal chromatin appears in red (propidium iodide labelling is dominant). After fertilization, maternal and paternal pronuclei migrated toward each other and apposed (documented embryos with confocal microscopy images n = 4, Fig 1A). Then, paternal and maternal chromatins condensed and entered into first mitotic division (n = 3, Fig 1B). During the first division, paternal and maternal chromosomes aligned in separate region at the metaphase plate (n = 1, Fig 1C). Both sets of chromosomes segregated equally during anaphase (n = 3, Fig 1D) to produce two diploid nuclei (n = 1, Fig 1E) that proliferate mitotically (n = 16, Fig 1F). After 24 hours of development, organogenesis was ongoing and segmentation was clearly visible (n = 2, Fig 1G).
In sterile crosses between two infected incompatible C. pipiens lines (Fig 2) as well as in crosses between infected males and uninfected females (Fig 3), paternal and maternal pronuclei migrated and apposed normally (n = 2, Figs 2A and 3A). However, during the early prophase, paternal chromatin appeared under-condensed compared to maternal chromatin (n = 2, Fig 2B and 2C). Then the paternal chromatin failed to segregate properly during anaphase (n = 16, Figs 2D, 2E, 3B and 3C). In telophase paternal chromatin can either i) form chromatin bridges between the two maternal nuclei (n = 10, Figs 2D and 3B), certainly causing the early arrest of embryogenesis and production of undeveloped embryos (Figs 2G1 and 3E), or ii) appear fully excluded (n = 6, Figs 2E and 3C), allowing maternal chromatin to Normal embryogenesis in C. pipiens: From first nuclear divisions to segmentation. Paternal chromatin appears in green/yellow (acetylated histone H4 labelling is dominant) and maternal chromatin appears in red (propidium iodide labelling is dominant). (A) apposition of maternal and paternal pronuclei, (B) chromatin under condensation, (C) condensed chromatin, (D) first mitotic division anaphase (maternal and paternal chromosome segregate independently), (E) two nuclei following the first division, (F) normal diploid development 1 hour after oviposition, six diploid nuclei are visible after 1h development, (G) segmented embryo after 24 hours of development. White arrows indicate the paternal chromatin. Confocal stacks were obtained on embryos from several fertile intra-line crosses due to the difficulty to obtain all the early embryonic stages from each cross (S2 Table). Scale bar is 10μm.
https://doi.org/10.1371/journal.ppat.1007364.g001 CI embryos from incompatible infected parents in C. pipiens: From first nuclear divisions to segmentation. Paternal chromatin appears in green/yellow (acetylated histone H4 labelling is dominant) and maternal chromatin appears in red (propidium iodide labelling is dominant) (A) apposition of maternal and paternal pronuclei, (B) delay in paternal chromatin condensation, (C) condensed maternal chromatin and under-condensed paternal chromatin, (D) paternal chromatin failed to segregate and form a chromatin bridge between segregating maternal chromatin, (E) two nuclei containing mainly maternal chromatin while paternal chromatin do not segregate, (F) haploid development 2 hours after oviposition, (G) the two possible fates of development after 48 hours (1) non-viable embryo with no visible development, and (2) unhatched developed embryo with visible segments. White arrows indicate the paternal chromatin. Confocal stacks (panels A,B,C,D,E,F) and optical images (panels G1 and G2) were obtained on embryos from several CI crosses between infected males and females due to the difficulty to obtain all the early embryonic stages for each cross (S2 Table). Green dots are background noises likely due to the presence of residual antibodies. Scale bar is 10μm. successfully segregate and eventually formed unhatched haploid developed embryos presenting eyes and segments ( Fig 2G2).
Eight distinct CI crosses were monitored between males and females infected with wPip strains belonging to different wPip groups, and two distinct CI crosses were monitored between infected males and uninfected females (S2 Table). However, despite this diversity of CI crosses, condensation and segregation defects of the paternal chromatin were the only observed cellular defects resulting in embryonic death, and were never observed in any embryo resulting from fertile crosses (seven intra-line fertile crosses, S2 Table).

Absence of Wolbachia in the oocytes blocks embryogenesis in CI embryos
2 hours after oviposition, some embryos resulting from CI crosses between infected lines pursued their embryogenesis (n = 2, Fig 2F), and after 48 hours these embryos exhibited visible development, as segmentation was clearly observable under optical microscope ( Fig 2G2). However, more than 99.9% of these developed embryos did not hatch. In the sterile cross between ♂ Slab x ♀ Ichkeul 13, unhatched developed embryos only displayed maternal markers (see Material and methods, "Ploidy determination in CI developed embryos", S1 Fig), showing that they were composed of only haploid maternal DNA, as previously described in  [44].
All the seven different crosses performed between males infected with different wPip strains and uninfected females from different TC-treated lines produced 100% of non-developed embryos (Fig 3E and S3 Table). Confocal observations of these embryos showed that only few White arrows indicate the paternal chromatin. Confocal stacks (panels A,B,C,D,) and optical images (panels E) were obtained on embryos from several CI crosses between infected males and uninfected females due to the difficulty to obtain all the early embryonic stages for each cross (S2 Table). Green dots are background noises likely due to the presence of residual antibodies. Scale bar is 10μm. and abnormal nuclei were observed in the cytoplasm 2 hours post oviposition (n = 5, Fig 3D), indicating an early arrest of the embryogenesis (S2 Table).

Males infected by different wPip with distinct mod profiles induced different CI defects intensities
It has been previously proposed that the production of haploid or aneuploid embryos in CI crosses represented a proxy of intensity of CI defects that leads to more or less complete paternal chromatin exclusion [29][30][31]44]. Severe defects would lead to the complete exclusion of the paternal chromatin during the first embryonic division (i.e. strong cellular CI intensity), which would allow maternal chromatin successful segregation and the production of a developed haploid embryo. Thus, unhatched developed haploid embryos reflect the occurrence during the first zygotic division of strong CI defects while unhatched non-developed embryos illustrate the occurrence of weak CI defects. We used this link between the degree of paternal chromatin exclusion (i.e. weak or strong cellular CI) and the proportion of unhatched developed embryos in eggs-rafts from incompatible crosses to investigate the variability of CI intensity (i. e. frequency of strong versus weak CI defects).
Using this proxy, we studied the variation in CI intensity between 20 incompatible crosses between infected lines (S3 Table). These 20 crosses involved i) males from four different isofemale lines (MAL lines) infected with wPip strains from different wPip groups all exhibiting distinct mod profiles, and ii) females from five isofemale lines (FEM lines) all harbouring wPip strains from the wPipIV group and exhibiting the same resc profile [36,42] (S4 Table). Significant differences were found regarding the proportion of unhatched developed embryos between these incompatible crosses (generalized linear model (GLM), χ 2 = 245.695, df = 19, p< 0.001, Fig 4, Table 1 and S3 Table). While no effect of FEM lines was detected on this proportion (GLMM, χ 2 = 2.508, df = 4, p = 0.643, Fig 4), the MAL lines involved in the crosses had a significant effect (GLMM, χ 2 = 16.211, df = 3, p = 0.001, Table 1 and Fig 4). Males from Tunis (wPipI mod ii) and Slab (wPipIII mod iii) lines induced the highest proportion of developed embryos (72% and 73%, respectively) but were not significantly different from one another (GLMM, χ 2 = 0.002, df = 1, p = 0.968); males from Utique (wPipI mod iv) and Lavar (wPipII mod vi) lines induced significantly different and lower proportions of unhatched developed embryos (respectively 42% and 18%, Table 1). The nuclear genetic background of the males seems not to be involved in the variability of CI defects intensity: males from backcrossed line Sl(wPipI-Tunis) and males from the Tunis line, which host the same wPipI strain in different genetic backgrounds, indeed induced similar unhatched developed embryos proportions when crossed with the five FEM lines (0.71 ± 0.22 and 0.72 ± 0.19 respectively; GLMM, χ 2 = 0.008, df = 1, p = 0.927). Consequently variability in CI defects intensity appears to be only dictated by the wPip strain harbored by the different males.

Genetic investigations of cellular CI intensity variation
The results from the previous section indicate that the proportion of unhatched developed embryos in CI crosses likely depended on variations in the males' mod profiles. To investigate the sources of such variation in CI defects, we tested the putative influence of several variables: i) the density of Wolbachia in the testes, ii) the copy numbers of cidA and cidB genes in the different wPip genomes, iii) the expression levels of cidA and cidB, and iv) the cidA and cidB variants repertoires in the genomes of the different wPip strains hosted by the males.
CidA and cidB variants repertoires were variable between wPip strains. Cloning and Sanger sequencing revealed that the four MAL lines with different mod were infected with Wolbachia strains that harbored different cidA and cidB variant repertoires (S2 and S3 Figs). No

Correlations between CI defects intensity and wPip genetic variations
Correlative analyses were conducted to assess the potential links between variations in CI defects intensity and genetic variations. We found no significant correlations between the proportion of unhatched developed embryos in CI crosses and i) Wolbachia density in the testes

Discussion
To investigate whether the high diversity of cidA/cidB variants within wPip could be responsible for variations in the cellular phenotype of CI, we studied the development of C. pipiens embryos resulting from various incompatible crosses. The early embryogenesis was assessed using fluorescence confocal microscopy in i) fertile intra-line crosses, ii) incompatible crosses between infected males and infected females, and iii) incompatible crosses between infected males and uninfected females. Despite the diversity of performed crosses between males and females infected with wPip strains harboring different cidA/cidB variants repertoires or uninfected female, a unique and recurrent embryonic phenotype was detected, consisting in paternal chromatin condensation and segregation defects during the first embryonic division (Figs 2B, 2E, 3B and 3C). This phenotype was never detected in any embryos derived from intra-line crosses (Fig 1). Hence the diversity of cidA/cidB variants repertoires describes in C. pipiens does not seem to influence the CI mechanism itself, which is consistent with all CidB variants carrying a conserved DUB domain [36]. Similar defects were already reported in both Drosophila and Nasonia [23,25], suggesting an universality of Cid induced-cellular CI mechanism whenever cid genes are diversified or not in the Wolbachia genome.
An unsolved question is the molecular pathway(s) targeted by CidA and CidB. Most protein domains within CidA and CidB remain to be characterized and how they interact with each other and host targets to induce CI remains unclear. However, a first tangible element is that the catalytically active DUB domain (involved in deubiquitination) in CidB proteins, which is considered as involved in the mod function, is necessary to induce CI in transgenic Drosophila [34]. Ubiquitination pathways have been shown to be crucial for many essential cellular processes, such as the regulation of the chromatin dynamics and the cell cycle progression [45]. Changes in ubiquitination could for instance directly or indirectly affect H3.3 histone incorporation after protamine removal and DNA replication as suggested by PCNA persistence on the paternal chromatin [22], which would result in an asynchronous mitotic entry of paternal and maternal pronuclear chromatin [30]. Interestingly, Cardinium, an endosymbiont phylogenetically distant from Wolbachia, induces CI with quite similar embryogenesis defects in the hymenoptera Encarsia suzanna [46]. Moreover, an ubiquitin specific protease USP classified as a DUB protein has also been detected in Cardinium genome, suggesting a convergent implication of DUB in CI induced by insect endosymbionts [47]. However, some Wolbachia strains able to induce CI do not carry DUB domain (i.e. no cid) in their genomes, but display instead a paralog gene with a nuclease domain called cinB [34,35,38]. DUB (Cid) and Nuclease (Cin) domains do not have the same predicted functions suggesting that distinct molecular pathways may be responsible for CI [34,38]. The CI cellular defects caused by Wolbachia strains harboring only cin genes remain unknown and could differ from the one induced by cid genes. Our study showed that wPip strains, which carry both cid and cin genes in their genomes, induce similar defects during embryogenesis as wMel, which carries only a cid gene. This suggests that the association of cid and cin does not change the cellular phenotype of CI, but the molecular mechanism induces by DUB and Nuclease which must be different due to the biochemical nature of the proteins might converge on a similar cellular defect (i.e. paternal chromatin condensation defect). However, the presence of DUB and Nuclease domains in the same Wolbachia genome could still contribute to CI by modifying its penetrance: wRi (D. simulans) and wPip (C. pipiens), which harbor both paralogs, have indeed a strong CI penetrance (almost no hatched embryos), while wNo and wHa (D. simulans), which carry either cin or cid genes, respectively, induce lower CI penetrance [13,38].
Our cytological investigation in C. pipiens evidenced a link between the paternal chromatin exclusion degree during the first zygotic division and the existence of two developmental fates following first-division defects. In fact, unhatched embryos can either reach advanced developmental stages, exhibiting segments and visible eyes, or display no visible development (Figs 2G1, 2G2 and 6) [26,44]. We confirmed  [44] findings that the unhatched developed embryos resulting from CI were haploid, and carried genetic material from maternal origin only (Fig 6 and S1 Fig). Confocal observations showed that such haploid development likely occurred when paternal chromatin was fully excluded during the first zygotic division, allowing the successful segregation of the isolated maternal chromatin (Fig 6). In contrast, unhatched non-developed embryos would be due to partial exclusion of the paternal chromatin, which would result in aneuploid nuclei and early arrest of embryogenesis ( Fig  6). It has been previously proposed for other arthropod models that the participation of paternal chromatin to the first division would depend on the intensity of paternal chromatin defects (i.e. improper condensation) [29,30,44,48]. Severe defects would lead to complete paternal exclusion (i.e. strong cellular CI) and to the production of haploid developed embryos, while less severe defects would lead to a partial paternal chromatin exclusion (i.e. weak cellular CI) and to the production of aneuploid non-developed embryos. We used this link between the degree of paternal chromatin exclusion and the ratio of unhatched developed and non-developed embryos in eggs-rafts from incompatible crosses to investigate the variability of cellular CI intensity between different incompatible crosses.
We first studied the variability of CI intensity using males and females both infected with incompatible wPip strains. Developed embryos were observed in all these incompatible crosses, with two possible outcomes: i) less than one per thousand of these embryos were apparently not affected by CI and hatched into diploid larvae [14,44], and ii) from 11% to 85% of the unhatched embryos, depending on the crosses, reached late embryonic developmental stages showing that they experienced strong CI defects (Figs 4 and 6). We then studied the influence of the absence of Wolbachia in the oocytes on the cellular CI intensity. As in  [44], i) we confirmed that not a single larvae was produced in such crosses, and ii) all the seven CI crosses between infected males and uninfected females (TC lines) The two MAL lines Slab and Lavar have been chosen to illustrate the variability in CI defects intensity (i.e. frequency of strong and weak cellular CI). During spermatogenesis, the Wolbachia in male testes produce a toxin (mod factor). After fertilization, if females and males are infected with the same Wolbachia (Panel A), they can prevent the toxicity (resc function) and allow normal embryogenesis and the production of living diploid larvae. However, if the Wolbachia in the females are incompatible with the Wolbachia in the males (Panel B) or absent (Panel C) then the toxicity would not be prevented and paternal chromatin condensation delay and segregation defects occur. Two outcomes of the first zygotic division are possible regarding the degree of paternal chromatin exclusion. If the paternal chromatin is partially excluded, chromatin bridges would be formed resulting in aneuploid nuclei which might go through few mitotic divisions but will arrest the embryogenesis at early stages producing embryos with no visible development. If the paternal chromatin is fully excluded from the first zygotic division, maternal chromatin can segregate and produce two haploid nuclei which will divide further to produce non-viable haploid embryos exhibiting advanced development stages with eyes and segmentation clearly visible. The proportion of unhatched haploid embryos is influenced by different wPip with distinct mod profiles in the MAL lines involved in the crosses. However when the egg is not infected by Wolbachia (Panel C), all embryos exhibited an absence of further development after the first zygotic divisions whatever the mod induced by the wPip hosted by the MAL lines. resulted in 100% of non-developed embryos suggesting that in such crosses, CI phenotype was always weak (Fig 6).
In crosses between infected individuals, it clearly appeared that MAL lines harboring Wolbachia from different wPip groups (wPip I, II, III) and displaying distinct mod induced significant variation in CI defects intensity when crossed with females harboring distinct wPipIV strains displaying the same resc (Figs 4 and 6). Variation in CI defects intensity has already been reported in Nasonia species, where the production of haploid viable males in N. vitripenis was interpreted as resulting from severe paternal chromatin defects, while the production of unviable aneuploid embryos in N. longicornis and N. giraulti was interpreted as resulting from weak paternal chromatin defects. However, variation of CI intensity in these host species was not associated with the different Wolbachia strains, but to variation in host genetic backgrounds [19]. The backcross experiment performed in the present study suggests that CI intensity is not impacted by nuclear genetic variations in C. pipiens. While it was already established that Wolbachia drives alone the observed variation in crossing types in C. pipiens [11,49,50], Wolbachia also seems to dictate the intensity of CI defects. Consequently, the variation in CI intensity observed when two infected individuals are crossed seems to be under the major influence of the wPip strain infecting the MAL line via the degree of paternal chromatin exclusion they trigger.
In C. pipiens, when females from tetracycline-cleared lines (TC females) were crossed with the four MAL lines, 100% of unhatched non-developed embryos only exhibiting few degenerated nuclei were observed, even 2 hours after oviposition (Figs 3D and 6). Such CI phenotype suggests that the defects caused by the wPip infecting all the MAL lines are always weak (Fig 6). This result is counter intuitive because one would expect that when Wolbachia is absent from the eggs CI should be always strong and many haploid embryos should be produced. We mentioned above that all the MAL lines can induce strong CI defects in variable proportion of the embryos when crossed with infected females. Consequently, the constant weak CI phenotype observed when females are not infected is linked to the absence of Wolbachia during egg maturation. Our results suggest that in incompatible crosses between infected C. pipiens individuals, the presence of maternal Wolbachia somehow interferes with early embryogenesis allowing haploid development to occur. It seems very unlikely that the presence of incompatible Wolbachia in the egg would enhance the mechanisms leading ultimately to paternal chromosome condensation defects (i.e. accentuate the mod function) to result in its total exclusion during the first embryonic division. Instead, the presence of incompatible Wolbachia in the eggs may have an additive effect on the incompatibility between pronuclei, not by directly affecting the paternal chromatin but by influencing the cell cycle timing. For instance, maternal Wolbachia could modulate the maternal kinetics for DNA replication or the mitotic entry during early development, increasing the incompatibility between pronuclei and therefore favoring the haploid development. Thus, while paternal Wolbachia-induced CI defects always occur regardless of the infection status of the eggs, the absence of incompatible maternal Wolbachia would block haploid development resulting in weak CI phenotype.
We then investigated the putative genetic determinism of CI intensity variation in embryos derived from infected parents. We assessed whether it could result from difference in Wolbachia density, cidA-cidB gene expression, copy numbers, or variant diversity between the wPip strains. As previously described in Drosophila [35,38], we found in C. pipiens that cidA was always significantly more expressed than cidB, whatever the wPip strain (Table 1). This is in accordance with the hypothesis that cidA and cidB form a toxin-antidote system where CidA is the antidote of CidB [34,36]. Indeed, in such system the antidote was always found more expressed than the toxin to prevent the host from toxicity [51]. No significant difference between MAL lines was found for cidA and cidB expression levels per Wolbachia cell ( Table 1), suggesting that the cidA and cidB expression does not influence CI defects intensity. However, while the cidA and cidB expression levels per Wolbachia cell did not significantly vary between C. pipiens lines, the total amount of CidA and CidB proteins in the host mainly depends on the density of Wolbachia. Since the mod factors are most likely deposited on the sperm in the testes during spermatogenesis [32,33], we measured the density of Wolbachia in the male gonads. We found that Lavar males hosted significantly less Wolbachia in their testes than males from the three other lines (Fig 5); Lavar males were also those that generated the lowest proportion of unhatched developed embryos in their offspring, whatever the FEM lines (Fig 4). Due to lower Wolbachia density in the testes, the global amount of CidB protein could be lower in Lavar line compared to the other lines. This low dosage of CidB would more likely result in weak CI defects leading to only few haploid development. However, this hypothesis relies on a single line and requires more C. pipiens lines with distinct testicular Wolbachia densities to be confirmed. Lavar was also the line with the highest cidA expression relatively to cidB (Table 1); as CidA has been proposed as the CidB antidote [34,36], its overexpression could reduce CidB-induced CI defects, and contribute to the low frequency of developed haploid embryos observed in crosses involving males from Lavar line.
We previously demonstrated that the amplification followed by the diversification of cidA and cidB variants in wPip certainly constitutes the source for CI diversity profiles in C. pipiens while cinA and cinB did not exhibit any polymorphism [36]. Indeed, specific variations in cidA and cidB repertoires (number and/or nature of the variants) clearly seemed to determine the compatibility outcome of crossings between wPipIV-infected males and any infected females, pointing out the putative role of these variations in the prodigious CI complexity recorded in this species [36,42]. Here, we tested the putative consequence of cidA and cidB gene amplification (i.e. number of copies per genome) on variation of CI defects intensity, and demonstrated no significant correlation between the two parameters. When the quantification of genomic copies obtained by q-PCR are put in relation to the number of different variants in the same isofemale line obtained by cloning-sequencing, some of cidA results appear discordant. This is especially true for the Slab line, which exhibits ten distinct cidA variants for~5 copies per genomes quantified (Table 1 and S2 Fig). Even taking into account technical limits of q-PCR to quantify high level of gene amplification, this discordance suggests that, at least in the Slab line, some of the Wolbachia cells do not harbor the same cidA variants.
We found that the different wPip strains carried by the four MAL lines exhibiting different mod profiles harbored distinct cidB variants. Any variant of this gene could certainly trigger CI alone, as the DUB domain is perfectly conserved between all variants [36]. However, their diversity can modulate CI defects intensity. We thus tested whether cidB repertoire diversity could play a role in CI intensity variability. Supporting this hypothesis, we found that males from the two C. pipiens lines harboring wPip strains with four different cidB variants induced higher proportions of unhatched developed embryos compared to lines harboring wPip with only two different cidB variants (S4 Fig). Each distinct cidB variants could differentially impact the paternal chromatin (i.e. like different locks), putatively leading to an additive mod effect: the more different cidB variants present in a wPip strain, the more likely strong CI defects. However, more wPip strains varying in their diversity of cidB are required to further test this hypothesis.
In conclusion, despite the diversity of crossing types observed in C. pipiens, linked to the diversity of cidA/cidB variants repertoires, a single cellular phenotype of CI, was observed in this species. In all crosses (i.e. uni-bidirectionnal), CI results in early developmental defects in the paternal chromatin condensation and segregation during the first zygotic division similar to that observed in other insects. Our study demonstrates that in CI crosses between two infected individuals, the CI intensity (i.e. frequency of strong and weak CI defects) is influenced by the male-carried wPip. However, when the female is not infected, and despite the variability of the distinct wPip strains carried by the males, no unhatched developed embryos (strong cellular CI) were ever found, suggesting that the weak CI phenotype observed in such crosses is instead due to the absence of Wolbachia in the eggs. Genetic investigation reveals that the variability of CI defects intensity may be linked to cidB variant diversity in wPip strains. While the putative functional role and the singularity of cidB amplification and diversification in wPip remains yet to be fully solved, it clearly appears that it deeply modifies the wPip-induced CI phenotype at different scales, from crossing types [36] to its intensity at the cellular level.

Materials and methods
Culex pipiens lines C. pipiens lines hosting different Wolbachia strains or without Wolbachia. Ten laboratory isofemale lines belonging to C. pipiens s.l., from our laboratory, were used; they differ in their geographical origins and in the Wolbachia strain hosted (S1 Table). To determine the mod and resc profiles of the different mosquito lines, crosses with four reference lines (4-ref cytotypes) were realized similarly to Atyame et al. (2014) [42] (S4 Table). To study the effect of the absence of Wolbachia on embryogenesis, tetracycline-treated Wolbachia-free lines (TC lines) SlabTC, IstanbulTC, Ichkeul 21TC, and Ichkeul 13TC were obtained respectively from Slab, Istanbul, Ichkeul 21 and Ichkeul 13 wPip infected lines, as described in  [11]. After TC treatment, PCR amplifications of a fragment of wsp gene using the primers designed in Berticat et al. (2002) [52] allowed controlling the absence of Wolbachia in DNA extracted from a larvae pool (Dneasy Blood & Tissue Spin-Column protocol Kit; Qiagen; Bench protocol: Animal Tissues). To prevent possible side-effects of the treatment, TC-treated lines were raised in standard laboratory conditions for at least four generations without tetracycline before the beginning of experiments.
C. pipiens lines with the same host genetic background but different Wolbachia strains. wPipI strain from Tunis line was introduced into Slab line nuclear genetic background through 8 backcrosses. For the first generation, 200 females from Tunis line were crossed with 100 males of the SlabTC line. Then, for each of the seven following generations, 200 females from the previous generation were crossed with 100 males from the SlabTC line. This led to a progressive replacement (over 97%) of the maternal nuclear genome (Tunis) by the paternal nuclear genome (SlabTC), with retention of the maternal cytoplasm, including the Tunis Wolbachia strain [this line was called Sl(wPipI-Tunis)].

Cellular study of early embryogenesis
To characterize CI cellular phenotype(s) in C. pipiens, several crosses were performed (S2 Table). For every crosses, to avoid confounding age effects, two-day old adults were released in cages. Cages containing 100 females and 50 males were then put into a closet at 25˚C where day-night cycle was inverted to allow collection of early developmental stage eggs during the day. After six days in these cages, females were fed with turkey blood in heparin sodium (bcl Wholly Wild World) using a Hemotek membrane feeding system (Discovery Workshops, United Kingdom). Five days after blood meal, water-pots were placed into the cages to collect the eggs-rafts. For C. pipiens eggs, at 25˚C, the meiosis is approximatively completed 30 minutes after the oviposition and the first mitotic nucleus division 15 minutes after the end of the meiosis, while four hours after oviposition the embryos normally reach the syncytial blastoderm stage [53]. Since, the CI defects described in D. simulans [25] and N. vitripenis [23,24] occurred during the first nucleus mitotic division, we mainly collected eggs aged from 30 minutes to 1hour. Older eggs were also harvested to monitor further developmental stages in both fertile and sterile crosses. Eggs-rafts were then placed into commercial bleach (active ingredient, 9.6% of sodium hypochlorite) to dissociate eggs, and then washed in distilled water. They were then fixed by being shaken for 2 hours in a solution of 3.2% para-formaldehyde in PBS 1X with Tween 0,02% (PBS-T) and washed with PBS 1X. For each fixed egg, the chorion was removed manually with a needle under an optical microscope (Leica MZ 8). Dechorionated embryos were then collected and treated with RNAse A (10 mg/mL, Sigma) overnight.
To differentially visualize the paternal from the maternal chromatin, we used propidium iodide to mark both chromatin and an anti-acetylated histone H4 labelling that preferentially marks the de novo assembled paternal chromatin after protamine removal [22]. Thus maternal and paternal chromatin will be respectively predominantly marked with propidium iodide (mosty red fluorescence) and with anti-acetylated histone H4 antibodies (mostly green fluorescence). For immunolabeling, embryos were first incubated overnight at 4˚C with primary antibodies (Polyclonal anti-acetylated histone H4 primary antibody (1:1000, Upstate)), washed during one day with PBS-T 1X, then incubated overnight at 4˚C with the secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG secondary antibodies (1:250, Invitrogen)) then washed with PBS-T 1X. Embryos were then incubated in PBS-T 1X for 20 minutes with propidium iodide a DNA intercalating agent (Molecular Probes, 10μL/1mL). Finally, embryos were washed for 5 minutes and mounted between slide and coverslip in Fluoroshield Mounting Medium (Vector). Confocal microscope images were captured on an inverted photoscope (DMIRB; Leitz) equipped with a laser confocal imaging system (TCS SP5; Leica) using an HCX PL APO 1.4 NA 63 oil objective (Leica). Images from fixed, immunostained embryos are merged confocal z-stacks taken sequentially in the green and red channels for the anti-acetylated histone H4 labelling and the propidium iodide signal respectively. Crosses from which confocal microscope images were obtained (Figs 1-3) are listed in S3 Table.

Proportion of unhatched developed embryos in CI crosses
To study the proportion of unhatched developed embryo in CI crosses, we performed a total of 32 crosses: 20 crosses involving four lines for the males (MAL lines) and five lines for the females (FEM lines), 5 involving Sl(wPipI-Tunis) for the males and the five FEM lines, and 7 involving the four MAL lines and females from different TC lines (S2 Table). All these crosses were performed using 50 females and 25 males. After 6 days in the cages, females were bloodfed and after 5 days eggs-rafts were collected in water pot and deposited into 24 wells plates. As hatching normally occurs approximately 48 hours after oviposition, developmental status in non-viable rafts was characterized at least two days after eggs-rafts collection. To attribute a developmental status to each egg, eggs-rafts were mounted between slide and coverslip, observed and documented with an optic microscope (Axiophot2 equipped with a CCD camera, Zeiss). Two developmental statuses were discriminated i) unhatched embryos harboring no visible development (Fig 2G1), or ii) unhatched embryos with visible development ( Fig  2G2). For each cross, we calculated the proportion of embryos showing development for 50 embryos per eggs-raft in 10 eggs-rafts (total of 500 eggs observed per cross).

Ploidy determination in CI developed embryos
To assess the ploidy status in unhatched developed embryos, we used a PCR/RFLP diagnosis kdr/ RsaI that allowed discriminating between C. pipiens and C. quinquefasciatus lines, as previously described in  [44]. Slab (C. quinquefasciatus) and Ichkeul 13 (C. pipiens) were chosen because they exhibit an unidirectional sterile cross: fertile in the direction (♂Ichkeul 13 x ♀Slab) and sterile in the other direction (♂Slab x ♀Ichkeul 13). This PCR/RFLP test was performed on DNA extracted as describe above from i) a pool of larvae from Slab and Ichkeul 13 parental lines and ii) from eggs-rafts resulting from the two reciprocal crosses between those two lines.

Real Time Quantitative PCR
Quantification of Wolbachia density in male testes. In order to test the influence of Wolbachia densities in testes on the CI cellular intensity, we quantified them with Real Time Quantitative PCR using the LightCycler 480 system (Roche). Specific primers and procedures were described in Berticat et al. (2002) [52]. Testes from 6-day old males of Tunis, Utique, Lavar and Slab lines were sampled. Each DNA template were obtained from pools of three testis pairs and extracted as described above. Five independent DNA templates were realized for each line. To estimate the number of Wolbachia per mosquito testes, we amplified two different genes on each sample, the C. pipiens specific ace-2 locus [54] and the Wolbachia specific monocopy wsp locus [52]. Standard curves were performed using dilutions of a pBluescriptKS vector containing a unique ace-2 and wsp gene copy. Each DNA template was analyzed in triplicate for both wsp and ace-2 quantification. As both genes are present as single copies per haploid genome, the ratio of wsp over ace-2 signals allowed estimating the relative number of Wolbachia genomes per Culex genome, thus correcting for mosquito size and DNA quality.
Amplification of cidA and cidB genes within wPip genome. For each C. pipiens-Wolbachia line (Tunis, Utique, Lavar and Slab hosting a different wPip strains belonging to group I, II or III) and exhibiting different mod profiles, quantitative PCRs were carried out to estimate the number of copies of cidA and cidB genes per wPip genome. Three different quantitative PCRs were performed on DNA samples extracted from ten 6-day old males per line following the procedure described in Berticat et al. (2002) [52]: i) specific of the locus wsp, ii) specific of a 189bp fragment of the cidA gene conserved between all wPip strains using primers wPip_ 0282_QPCR_2_Dir (5'-AGG-TCC-TGT-ATT-TGA-TTT-CTG-GA) and wPip_0282_ QPCR_2_Rev (5'-TGA-ACG-CGA-GAA-AGA-GCA-AG), and iii) specific of a 135bp fragment of the cidB gene conserved between all wPip strains using primers wPip_0283_ QPCR_1_Dir (5'-TGA-GTG-TTT-GGA-GAA-TGA-AGG-A) and wPip_0283_QPCR_1_Rev (5'-TTC-CCA-AAA-GCA-AAA-CCA-GTT). Standard curves of cidA, cidB and wsp genes were performed using dilution of the PCR product of these three genes previously quantified using the flurorometre-QuBit (Invitrogen). Each DNA template was analysed in triplicate for wsp, cidA and cidB locus. CidA and cidB copy numbers were estimated using the ratio of cidA or cidB estimated copy number over wsp estimated copy number, to obtain a copy number per Wolbachia genome since wsp is present in one copy in Wolbachia genome.
Expression of cidA and cidB genes. For each line Tunis, Utique, Lavar and Slab, ten 6-day old males were used for RNA extraction with Trizol (Life Technologies) and treated with DNase with the TURBO DNA-free Kit (Life Technologies), in accordance with the manufacturer's instructions. 2-5 μg of each total RNA sample were reverse-transcribed into cDNA with the SuperScript III Reverse Transcriptase Kit and 30 ng of random oligomer primers ((RP)10; Invitrogen, Life Technologies). Three different quantitative PCRs were performed: i) specific of wsp locus, ii) specific of cidA locus, and iii) specific of cidB locus as describe above. Each DNA template was analysed in triplicate for wsp cidA and cidB locus. Levels of expression of cidA and cidB genes were estimated relatively to wsp genes by using the ratio of expression of these two genes over wsp.

Determination of cidA/cidB variants repertoire in the different wPip hosted by males
To describe the diversity of cidA wPip /cidB wPip repertoires for the two C. pipiens lines Utique and Slab not yet investigated, cloning and Sanger sequencing of the cidA and cidB variants were performed as described in Bonneau et al. (2018) [36] on DNA from pools of larvae extracted as described above. Variant sequences were aligned, using the Muscle algorithm implemented in Seaview 6.4.1 software [55].

Statistical analysis
Variability of unhatched developed embryo proportion in sterile crosses was analyzed using a generalized linear model (GLM): UDEP = CROSS + ε, with UDEP the proportion of unhatched developed embryos for each cross (CROSS, which represent the interaction between the MAL and FEM lines) and ε the error parameter, following a binomial distribution. To test the specific effect of the four MAL lines and the five FEM lines separately, GLMs with mixed effects (GLMM) were used: UDEP = MALE + FEMALE + 1|CROSS + ε with MALE and FEMALE respectively the MAL and FEM lines involved in each cross as fixed effects, with CROSS as the interaction between MAL and FEM lines as a random effect (as crosses to produce embryos necessary require an interaction between females and males), and ε the error parameter, following a binomial distribution. To test for a specific effect of the host genetic background in crosses involving males from Sl(wPipI-Tunis) and Tunis lines which host the same Wolbachia in two different genetic background we used a GLMM: UDEP = MALBACK + FEMALE + 1|CROSS + ε with UDEP the unhatched developed embryos proportion for each cross involving males from Sl (wPipI-Tunis) and Tunis lines (MALBACK) and the five FEM lines (FEMALE) as fixed effects, with CROSS as a random effect, and with ε the error parameter, following a binomial distribution.
For several variables (Wolbachia density in testes, cidA and cidB expressions and copy number) obtained with q-PCR, variability between the four MAL lines was analyzed using GLMs in the form VAR = MALE + ε, with VAR one of the estimated variable of the MAL line (MALE) and ε the error parameter, following a Gaussian distribution.
Spearman correlation tests [56] were used to test for correlation between these variables (Wolbachia density in testes, cidA and cidB expressions and copy number) and the proportion of unhatched developed embryos for each MAL line. We did the same for the relation between the number of different cidA variants and the proportion of unhatched developed embryos for each MAL line. Finally, Wilcoxon test [57] was used to compare mean proportions of unhatched developed embryos between the two MAL lines harboring only two different cidB variants and the two MAL lines harboring four different cidB variants.
All computations were performed using the R version 3.4.4 [58]. Computed models were simplified by testing the significance of the different terms using likelihood ratio tests (LRT) and starting from the higher-order terms, as described in Crawley [59]. Factor levels of qualitative variables that were not different in their estimates (using LRTs) were grouped as described by Crawley [59]. The normality of the residuals was tested using Shapiro test for models with Gaussian error [60]. For models with Binomial error, overdispersion was calculated using the "dispersion_glmer" function from the package blmeco for GLMM, and by dividing the residual deviance by the residuals degree of freedom of the model for GLM [61]; when detected, overdispersion was taken into account in the LRTs [62,63].
Supporting information S1 Table. Presentation of the ten C. pipiens lines used in this study. (DOCX) S2 Table. The different crosses from which the CI cellular phenotype in C. pipiens was studied. Three different types of crosses were performed to study the cellular phenotype responsible for embryonic death in sterile crosses: i) sterile crosses between males and females infected with different Wolbachia strains, ii) sterile crosses between infected males and uninfected females, and iii) fertile crosses between males and females from the same mosquito line infected or not by Wolbachia. Crosses from which confocal and optical microscopy pictures were taken are indicated next to the cross (Figs 1-3). Table. Proportion of unhatched developed embryos from CI crosses involving either infected or uninfected (TC) females. Proportion of unhatched developed embryos are given as the mean proportion measured on fifty eggs for 10 rafts per cross (500 eggs observed per cross) ± standard deviation. No unhatched developed embryos were found in any of the seven different crosses performed between infected males and uninfected females. Protein sequences alignment of the CidA variants found in the four Wolbachia strains wPipI-Tunis, wPipI-Utique, wPipII-Lavar and wPipIII-Slab (MAL lines). The first sequence is used as a reference to determine the polymorphic region. For more clarity, only polymorphic positions are represented, thus amino-acid positions are not continuous. When more than two contiguous amino-acids were variable the "-"symbol was used between the first and the last variable position of the zone. Colors show polymorphic blocks of residues present in variants regardless of their phylogenetic wPip group (I to III). No cidA or cidB nucleotide sequence variant was shared between the three wPip groups. However, the wPipII-Lavar CidA_II(α/1) variant and the wPipIII-Slab CidA_III(β/8) variant presented the same amino-acid sequence. Based on their nucleotide sequences wPipIII-Slab exhibited ten variants of cidA, wPipII-Lavar three, wPipI-Tunis four and wPipI Utique seven. However, wPipIII-Slab exhibited only seven variants that differ in their amino-acid sequences since cidA_III(χ/6) and cidA_III(χ/7), cidA_III (δ/6) and cidA_III(δ/7), cidA_III(δ/8) and cidA_III(δ/9) have respectively identical amino-acid sequences (i.e. nucleotide polymorphic positions between them are synonymous). (TIF)

S3 Fig. The repertoire of CidB protein variants in the four MAL line wPip strains.
Protein sequences alignment of the CidB variants found in the four Wolbachia strains wPipI-Tunis, wPipI-Utique, wPipII-Lavar and wPipIII-Slab (MAL lines). The first sequence is used as a reference to determine the polymorphic region. For more clarity, only polymorphic positions are represented, thus amino-acid positions are not continuous. When more than two contiguous amino-acids were variable the "-"symbol was used between the first and the last variable position of the zone. Colors show polymorphic blocks of residues present in variants regardless of their phylogenetic wPip group (I to III). However, no variant (i.e. complete CidB sequence) is common to wPip strains from different groups.