# Wolbachia transinfections in Culex quinquefasciatus generate cytoplasmic incompatibility

Thomas H. Ant, C. Herd, Faustine Louis, Anna-Bella Failloux, Steven P. Sinkins

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Thomas H. Ant, C. Herd, Faustine Louis, Anna-Bella Failloux, Steven P. Sinkins. Wolbachia transinfections in Culex quinquefasciatus generate cytoplasmic incompatibility. Insect Molecular Biology, 2019, 10.1111/imb.12604. pasteur-02160213

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# Wolbachia transinfections in Culex quinquefasciatus generate cytoplasmic incompatibility

Thomas Ant, ; Herd, ; Louis, ; Failloux, Anna -Bella, ; Sinkins, C. Herd, Faustine Louis, Anna-Bella Failloux, C Sinkins

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## To cite this version:

Thomas Ant, ; Herd, ; Louis, ; Failloux, Anna -Bella, et al.. Wolbachia transinfections in Culex quinquefasciatus generate cytoplasmic incompatibility. Insect Molecular Biology, Wiley, 2019, 10.1111/imb.12604. pasteur-02160213

HAL Id: pasteur-02160213

https://hal-pasteur.archives-ouvertes.fr/pasteur-02160213

Submitted on 19 Jun 2019

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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# Title: *Wolbachia* transinfections in *Culex quinquefasciatus* generate cytoplasmic incompatibility

Authors: Ant, Thomas H.<sup>1,2</sup>; Herd C<sup>1,2</sup>; Louis, Faustine<sup>3</sup>; Failloux, Anna-Bella<sup>3</sup>; Sinkins, Steven p.<sup>1,2*</sup>

1. MRC-University of Glasgow Centre for Virus Research; University of Glasgow; Glasgow; UK.

2. Biomedical and Life Sciences; Lancaster University; Lancaster; UK.

3. Department of Virology, Arboviruses and Insect Vectors, Institut Pasteur, Paris, France

*Correspondence to: steven.sinkins@glasgow.ac.uk

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/imb.12604
Abstract

*Culex quinquefasciatus* is an important mosquito vector of a number of viral and protozoan pathogens of humans and animals, and naturally carries the endosymbiont *Wolbachia pipientis*, strain *wPip*. *Wolbachia* are being used in two distinct vector control strategies: firstly, population suppression caused by mating incompatibilities between mass-released transinfected males and wild females; and secondly, the spread of pathogen transmission-blocking strains through populations. Using embryonic microinjection, two novel *Wolbachia* transinfections were generated in *Cx. quinquefasciatus* using strains native to the mosquito *Aedes albopictus*: a *wAlbB* single infection, and a *wPip* plus *wAlbA* superinfection. The *wAlbB* infection showed full bi-directional cytoplasmic incompatibility (CI) with wild-type *Cx. quinquefasciatus* in reciprocal crosses. The *wPipwAlbA* superinfection showed complete unidirectional CI, and therefore population invasion potential. While the *wAlbB* strain showed comparatively low overall densities, similar to the native *wPip*, the *wPipwAlbA* superinfection reached over 400-fold higher densities in the salivary glands compared to the native *wPip*, suggesting it may be a candidate for pathogen transmission blocking.
Introduction

*Culex quinquefasciatus* (Say), the southern house mosquito, transmits a number of important human and animal pathogens, including arboviruses such as West Nile (WNV) and Rift Valley Fever (RVFV), and the filarial nematode *Wuchereria bancrofti* (Sudomo et al. 2010). It is also significant from the perspective of wildlife conservation, since it transmits avian malaria (*Plasmodium relictum*) and avian pox virus on the Hawaiian Islands, where it has been incriminated in decline of several endangered bird species (van Riper et al. 1986). *Cx. quinquefasciatus* exhibits plasticity in host choice, frequently biting humans and other mammals as well as birds, and as such has the potential to act as a bridge vector for zoonotic pathogens (Farajollahi et al. 2011). As a cosmopolitan species, it has a wide distribution throughout the tropics and subtropics where it is frequently associated with urban areas. The larval stages thrive in domestic water bodies polluted with organic matter, such as pit latrines, blocked drainage ditches, and shallow wells. Vector control is generally limited to insecticide treatments and larval-source management. Due to predominantly night-time biting and indoor resting, the distribution of insecticide-treated nets (ITNs) and the use of indoor residual spraying (IRS) for the control of malaria-transmitting *Anopheles* species has applied concomitant selection on *Cx. quinquefasciatus* populations, with high levels of insecticide resistance reported in Africa (Jones et al. 2012; Norris and Norris 2011; Yadouléton et al. 2015) and Asia (Yanola et al. 2015).

*Cx. quinquefasciatus* is a member of the *Culex pipiens* species complex, almost all populations of which are infected at close to 100% frequency with the maternally-inherited intracellular endosymbiont *Wolbachia pipientis*, strain wPip. *Wolbachia* is widespread throughout the phylum Arthropoda, where different strains induce a variety of reproductive manipulations to facilitate host population invasion. A common variant found in mosquitoes

Accepted Article
and other diptera is a modification of the infected male germline that results in sterility unless a compensatory *Wolbachia*-secreted rescue factor is present in the germline of infected females. This coupling of CI rescue with maternal transmission results in a relative reproductive advantage for *Wolbachia*-infected females, providing a population invasion potential, with frequency thresholds for spread largely determined by the balance between the positive fitness effects of CI and negative effects on life-history traits (Hancock et al. 2011; Hancock et al. 2016). In the *Cx. pipiens* species group, strain wPip induces a particularly complex pattern of crossing types between populations, with both unidirectional and bidirectional CI observed at varying levels of penetrance (Barr 1980; Bonneau et al. 2018; Guillemaud et al. 1997; Magnin et al. 1987; O'Neill and Paterson 1992; Sinkins et al. 2005; Walker et al. 2009).

Cytoplasmic incompatibility provides a mechanism of sterility that can be used to reduce the reproductive potential of a population through the mass-release of males (Atyame et al. 2016; Atyame et al. 2011; Calvitti et al. 2012; Chen et al. 2013; Dobson et al. 2002; Laven 1967; Zabalou et al. 2004); the development of highly efficient automated sex separation technology makes this feasible on a large scale (Gilbert and Melton 2018). The natural incompatibilities between wPip variants within the complex could in theory be utilised for sterile male releases; however, it would be highly desirable for practical purposes to select a single 'universal' line adapted to mass rearing that generates sterility with the females of all target populations. To do so, it will be necessary to create transinfections with *Wolbachia* strains introduced from other host species.

*Wolbachia* has also been shown to possess a strong pathogen-blocking capacity when some novel *Wolbachia*-host combinations are generated (Ant et al. 2018; Bian et al. 2010;
Blagrove et al. 2012; Kambris et al. 2010; Moreira et al. 2009; Walker et al. 2011). *Aedes aegypti* transinfected with the *wAlbB Wolbachia* strain, for example, show strong transmission blocking of a number of arboviruses (Ant et al. 2018; Bian et al. 2010), including dengue, while *wAlbB* transinfected *Anopheles stephensi* show reduced *Plasmodium falciparum* oocyst and sporozoite loads (Bian et al. 2013). Artificial germline transinfection with *Wolbachia* has so far been limited to *Ae. aegypti* (Blagrove et al. 2012; Moreira et al. 2009; Walker et al. 2011; Xi et al. 2005), *Ae. albopictus* (Ant and Sinkins 2018; Blagrove et al. 2012), and *An. stephensi* (Bian et al. 2013). The extension of *Wolbachia* transinfection generation to *Culex* or other vector species, to allow the exploration of either transmission blocking for replacement strategies or the generation of sterile males for suppression, has been encumbered by the technical challenges inherent in generating stable infections in the laboratory. Here we report the generation of two novel transinfections in *Cx. quinquefasciatus* with *Wolbachia* strains native to *Aedes albopictus*, including a native-plus-novel strain superinfection. The relative densities achieved by the transinfections, CI crossing patterns, the effects of the novel strains on host fecundity and immune gene expression are presented.

## Results

### Generation of *wAlbB* and *wPipwAlbA* lines in *Cx. quinquefasciatus*

A *Wolbachia*-free *Cx. quinquefasciatus* line PelU was previously created by antibiotic treatment of the wild-type *wPip*-carrying Sri Lankan PelA colony (Pinto et al. 2013). A *wAlbB* transinfection was generated by transferring cytoplasm from eggs of a *wAlbB*-carrying *Ae. aegypti* line to PelU embryos. A total of 420 PelU embryos were microinjected with *wAlbB* (Table 1). The *wAlbB*-carrying *Cx. quinquefasciatus* line was generated from a single G₀
female. Females of the wAlbB line were outcrossed to PelU males for five generations before a stable inbreeding colony was established. Maternal transmission rates of wAlbB when PelU males were crossed to wAlbB females (i.e. in the absence of CI) were found to be 100% from 200 progeny assessed.

A superinfected *Cx. quinquefasciatus* line carrying both wAlbA and wPip was established through transfer of cytoplasm from the eggs of wAlbA transinfected *Ae. aegypti* to embryos of the PelA (wild-type wPip-infected) colony. A total of 580 embryos were microinjected with wAlbA (Table 1). The wPipwAlbA line was established from the progeny of a single superinfected G₀ female. Females from this line were backcrossed for five generations to males of the wPip line before a stable inbreeding colony was established. wPipwAlbA females were crossed to PelU males to evaluate rates of maternal inheritance in the absence of CI. Strain-specific PCR indicated that the superinfection was transmitted at 100% fidelity from 200 progeny assessed.

Attempts were also made to generate a line carrying wMel, a *Wolbachia* strain native to the fruit fly *Drosophila melanogaster*. Embryos from a transinfected strain of *Ae. aegypti* were used as the source of wMel, and although more than 1,700 embryos of the wPip and PelU lines were injected, far more than for wAlbA and wAlbB, no stable transinfection was generated (Table 1).

CI crossing patterns and fecundity

Crosses were set-up between the transinfected, wPip (wildtype) and PelU lines. No eggs hatched from reciprocal crosses between the wAlbB line and the wPip line, displaying a classical pattern of complete bi-directional CI (Figure 1). Egg hatch rates from crosses between PelU males and wAlbB females were not significantly different from wild-type
hatch rates (*p*=0.077, Fisher's exact test), suggesting little effect of *wAlbB* on embryonic viability.

When males of the *wPipwAlbA* line were crossed to *wPip* females no egg hatching was observed, while *wPipwAlbA* females were fully compatible with *wPip* males and displayed no reduction in hatch rates compared to *wPip* within-strain crosses (*p*=0.586, Fisher's exact test), suggesting full *wPip* CI rescue (Figure 2). The *wPipwAlbA* line therefore displayed a classical pattern of complete unidirectional CI with wild-type *Cx. quinquefasciatus*. Eggs resulting from crosses between females of the *wPipwAlbA* line and *Wolbachia*-free males showed similar hatch rates to those seen for the *wPip* colony (*p*=0.238, Fisher's exact test), suggesting little or no negative effects of the *Wolbachia* superinfection on embryo hatch rates in non-CI crosses.

The effects of *Wolbachia* infection status on the mean number of eggs produced by a female in an egg-raft was assessed. No significant effect of *Wolbachia* infection status or strain was detected (Figure 3) (*p*>0.4 for all comparisons, 1-way ANOVA with Dunnett's), indicating that the presence of non-native *Wolbachia* did not result in a reduction in fecundity, at least over the first gonotrophic cycle.

### *Wolbachia* densities

Total *Wolbachia* densities were measured in five-day-old whole female carcasses, dissected salivary glands and ovary tissue (Figure 4). The *wAlbB* line displayed the lowest whole carcass density, with a mean of 1.64 (±1.11 SD) *Wolbachia* per host genome copies, significantly lower than the 4.34 (±1.68 SD) *Wolbachia* per host genome for the native *wPip* strain (*p*=0.014, 1-way ANOVA with Dunnett's). The *wPipwAlbA* superinfection reached a
significantly higher density than wild-type with a mean of 13.45 (±6.19 SD) *Wolbachia* per
host genome copies (*p*=0.00765, 1-way ANOVA with Dunnett's). Densities of *Wolbachia* in
the ovaries were not found to vary between the transinfected and wild-type line (*p*>0.075
for both comparisons, 1-way ANOVA with Dunnett's). For the salivary glands, however, a
significantly higher mean density was observed for the *wPipwAlbA* superinfection compared
to the *wPip* strain alone (*p*<0.0001, 1-way ANOVA with Dunnett's), with 200.84 (±47.31 SD)
compared to 0.494 (±0.36 SD) *Wolbachia* per host genome copies, respectively. The *wAlbB*
strain showed a mean salivary-gland density of 8.59 (±7.23 SD) *Wolbachia* per host genome,
a non-significant difference compared to *wPip* (*p*=0.072, 1-way ANOVA with Dunnett's).

Immune gene expression

The transcription of a selection of immune genes was measured in whole adult females of the wAlbB and wPipwAlbA lines and was compared to transcription levels in wPip females. Immune genes investigated were: Rel1 and Rel2, regulators of the Toll and IMD pathways respectively, Defensin1 which can be activated through both Toll and IMD signalling, and LRIM1, part of the complement-like pathway. No significant effect of either *Wolbachia* strain was found on immune gene transcription (*p*>0.2, 1-way ANOVA with Dunnett's) (Figure 5).

Discussion

The two novel *Wolbachia* transinfections in *Cx. quinquefasciatus* reported here could
potentially contribute to control in two ways: by providing a source of sterile males for
population suppression, and through pathogen transmission blocking via population
replacement. Males of the wAlbB-only infection and the wPipwAlbA superinfection both
caused fully penetrant CI when crossed to wild-type females. Females of the wAlbB line

Accepted Article
were also incompatible with wild-type males, a bi-directional CI pattern resulting in high
invasion thresholds, ideal for a suppression strain. No significant effect of wAlbB was
observed on host fecundity. This suggests the line is relatively fit compared to wild-types, an
important factor given successful suppression would depend on the mass-rearing and
release of large numbers of fit, competitive incompatible males. Females of the wPipwAlbA
line were fully compatible with wild-type males. The superinfection is thus expected to have
the capacity to invade and establish in wild populations of the same crossing type as the Sri
Lankan Pel wildtype line. Although novel *Wolbachia* transinfections have been shown to
decrease fecundity in some instances (Hoffmann et al. 2014), likely reducing strain
invasiveness (Schmidt et al. 2017), no significant effects of the wPipwAlbA superinfection
were found on fecundity - although an impact of the infection on other life-history traits
such as longevity cannot be ruled out. wPipwAlbA in *Cx. quinquefasciatus* provides a further
example of additive CI, with modification and rescue of co-infecting strains expressed
independently (Dobson et al. 2004; Joubert et al. 2016). However, additive superinfection CI
is not always stable; a *Wolbachia* triple infection in *Ae. albopictus* suggested co-infecting
strain interaction, affecting densities and CI rescue of co-infecting strains (Ant and Sinkins
2018). Attempts to generate a wMel infection in *Cx. quinquefasciatus* were unsuccessful.
The relatively high numbers of positive G0 females generated with no resulting G0-G1
transmission suggests that there may be factors limiting the transmissibility of wMel in this
species.

*Wolbachia* intracellular density correlates positively with levels of pathogen inhibition (Lu et al. 2012), although there is considerable between-strain variability in blocking capacity (Ant et al. 2018; Martinez et al. 2014). Surprisingly, we found lower average densities for wAlbB compared to the native wPip infection. This was unexpected as novel transinfections tend to
show greater somatic tissue dispersal (McGraw et al. 2002), and thereby higher overall densities than native strains. As somatic infections can have deleterious effects on fitness, co-evolutionary pressures acting on both host and symbiont are expected to favour mechanisms that restrict tissue tropism to the testes and ovaries given CI and transovarial transmission. These factors appear to be strain and host-specific; the native *Wolbachia* strains in female *Ae. albopictus* for example, particularly wAlbA, are largely localised to the ovaries and testes, while the non-native wMel can be found at high density in somatic tissues (Ant and Sinkins 2018). A possible explanation for the low density of wAlbB in *Cx. quinquefasciatus* is the close phylogenetic relationship of wAlbB and wPip (Ellegaard et al. 2013), with mechanisms selected to restrict wPip in somatic tissues also functioning with wAlbB. As high densities also tend to result in reduced fitness (Ant et al. 2018; Chrostek et al. 2013; Fraser et al. 2017; Sinkins 2013), the finding that wAlbB achieves low densities in *Cx. quinquefasciatus* suggests that any fitness costs in this line may be minimal, important for mass-rearing and mate competition; however, it does also suggest that there will be limited pathogen inhibition potential.

The wPipwAlbA transinfection was found to have an approximately three-fold greater whole carcass density than the wPip-only native infection in the PelA line. This appears to be due to a greater distribution of *Wolbachia* in somatic tissues, with a 400-fold higher density observed in the salivary glands. A high wAlbA density is consistent with previous results from a transinfection in *Ae. aegypti*, where wAlbA was found to reach higher densities than a range of other strains, including wAlbB (Ant et al. 2018). This contrasts with the relative densities of the two strains in the native *Ae. albopictus*, where wAlbA reaches approximately 10% the density of wAlbB (Dutton and Sinkins 2004); again, co-evolutionary pressures have likely selected for reproductive tissue localization in the native host.
Experiments carried out in *Ae. aegypti* showed a low virus inhibition potential for wAlbA against the model arbovirus Semliki Forest Virus (SFV) (Ant et al. 2018) following intrathoracic viral challenges, but it is nevertheless able to block transmission of Zika using oral challenges (Chouin-Carneiro et al. 2019). West Nile and Zika are related Flaviviruses, and thus wAlbA may have transmission-blocking potential in *Culex*.

*Culex quinquefasciatus* is a competent vector for a wide variety of pathogens, ranging from viruses including West Nile and Rift Valley Fever, to eukaryotes including the protozoan *Plasmodium relictum* and the filarial nematode *Wuchereria bancrofti*. Experimental results from a range of host species suggest that the mechanism of *Wolbachia*-mediated pathogen inhibition differs between viruses and eukaryotic parasites. *Plasmodium* and filarial inhibition likely depends at least in part on a priming of the host innate immune system (Bian et al. 2013; Kambris et al. 2010; Kambris et al. 2009). *Wolbachia* transinfections in *Ae. aegypti* activate a range of immune signalling pathways, including the Toll, Imd, and complement-like pathways (Kambris et al. 2009; Moreira et al. 2009; Rancès et al. 2012). *An. gambiae* somatically infected with *wMelPop* block *Plasmodium berghei* development, which can be restored by knock-down of the Tep1 opsonin (Kambris et al. 2010). No immune priming was detected in the transinfections of *Cx. quinquefasciatus* presented here, which included examining defensin, an anti-microbial peptide that was very highly upregulated in *wMelPop*, *wMel* and *wAlbB* infected *Ae. aegypti* (Bian et al. 2010; Rancès et al. 2012). This lack of immune upregulation suggests that any blocking of eukaryotic parasites in these *Wolbachia* transinfections may be limited. In contrast, *Wolbachia*-mediated blocking of viruses does not appear to require immune priming (Blagrove et al. 2012; Molloy and Sinkins 2015; Rances et al. 2013; Rances et al. 2012). Evidence from *Ae. aegypti* cells infected with *wMelPop* and challenged with dengue suggest that blocking is due to a
disruption of host cell lipid homeostasis and accumulation of cholesterol in lipid droplets (Geoghegan et al. 2017). A previous study investigating the immune priming of a transinfection of *wMel* in *Ae. albopictus* also found very low levels of immune gene upregulation (Blagrove et al. 2012; Molloy and Sinkins 2015), suggesting that the immune response of natively infected species may have an innate desensitisation to the presence of *Wolbachia*. The demonstration of strong dengue and chikungunya blocking by the high density *wMel* infection in *Ae. albopictus* in the absence of immune priming is encouraging for the potential for viral inhibition in the *wPipwAlbA Cx. quinquefasciatus* line presented here.
Experimental procedures

Lines and rearing

The *Cx. quinquefasciatus* wild-type was the Pel line originally colonized in Sri Lanka. The *Wolbachia*-free PelU line was created by antibiotic treatment (Pinto et al. 2013). The source of *wAlbA* and *wAlbB Wolbachia* for cytoplasmic transfers was from transinfected *Ae. aegypti* colonies (Ant et al. 2018). All mosquito colonies were maintained at 27°C and 70% relative humidity with a 12-hour light/dark cycle. Larvae were fed tropical fish pellets (Tetramin, Tetra, Melle, Germany) and adults were given access to a sucrose meal *ad libitum*. Blood meals were provided using a Hemotek artificial blood-feeding system (Hemotek, UK) using defibrinated sheep blood (TCS Biosciences, UK). Eggs were collected by providing a bowl of water for oviposition 3-4 days post blood-feeding.

Transinfection generation

The *wAlbB Cx. quinquefasciatus* line was generated by transferring cytoplasm from *wAlbB*
infected *Ae. aegypti* into embryos derived from the PelU colony. The *wPipwAlbA*
superinfection was generated by transferring cytoplasm from *wAlbA* infected *Ae. aegypti*
into embryos derived from the wild-type PelA colony. Microinjections were performed using
methods described previously (Blagrove et al. 2012) adapted for *Culex* mosquitoes. Briefly,
~30-minute-old egg rafts were collected and individual eggs lined against a damp
nitrocellulose membrane fixed to a glass microscope slide. Eggs were briefly dried (~1-
minute) and covered in Voltalef 10s oil for injection. Injected eggs were monitored for 24
hours, and neonate larvae removed from oil using a fine paint brush and placed in a bowl of
water for development. Female *G₀* survivors were back-crossed to wild-type males, blood-
fed and separated individually for oviposition. G₀ females were analysed for *Wolbachia* infection by strain specific PCR and eggs from *Wolbachia* negative G₀ females were discarded. Eggs of *Wolbachia*-positive females were hatched and G₁'s were assessed for *Wolbachia* G₀-G₁ germ-line transmission. In generating both the *wAlbB* and *wPipwAlbA* lines, two separate G₀ females with G₁ transinfection transmission were derived. As duplicate transinfections carried the same *Wolbachia* strains in the same host background, only one line of each was carried forward for characterisation – in both instances the G₃ colony with the greatest number of individuals was chosen. Individual *Wolbachia* strains were screened using strain-specific primers: 183F+691R for *wPip*; *wAlbAF*+*wAlbAR* for *wAlbA*; *wAlbBF*+*wAlbBR* for *wAlbB*. For sequences see Table 2.

## Maternal inheritance, CI crosses, and fecundity

To assess rates of maternal inheritance, females from the *Wolbachia* transinfected lines were crossed to PelU males in pools of 30 males and 15 transinfected females. A blood-meal was provided and egg rafts individualised and hatched. DNA from a selection of 10 larvae resulting from each egg raft (100 larvae assessed for each line in total) was extracted at the pupal stage and a PCR for *Wolbachia* was performed.

Rates of CI induction and rescue both with wild-type mosquitoes and between infected lines were assessed by crossing 30 males and 15 females of each line. A blood-meal was provided and egg rafts collected and individualised. Eggs were counted to assess female fecundity. Resulting larvae were counted at the L2-L3 stage to provide hatch rates. Females with no egg hatch were dissected to check spermathecae for successful mating. Unmated females were excluded from hatch rate evaluations.

Accepted Article
### Density assessment

For qPCR analysis, genomic DNA was extracted from mosquitoes using phenol/chloroform. Mosquitoes used in density experiments were adults 5-days post pupal eclosion. gDNA was diluted to 100ng/µl using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA). A BioRad CFX-96 real-time PCR detection system was used (BioRad, Hercules, California, USA) with 2 x SYBR-Green mastermix (Biotool, Houston, Texas, USA). Total *Wolbachia* density was analysed by relative quantification of the *Wolbachia* surface protein (wsp) against the mosquito homothorax (HTH) gene.

### Immune gene expression

Adult female RNA was extracted from 4-5 adult mosquitoes using TRIzol Reagent (Invitrogen-Life Technologies) following manufacturer's instructions. TRIzol-extracted RNA was DNase I treated and purified via standard phenol/chloroform extraction. cDNA synthesis was performed in 10 µl, using the iScript cDNA synthesis kit (BioRad). A BioRad CFX-96 real-time PCR detection system was used (BioRad, Hercules, California, USA) with 2 x SYBR-Green mastermix (Biotool, Houston, Texas, USA). Primers Def1-F + Def1-R, Rel1-F + Rel1-R, Rel2-F + Rel2-R and LRIM1-F + LRIM1-R were used to assess levels of defensin 1, Rel1, Rel2, and LRIM1, respectively. Levels of target RNA sequences were normalized to the 18S rRNA house-keeping gene using the Pfaffl method. Primer sequences can be found in Table 2.

### Conflict of interest statement

The authors declare no conflict of interest.

Accepted Article
## Acknowledgements

This work was supported by the EDIMA ERA Net (contract N° 219235) and Wellcome Trust awards 108508 and 202888.

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Accepted Article
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**Figure 1.** Percentage egg hatch rates from individual egg rafts resulting from crosses between wild-type *Wolbachia* *wPip* line, *wAlbB*, and *Wolbachia* -ve (PelU, antibiotic-treated) lines. Boxplots show median values and interquartile ranges. Dots show hatch rates from individual egg rafts.

**Figure 2.** Percentage egg hatch rates from individual egg rafts resulting from crosses between wild-type *wPip* line, *wAlbAwPip*, and *Wolbachia*-ve (PelU, antibiotic-treated) lines. Boxplots show median values and interquartile ranges. Dots show hatch rates from individual egg rafts.

**Figure 3.** Average egg number per egg raft from *Wolbachia*-transinfected, *wPip*, and PelU lines over the first gonotrophic cycle. Eggs from the rafts of 12-15 females were counted. Error bars show standard deviation.

**Figure 4.** *Wolbachia* densities in ovary (O), salivary glands (SG), and whole female carcasses (WC) for the *wAlbAwPip*, *wAlbB* and *wPip* lines. Barcharts show mean densities and error bars show standard deviation. Each bar summarises data from 5 biological repeats, each with either 3 whole female carcasses, or the dissected tissues from 5 females.

**Figure 5.** Expression of immune genes in the *wAlbB* and *wAlbAwPip* lines normalised initially to the 18s rRNA house-keeping gene and then to expression in the *wPip* line. Error bars show 95% CI from 5 biological replicates, each containing cDNA from a pool of 3 females.
<table><caption>**Table 1.** Microinjection statistics for strain generation. 'Total embryos injected' is the number of *Culex quinquesfasciatus* embryos microinjected with each *Wolbachia* strain for each of the wPip and PelU lines. 'Total adults emerged' is the number of microinjected embryos surviving to produce adults, with parentheses showing percentage. 'Total positive G0 females' is the number of resulting adult female mosquitoes that were PCR positive for the transinfecting *Wolbachia* strain. 'Total G0-G1 maternal transmission' shows numbers of G0 females that successfully produced progeny positive for the transinfecting *Wolbachia* strain, with parentheses showing percentage of females displaying transmission out of total positive G0 females.</caption><thead><tr><th>Wolbachia strain</th><th colspan="2">wAlbB</th><th colspan="2">wAlbA</th><th colspan="2">wMel</th></tr><tr><th>Donor species</th><th colspan="2">Ae. aegypti</th><th colspan="2">Ae. aegypti</th><th colspan="2">Ae. aegypti</th></tr><tr><th>Recipient <i>Culex quinquefasciatus</i> strain</th><th>wPip</th><th>PelU</th><th>wPip</th><th>PelU</th><th>wPip</th><th>PelU</th></tr></thead><tbody><tr><td>Total embryos injected</td><td>680</td><td>420</td><td>580</td><td>660</td><td>780</td><td>940</td></tr><tr><td>Total adults emerged (%)</td><td>111 (16)</td><td>78 (19)</td><td>58 (10)</td><td>36 (5)</td><td>102 (13)</td><td>107 (11)</td></tr><tr><td>Total positive G0 females</td><td>20</td><td>18</td><td>8</td><td>4</td><td>12</td><td>18</td></tr><tr><td>Total G0-G1 maternal transmission (%)</td><td>0</td><td>2 (11)</td><td>2 (25)</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>**Table 2.** List of primer sequences used in this study.</caption><thead><tr><td>Primer name</td><td>5'-3' sequence</td></tr></thead><tbody><tr><td>Rel1-F</td><td>GCGACTTTGGCATCAAGCTC</td></tr><tr><td>Rel1-R</td><td>GTTCGACCGGAGCGTAGTAG</td></tr><tr><td>Rel2-F</td><td>GTCGAGATGGCCAAAACGATG</td></tr><tr><td>Rel2-R</td><td>TCATATTGTTGATGGCATT</td></tr><tr><td>LRIM1-F</td><td>CGTAATGGTGCCAAGAGACA</td></tr><tr><td>LRIM1-R</td><td>GGCGTAAGGTGCTGATGATT</td></tr><tr><td>Def1-F</td><td>GGTCCAATACTTCGCCAATAC</td></tr><tr><td>Def1-R</td><td>GATTGGGCGTCAACGATAGT</td></tr><tr><td>qWSP-F</td><td>ATCTTTTATAGCTGGTGGTGGT</td></tr><tr><td>qWSP-R</td><td>AAAGTCCCTCAACATCAACCC</td></tr><tr><td>qHTH-F</td><td>TGGTCCTATATTGGCGAGCTA</td></tr><tr><td>qHTH-R</td><td>TCGTTTTTGCAAGAAGGTCA</td></tr><tr><td>18S rRNA-F</td><td>CGCGGTAATTCCAGCTCCACTA</td></tr><tr><td>18S rRNA-R</td><td>GCATCAAGCGCCACCATATAGG</td></tr><tr><td>183F (Zhou et al. 1998)</td><td>AAGGAACCGAAGTTCATG</td></tr><tr><td>691R (Zhou et al. 1998)</td><td>AAAAATTAAACGCTACTCCA</td></tr><tr><td>wAlbB-F</td><td>GCAATACCTATGCCGTTTA</td></tr><tr><td>wAlbB-R</td><td>GACGAAGGGGATAGGTTAATATC</td></tr><tr><td>wAlbA-F</td><td>GTAGTATTTACCCCAGCAG</td></tr><tr><td>wAlbA-R</td><td>ATCTGCACCAGTAGTTTCG</td></tr></tbody></table>
<figure><img src="image_4.png" /></figure>

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<figure><img src="image_6.png" /></figure>

Percentage hatch
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<figure><img src="image_9.png" /></figure>

Percentage hatch
Accepted Article

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /></figure>

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wsp/host genome
<figure><img src="image_14.png" /></figure>