# Diverse novel Wolbachia bacteria strains and widespread co-infections with Asaia bacteria in Culicine mosquitoes from ecologically diverse regions of Cameroon

Aina Mercant Osuna, Alexandra Gidley, Marie Paul Audrey Mayi, Roland

Bamou, Vishaal Dhokiya, Christophe Antonio-Nkondjio, Claire Louise

Jeffries, Thomas Walker

► To cite this version:

Aina Mercant Osuna, Alexandra Gidley, Marie Paul Audrey Mayi, Roland Bamou, Vishaal Dhokiya, et al.. Diverse novel Wolbachia bacteria strains and widespread co-infections with Asaia bacteria in Culicine mosquitoes from ecologically diverse regions of Cameroon. Wellcome Open Research, 2023, 8, pp.267. 10.12688/wellcomeopenres.18580.1. hal-04543674

HAL Id: hal-04543674

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# Diverse novel Wolbachia bacteria strains and widespread co-infections with Asaia bacteria in Culicine mosquitoes from ecologically diverse regions of Cameroon

Aina Mercant Osuna, Alexandra Gidley, Marie Paul Audrey Mayi, Roland Bamou, Vishaal Dhokiya, Christophe Antonio-Nkondjio, Claire Louise Jeffries, Thomas Walker

<figure><img src="image_1.png" /></figure>

## To cite this version:

Aina Mercant Osuna, Alexandra Gidley, Marie Paul Audrey Mayi, Roland Bamou, Vishaal Dhokiya, et al.. Diverse novel Wolbachia bacteria strains and widespread co-infections with Asaia bacteria in Culicine mosquitoes from ecologically diverse regions of Cameroon. Wellcome Open Research, 2023, 8, pp.267. 10.12688/wellcomeopenres.18580.1. hal-04543674

HAL Id: hal-04543674

https://amu.hal.science/hal-04543674v1

Submitted on 12 Apr 2024

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.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_2.png" /></figure>

Check for updates

RESEARCH ARTICLE

REVISED

# Diverse novel *Wolbachia* bacteria strains and genera-specific co-infections with *Asaia* bacteria in Culicine mosquitoes from ecologically diverse regions of Cameroon
## [version 2; peer review: 3 approved]

Aina Mercant Osuna<fnref n="1" />*, Alexandra Gidley<fnref n="1" />*, Marie Paul Audrey Mayi<fnref n="2" />,<fnref n="3" />,

Roland Bamou ID <fnref n="4" />⁻<fnref n="8" />, Vishaal Dhokiya ID <fnref n="1" />,<fnref n="9" />, Christophe Antonio-Nkondjio ID <fnref n="8" />,<fnref n="9" />,

Claire Louise Jeffries<fnref n="1" />, Thomas Walker ID <fnref n="1" />,<fnref n="10" />

v2

First published: 21 Jun 2023, 8:267

https://doi.org/10.12688/wellcomeopenres.18580.1

Latest published: 26 Sep 2023, 8:267

https://doi.org/10.12688/wellcomeopenres.18580.2

## Abstract

**Background:** The endosymbiotic bacterium *Wolbachia* infects numerous species of insects and *Wolbachia* transinfection of *Aedes* mosquito species is now being used for biocontrol programs as *Wolbachia* strains can both inhibit arboviruses and invade wild mosquito populations. The discovery of novel, resident *Wolbachia* strains in mosquito species warrants further investigation as potential candidate strains for biocontrol strategies.

**Methods:** We obtained mosquito specimens from diverse Culicine mosquitoes from Cameroon including ecologically diverse locations in the Central and West Regions. *Wolbachia* prevalence rates were assessed in addition to the environmentally acquired bacterial species *Asaia* in major Culicine genera. PCR-based methods were also used with phylogenetic analysis to confirm identities of host mosquito species and *Wolbachia* strains were classified using multi-locus sequence typing (MLST).

## Open Peer Review

Approval Status

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26 Sep 2023

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1. **Eric P. Caragata** ID, University of Florida, Gainesville, USA

2. **Guido Favia**, University of Camerino, Camerino, Italy

<footnote n="1">¹Department of Disease Control, London School of Hygiene & Tropical Medicine, London, UK</footnote>

<footnote n="2">²Department of Microbiology, University of Yaoundé 1, Yaoundé, Cameroon</footnote>

<footnote n="3">³School of Biosciences & Veterinary Medicine, University of Camerino, Camerino, Italy</footnote>

<footnote n="4">⁴Laboratory of Malaria and Vector Research, NIAID, NIH, Rockville, Maryland, USA</footnote>

<footnote n="5">⁵IHU Méditerranée Infection, Marseille, France</footnote>

<footnote n="6">⁶Vecteurs-Infections Tropicales et Méditerranéennes (VITROME), Aix Marseille University, Marseille, France</footnote>

<footnote n="7">⁷Vector Borne Diseases Laboratory of the Research Unit of Biology and Applied Ecology (VBID-RUBAE), Department of Animal Biology, University of Dschang, Dschang, Cameroon</footnote>

<footnote n="8">⁸Organisation de Coordination pour la lutte Contre les Endémies en Afrique Centrale (OCEAC), Yaoundé, Cameroon</footnote>

<footnote n="9">⁹Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK</footnote>

<footnote n="10">¹⁰School of Life Sciences, University of Warwick, Coventry, UK</footnote>

<footnote>* Equal contributors</footnote>
<figure><img src="image_3.png" /></figure>

Check for updates

RESEARCH ARTICLE

**REVISED**
# Diverse novel *Wolbachia* bacteria strains and genera-
specific co-infections with *Asaia* bacteria in Culicine
mosquitoes from ecologically diverse regions of Cameroon
[version 2; peer review: 3 approved]

Aina Mercant Osuna<fnref n="1" />*, Alexandra Gidley<fnref n="1" />*, Marie Paul Audrey Mayi<fnref n="2" />,<fnref n="3" />,
Roland Bamou ID<fnref n="4" />⁻<fnref n="8" />, Vishaal Dhokiya ID<fnref n="1" />,<fnref n="9" />, Christophe Antonio-Nkondjio ID<fnref n="8" />,<fnref n="9" />,
Claire Louise Jeffries<fnref n="1" />, Thomas Walker ID<fnref n="1" />,<fnref n="10" />

**v2** First published: 21 Jun 2023, **8**:267
https://doi.org/10.12688/wellcomeopenres.18580.1

Latest published: 26 Sep 2023, **8**:267
https://doi.org/10.12688/wellcomeopenres.18580.2

## Abstract

**Background:** The endosymbiotic bacterium *Wolbachia* infects numerous species of insects and *Wolbachia* transinfection of *Aedes* mosquito species is now being used for biocontrol programs as *Wolbachia* strains can both inhibit arboviruses and invade wild mosquito populations. The discovery of novel, resident *Wolbachia* strains in mosquito species warrants further investigation as potential candidate strains for biocontrol strategies.

**Methods:** We obtained mosquito specimens from diverse Culicine mosquitoes from Cameroon including ecologically diverse locations in the Central and West Regions. *Wolbachia* prevalence rates were assessed in addition to the environmentally acquired bacterial species *Asaia* in major Culicine genera. PCR-based methods were also used with phylogenetic analysis to confirm identities of host mosquito species and *Wolbachia* strains were classified using multi-locus sequence typing (MLST).

## Open Peer Review

Approval Status

<figure><img src="image_4.png" /></figure>

1 2 3

version 2

(revision)

26 Sep 2023

<figure><img src="image_5.png" /></figure>

view

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

<figure><img src="image_7.png" /></figure>

view

<figure><img src="image_8.png" /></figure>

version 1

21 Jun 2023

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view

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

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1. **Eric P. Caragata** ID, University of Florida,
Gainesville, USA

2. **Guido Favia**, University of Camerino,
Camerino, Italy

<footnote n="1">¹Department of Disease Control, London School of Hygiene & Tropical Medicine, London, UK</footnote>

<footnote n="2">²Department of Microbiology, University of Yaoundé 1, Yaoundé, Cameroon</footnote>

<footnote n="3">³School of Biosciences & Veterinary Medicine, University of Camerino, Camerino, Italy</footnote>

<footnote n="4">⁴Laboratory of Malaria and Vector Research, NIAID, NIH, Rockville, Maryland, USA</footnote>

<footnote n="5">⁵IHU Méditerranée Infection, Marseille, France</footnote>

<footnote n="6">⁶Vecteurs-Infections Tropicales et Méditerranéennes (VITROME), Aix Marseille University, Marseille, France</footnote>

<footnote n="7">⁷Vector Borne Diseases Laboratory of the Research Unit of Biology and Applied Ecology (VBID-RUBAE), Department of Animal Biology, University of Dschang, Dschang, Cameroon</footnote>

<footnote n="8">⁸Organisation de Coordination pour la lutte Contre les Endémies en Afrique Centrale (OCEAC), Yaoundé, Cameroon</footnote>

<footnote n="9">⁹Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK</footnote>

<footnote n="10">¹⁰School of Life Sciences, University of Warwick, Coventry, UK</footnote>

<footnote>* Equal contributors</footnote>
**Results:** We report high *Wolbachia* prevalence rates for Culicine species, including in a large cohort of *Aedes africanus* collected from west Cameroon in which 100% of mono-specific pools were infected. Furthermore, co-infections with *Asaia* bacteria were observed across multiple genera, demonstrating that these two bacteria can co-exist in wild mosquito populations. *Wolbachia* strain MLST and phylogenetic analysis provided evidence for diverse *Wolbachia* strains in 13 different mosquito species across seven different genera. Full or partial MLST profiles were generated from resident *Wolbachia* strains in six *Culex* species (*quinquefasciatus*, *watti*, *cinerus*, *nigripalpus*, *perexiguus* and *rima*), two *Aedes* species (*africanus* and *denderensis*) and in *Mansonia uniformis*, *Catageiomyia argenteopunctata*, *Lutzia tigripes*, *Eretmapodites chrysogaster* and *Uranotaenia bilineata*.

**Conclusions:** Our study provides further evidence that *Wolbachia* is widespread within wild mosquito populations of diverse Culicine species and provides further candidate strains that could be investigated as future options for *Wolbachia*-based biocontrol to inhibit arbovirus transmission.

**Keywords**

Wolbachia, bacteria, mosquitoes

3. Perran Ross ID, The University of Melbourne, Melbourne, Australia

Any reports and responses or comments on the article can be found at the end of the article.

**Corresponding author:** Thomas Walker (thomas.walker.1@warwick.ac.uk)

**Author roles:** **Mercant Osuna A:** Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; **Gidley A:** Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; **Mayi MPA:** Investigation, Methodology, Writing – Review & Editing; **Bamou R:** Investigation, Methodology, Writing – Review & Editing; **Dhokiya V:** Investigation, Methodology, Writing – Review & Editing; **Antonio-Nkondjio C:** Investigation, Methodology, Supervision, Writing – Review & Editing; **Jeffries CL:** Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; **Walker T:** Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

**Competing interests:** No competing interests were disclosed.

**Grant information:** This work was supported by Wellcome [101285, https://doi.org/10.35802/101285]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Copyright:** © 2023 Mercant Osuna A 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 work is properly cited.

**How to cite this article:** Mercant Osuna A, Gidley A, Mayi MPA et al. Diverse novel *Wolbachia* bacteria strains and genera-specific co-infections with *Asaia* bacteria in Culicine mosquitoes from ecologically diverse regions of Cameroon [version 2; peer review: 3 approved] Wellcome Open Research 2023, **8**:267 https://doi.org/10.12688/wellcomeopenres.18580.2

**First published:** 21 Jun 2023, **8**:267 https://doi.org/10.12688/wellcomeopenres.18580.1
**Results:** We report high *Wolbachia* prevalence rates for Culicine species, including in a large cohort of *Aedes africanus* collected from west Cameroon in which 100% of mono-specific pools were infected. Furthermore, co-infections with *Asaia* bacteria were observed across multiple genera, demonstrating that these two bacteria can co-exist in wild mosquito populations. *Wolbachia* strain MLST and phylogenetic analysis provided evidence for diverse *Wolbachia* strains in 13 different mosquito species across seven different genera. Full or partial MLST profiles were generated from resident *Wolbachia* strains in six *Culex* species (*quinquefasciatus*, *watti*, *cinerus*, *nigripalpus*, *perexiguus* and *rima*), two *Aedes* species (*africanus* and *denderensis*) and in *Mansonia uniformis*, *Catageiomyia argenteopunctata*, *Lutzia tigripes*, *Eretmapodites chrysogaster* and *Uranotaenia bilineata*.

**Conclusions:** Our study provides further evidence that *Wolbachia* is widespread within wild mosquito populations of diverse Culicine species and provides further candidate strains that could be investigated as future options for *Wolbachia*-based biocontrol to inhibit arbovirus transmission.

**Keywords**

Wolbachia, bacteria, mosquitoes

3. Perran Ross ID, The University of Melbourne, Melbourne, Australia

Any reports and responses or comments on the article can be found at the end of the article.

**Corresponding author:** Thomas Walker (thomas.walker.1@warwick.ac.uk)

**Author roles:** **Mercant Osuna A:** Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; **Gidley A:** Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; **Mayi MPA:** Investigation, Methodology, Writing – Review & Editing; **Bamou R:** Investigation, Methodology, Writing – Review & Editing; **Dhokiya V:** Investigation, Methodology, Writing – Review & Editing; **Antonio-Nkondjio C:** Investigation, Methodology, Supervision, Writing – Review & Editing; **Jeffries CL:** Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; **Walker T:** Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

**Competing interests:** No competing interests were disclosed.

**Grant information:** This work was supported by Wellcome [101285, https://doi.org/10.35802/101285]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Copyright:** © 2023 Mercant Osuna A 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 work is properly cited.

**How to cite this article:** Mercant Osuna A, Gidley A, Mayi MPA et al. Diverse novel *Wolbachia* bacteria strains and genera-specific co-infections with *Asaia* bacteria in Culicine mosquitoes from ecologically diverse regions of Cameroon [version 2; peer review: 3 approved] Wellcome Open Research 2023, **8**:267 https://doi.org/10.12688/wellcomeopenres.18580.2

**First published:** 21 Jun 2023, **8**:267 https://doi.org/10.12688/wellcomeopenres.18580.1
**REVISED** Amendments from Version 1

We have taken into consideration all the very relevant reviewer comments and significantly improved all sections. Specifically, we have adjusted our title to reflect the 'genera-specific' associations seen between *Wolbachia* and *Asaia* prevalence rates, included more background information on using additional *Wolbachia* strains for dengue control in Malaysia and significantly increased the rationale for investigating *Asaia*-*Wolbachia* interactions by providing more context on previous studies looking at *Asaia* in mosquitoes. We have provided additional details on qPCR methodology and added an additional section in the methods for statistical analysis. Figure 2 now contains a more universally accessible colour scheme and we have significantly expanded our discussion to address some very valid and important reviewer queries. We have included more discussion around how our *Wolbachia* and *Asaia* prevalence rates results are more reflective of a 'genera-specific' pattern that is comparable to other previous studies and have included some limitations based on the challenging nature of mosquito morphological identification. Furthermore, we have expanded our discussion to highlight the limitations of our results based on both using whole bodies (rather than tissue-specific detection) and using monospecific pooling. We have now clearly defined what we would categorise as a 'novel' *Wolbachia* strain and acknowledged that even MLST allelic profiling may not represent the optimal loci to capture strain variation (despite being widely used to determine *Wolbachia* strain diversity).

Any further responses from the reviewers can be found at
the end of the article

**Introduction**

*Wolbachia* are endosymbiotic bacteria which reside within an estimated 40–70% of insect species¹,². These bacteria have been detected in numerous mosquitoes that transmit human pathogens including species within the *Aedes* (*Ae*.), *Culex* (*Cx*.) and *Anopheles* (*An*.) genera³⁻⁹. *Wolbachia* is now being used in applied mosquito biocontrol strategies as strains inhibit arboviruses and invade mosquito populations using the reproductive phenotype cytoplasmic incompatibility (CI). *Wolbachia*-induced CI was first used in mosquito release control trials in the 1960s in attempts to supress *Cx. quinquefasciatus* populations in Myanmar¹⁰. More recently, *Wolbachia*-infected *Aedes* males have been released to induce CI and the associated sterility from matings with wild-type females, resulting in inviable progeny. This method of suppressing the population, the incompatible insect technique (IIT), has seen field trials for species such as *Ae. polynesiensis* that contain natural resident strains¹¹,¹². Embryo microinjection has also resulted in transinfected strains in *Ae. aegypti*¹³⁻¹⁶. These transinfected strains, including wMel, have been shown to invade wild mosquito populations and also inhibit major arbovirus transmission, such as dengue virus (DENV)¹³,¹⁵,¹⁷⁻¹⁹. The wMel *Ae. aegypti* line, through release programmes, is now present in more than 10 countries and encouragingly, a randomised control trial in Indonesia resulted in a 77% DENV inhibition²⁰. The wMel strain is being continually released into additional DENV endemic countries, and based on mathematical modelling, has the capacity to reduce the DENV R0 (basic reproduction number) from 66–75%²¹. The wMel strain also inhibits other medically important arboviruses such as chikungunya virus (CHIKV)²²,

Yellow fever virus (YFV)²³ and Zika virus (ZIKV)²⁴. There are other *Wolbachia* strains also being used in applied strategies with the *wAlbB* strain²⁵ now established in Malaysian *Ae. aegypti* populations and having an impact on dengue incidence²⁶.

*Wolbachia* strains that naturally reside within mosquito populations can provide important comparative data to complement biocontrol strategies. For example, whether *Wolbachia* strains in natural populations are found at a high prevalence, and whether strains are capable of inducing CI to allow rapid population invasion. This first requires the identification of strains using molecular strain typing from diverse species which would then allow more comprehensive phenotypic characterization. Intra-genera transinfection has been shown to be successful without the need for adapting *Wolbachia* strains to insect cell lines so identifying strains residing in natural mosquito populations could provide additional candidate strains for bio-control strategies. The presence of other bacteria, such as the a-Proteobacterium *Asaia*, has been postulated to compete with *Wolbachia* to colonise the reproductive tissues of mosquitoes, including the ovaries in females²⁷,²⁸. *Asaia* has also been proposed for biocontrol strategies as this genus of bacteria forms stable associations with numerous insects that sugar feed²⁹ and can rapidly colonize the midgut and spread to other insect tissues after ingestion from either a sugar or blood meal³⁰. *Asaia* is particularly well studied in *Anopheles* (vectors of malaria parasites) and can stably associate with multiple species and be the dominant bacteria within some species such as *An. stephensi*³¹. Unlike *Wolbachia*, *Asaia* can be cultured in cell free media and has been genetically transformed³¹. *Asaia* can also be both horizontally and vertically transmitted providing a mechanism to invade mosquito populations.

Therefore, examining the possibility of co-infections in diverse mosquito populations will investigate if any antagonistic associations between these two common bacteria are present, as has been demonstrated in lab populations²⁷,²⁸. Numerous studies which have detected natural *Wolbachia* strains in Culicines have undertaken analysis of the 16S rRNA gene when looking at the wider microbiome³²,³³. However, a more targeted approach amplifying *Wolbachia*-specific genes is required to confirm a resident strain is present and phylogenetic analysis allows any newly discovered strains to be compared to existing strains. This then allows a more comprehensive assessment of *Wolbachia* tissue tropism (microscopy), whole genome sequencing and ultimately phenotypic characterisation. *Wolbachia* strains can be classified in Supergroups designated from A to H. A and B Supergroup strains are mostly found in arthropods, with only one strain per host, or multiple strains infecting the same host³⁴. Superinfection, the infection of more than one strain of *Wolbachia* in the same host – as can be seen in *Ae. albopictus* with the *wAlbA* and *wAlbB* strains³⁵ – can comprise strains of differing Supergroups, such as A and B group strain co-infections.

Cameroon is a country in West Africa in which outbreaks of arboviral diseases including DENV, YFV, CHIKV and Rift Valley fever virus (RVFV)³⁶ have been reported. For example, DENV IgM seroprevalence among febrile children was 14%
**REVISED** Amendments from Version 1

We have taken into consideration all the very relevant reviewer comments and significantly improved all sections. Specifically, we have adjusted our title to reflect the 'genera-specific' associations seen between *Wolbachia* and *Asaia* prevalence rates, included more background information on using additional *Wolbachia* strains for dengue control in Malaysia and significantly increased the rationale for investigating *Asaia*-Wolbachia interactions by providing more context on previous studies looking at *Asaia* in mosquitoes. We have provided additional details on qPCR methodology and added an additional section in the methods for statistical analysis. Figure 2 now contains a more universally accessible colour scheme and we have significantly expanded our discussion to address some very valid and important reviewer queries. We have included more discussion around how our *Wolbachia* and *Asaia* prevalence rates results are more reflective of a 'genera-specific' pattern that is comparable to other previous studies and have included some limitations based on the challenging nature of mosquito morphological identification. Furthermore, we have expanded our discussion to highlight the limitations of our results based on both using whole bodies (rather than tissue-specific detection) and using monospecific pooling. We have now clearly defined what we would categorise as a 'novel' *Wolbachia* strain and acknowledged that even MLST allelic profiling may not represent the optimal loci to capture strain variation (despite being widely used to determine *Wolbachia* strain diversity).

Any further responses from the reviewers can be found at
the end of the article

**Introduction**

*Wolbachia* are endosymbiotic bacteria which reside within an estimated 40–70% of insect species¹,². These bacteria have been detected in numerous mosquitoes that transmit human pathogens including species within the *Aedes* (*Ae*.), *Culex* (*Cx*.) and *Anopheles* (*An*.) genera³⁻⁹. *Wolbachia* is now being used in applied mosquito biocontrol strategies as strains inhibit arboviruses and invade mosquito populations using the reproductive phenotype cytoplasmic incompatibility (CI). *Wolbachia*-induced CI was first used in mosquito release control trials in the 1960s in attempts to supress *Cx. quinquefasciatus* populations in Myanmar¹⁰. More recently, *Wolbachia*-infected *Aedes* males have been released to induce CI and the associated sterility from matings with wild-type females, resulting in inviable progeny. This method of suppressing the population, the incompatible insect technique (IIT), has seen field trials for species such as *Ae. polynesiensis* that contain natural resident strains¹¹,¹². Embryo microinjection has also resulted in transinfected strains in *Ae. aegypti*¹³⁻¹⁶. These transinfected strains, including wMel, have been shown to invade wild mosquito populations and also inhibit major arbovirus transmission, such as dengue virus (DENV)¹³,¹⁵,¹⁷⁻¹⁹. The wMel *Ae. aegypti* line, through release programmes, is now present in more than 10 countries and encouragingly, a randomised control trial in Indonesia resulted in a 77% DENV inhibition²⁰. The wMel strain is being continually released into additional DENV endemic countries, and based on mathematical modelling, has the capacity to reduce the DENV R0 (basic reproduction number) from 66–75%²¹. The wMel strain also inhibits other medically important arboviruses such as chikungunya virus (CHIKV)²²,

Yellow fever virus (YFV)²³ and Zika virus (ZIKV)²⁴. There are other *Wolbachia* strains also being used in applied strategies with the *wAlbB* strain²⁵ now established in Malaysian *Ae. aegypti* populations and having an impact on dengue incidence²⁶.

*Wolbachia* strains that naturally reside within mosquito populations can provide important comparative data to complement biocontrol strategies. For example, whether *Wolbachia* strains in natural populations are found at a high prevalence, and whether strains are capable of inducing CI to allow rapid population invasion. This first requires the identification of strains using molecular strain typing from diverse species which would then allow more comprehensive phenotypic characterization. Intra-genera transinfection has been shown to be successful without the need for adapting *Wolbachia* strains to insect cell lines so identifying strains residing in natural mosquito populations could provide additional candidate strains for biocontrol strategies. The presence of other bacteria, such as the a-Proteobacterium *Asaia*, has been postulated to compete with *Wolbachia* to colonise the reproductive tissues of mosquitoes, including the ovaries in females²⁷,²⁸. *Asaia* has also been proposed for biocontrol strategies as this genus of bacteria forms stable associations with numerous insects that sugar feed²⁹ and can rapidly colonize the midgut and spread to other insect tissues after ingestion from either a sugar or blood meal³⁰. *Asaia* is particularly well studied in *Anopheles* (vectors of malaria parasites) and can stably associate with multiple species and be the dominant bacteria within some species such as *An. stephensi*³¹. Unlike *Wolbachia*, *Asaia* can be cultured in cell free media and has been genetically transformed³¹. *Asaia* can also be both horizontally and vertically transmitted providing a mechanism to invade mosquito populations.

Therefore, examining the possibility of co-infections in diverse mosquito populations will investigate if any antagonistic associations between these two common bacteria are present, as has been demonstrated in lab populations²⁷,²⁸. Numerous studies which have detected natural *Wolbachia* strains in Culicines have undertaken analysis of the 16S rRNA gene when looking at the wider microbiome³²,³³. However, a more targeted approach amplifying *Wolbachia*-specific genes is required to confirm a resident strain is present and phylogenetic analysis allows any newly discovered strains to be compared to existing strains. This then allows a more comprehensive assessment of *Wolbachia* tissue tropism (microscopy), whole genome sequencing and ultimately phenotypic characterisation. *Wolbachia* strains can be classified in Supergroups designated from A to H. A and B Supergroup strains are mostly found in arthropods, with only one strain per host, or multiple strains infecting the same host³⁴. Superinfection, the infection of more than one strain of *Wolbachia* in the same host – as can be seen in *Ae. albopictus* with the *wAlbA* and *wAlbB* strains³⁵ – can comprise strains of differing Supergroups, such as A and B group strain co-infections.

Cameroon is a country in West Africa in which outbreaks of arboviral diseases including DENV, YFV, CHIKV and Rift Valley fever virus (RVFV)³⁶ have been reported. For example, DENV IgM seroprevalence among febrile children was 14%
from a cross-sectional study performed in 961 children less than 15 years old attending public hospitals of Cameroon³⁷. Cases of arboviruses are reported throughout the country and there is concern that rapid urbanisation seen throughout Africa could exacerbate transmission through favouring the breeding conditions of urban-adapted mosquito species³⁸. Deforestation has a significant effect on the abundance and diversity of Cameroon mosquito species and could lead to spill-over transmission of additional circulating zoonotic viruses such as Semliki Forest, o'nyong'nyong and Bwamba viruses³⁹. Here, we obtained specimens from entomological surveys undertaken in Central and West Cameroon to determine if diverse Culicine species harbour resident *Wolbachia* strains and if co-infections exist with *Asaia*, given this bacterium can be environmentally acquired and can compete with *Wolbachia*. We extracted RNA from preserved samples and undertook qRT-PCR analysis to make any detection of *Wolbachia* strains more likely to be from actively expressed *Wolbachia* genes. A combination of phylogenetic analysis and strain typing using multi-locus sequence typing (MLST) revealed a diversity of newly discovered *Wolbachia* strains.

# Methods

## Mosquito collections

A variety of adult mosquito trapping methods were used in the Central Region of Cameroon in Yaoundé (3°52'22.2"N, 11°30'38.0"E) and Olama village (3°24'45.0"N 11°17'03.0"E) from June 2019 to July 2019. Yaoundé (the capital of Cameroon) is an urban location characterised by mosquito breeding sites such as tyres, containers, and temporary water

pools. In contrast, Olama village is rural, located along the Nyong River (Figure 1) and is characterised by houses built with corrugated metal roofs and mud walls. In Olama village, eight CDC miniature light traps were hung in four houses and two Stealth traps (model 14 which uses ultraviolet light for attraction) were hung in one house (one trap inside and one outside). BG Suna traps containing a carbon dioxide source from fermented yeast as an attractant were hung in trees nearby houses, approximately one metre above the ground. Traps were powered using a 12V battery over a period of 11 days from 17:00–07:00. Human landing catches were carried out in Olama Village as previously described⁴. In Yaoundé, BG Sentinel-2 traps containing a BG Sweetscent lure used as an attractant were used to collect mosquitoes for 15 days from 16:30–10:00. Traps were assembled using manufacturers guidelines and positioned nearby potential *Aedes* breeding sites under tree coverage. Larval collections using ladles and sieves were also carried out in Yaoundé in typical urban breeding sites from the following districts: Etoude, Nkolbissim and Briqueterie.

Mosquitoes were collected in the West Region from the Menoua division as previously described⁴⁰ and from two locations in the Dschang sub-division in a rural area; Fonakeukeu (05°24'73" N, 010°04'79" E) and a peri-urban area; Toutsang (05°25'65" N, 010°04'05" E). Collections were carried out in both the rainy season (from March to September 2019) and dry season (from November 2019 to February 2020) using sweep nets. Mosquitoes from Central Cameroon were morphologically identified to genera level, and from West Cameroon

<figure><img src="image_13.png" /><figcaption>**Figure 1.** Mosquito collection sites in Cameroon. Collection sites in the Western Province/region were Fonakeukeu (05°24'73" N, 010°04'79" E) and Toutsang (05°25'65" N, 010°04'05" E). Collection sites in the Central Province/regions included Yaoundé (3°52'22.2"N 11°30'38.0"E) and Olama village (3°24'45.0"N 11°17'03.0"E). Maps were produced using Mapchart licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.</figcaption></figure>
from a cross a cross-sectional study performed in 961 children less than 15 years old attending public hospitals of Cameroon³⁷. Cases of arboviruses are reported throughout the country and there is concern that rapid urbanisation seen throughout Africa could exacerbate transmission through favouring the breeding conditions of urban-adapted mosquito species³⁸. Deforestation has a significant effect on the abundance and diversity of Cameroon mosquito species and could lead to spill-over transmission of additional circulating zoonotic viruses such as Semliki Forest, o'nyong'nyong and Bwamba viruses³⁹. Here, we obtained specimens from entomological surveys undertaken in Central and West Cameroon to determine if diverse Culicine species harbour resident *Wolbachia* strains and if co-infections exist with *Asaia*, given this bacterium can be environmentally acquired and can compete with *Wolbachia*. We extracted RNA from preserved samples and undertook qRT-PCR analysis to make any detection of *Wolbachia* strains more likely to be from actively expressed *Wolbachia* genes. A combination of phylogenetic analysis and strain typing using multi-locus sequence typing (MLST) revealed a diversity of newly discovered *Wolbachia* strains.

# Methods

## Mosquito collections

A variety of adult mosquito trapping methods were used in the Central Region of Cameroon in Yaoundé (3°52'22.2"N, 11°30'38.0"E) and Olama village (3°24'45.0"N 11°17'03.0"E) from June 2019 to July 2019. Yaoundé (the capital of Cameroon) is an urban location characterised by mosquito breeding sites such as tyres, containers, and temporary water

pools. In contrast, Olama village is rural, located along the Nyong River (Figure 1) and is characterised by houses built with corrugated metal roofs and mud walls. In Olama village, eight CDC miniature light traps were hung in four houses and two Stealth traps (model 14 which uses ultraviolet light for attraction) were hung in one house (one trap inside and one outside). BG Suna traps containing a carbon dioxide source from fermented yeast as an attractant were hung in trees nearby houses, approximately one metre above the ground. Traps were powered using a 12V battery over a period of 11 days from 17:00–07:00. Human landing catches were carried out in Olama Village as previously described⁴. In Yaoundé, BG Sentinel-2 traps containing a BG Sweetscent lure used as an attractant were used to collect mosquitoes for 15 days from 16:30–10:00. Traps were assembled using manufacturers guidelines and positioned nearby potential *Aedes* breeding sites under tree coverage. Larval collections using ladles and sieves were also carried out in Yaoundé in typical urban breeding sites from the following districts: Etoude, Nkolbissim and Briqueterie.

Mosquitoes were collected in the West Region from the Menoua division as previously described⁴⁰ and from two locations in the Dschang sub-division in a rural area; Fonakeukeu (05°24'73" N, 010°04'79" E) and a peri-urban area; Toutsang (05°25'65" N, 010°04'05" E). Collections were carried out in both the rainy season (from March to September 2019) and dry season (from November 2019 to February 2020) using sweep nets. Mosquitoes from Central Cameroon were morphologically identified to genera level, and from West Cameroon

<figure><img src="image_14.png" /><figcaption>**Figure 1.** Mosquito collection sites in Cameroon. Collection sites in the Western Province/region were Fonakeukeu (05°24'73" N, 010°04'79" E) and Toutsang (05°25'65" N, 010°04'05" E). Collection sites in the Central Province/regions included Yaoundé (3°52'22.2"N 11°30'38.0"E) and Olama village (3°24'45.0"N 11°17'03.0"E). Maps were produced using Mapchart licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.</figcaption></figure>
to species level, using morphological identification keys, then
preserved in RNAlater (Invitrogen) and kept at below -20°C,
prior to being sent for molecular analysis at LSHTM.

RNA extraction and generation of complementary DNA
(cDNA)

From West Cameroon collections, mosquitoes of the same
species, from the same location and season, were pooled prior to
RNA extraction (650 mosquitoes, 192 pools, pool size range was
3–5 mosquitoes). From Central Cameroon collections, a sub-
sample (n=576) was selected for molecular analysis based on
diversity of genera and collection sites. Mosquito RNA was
extracted from either pools or individuals using Qiagen 96
RNeasy Kits and a Qiagen Tissue Lyser II (Hilden, Germany)
with 5 mm stainless steel beads (Qiagen) to homogenize mos-
quitoes. Resulting RNA was eluted in 45 µL of RNase-free water
and stored at –70°C. cDNA was generated from RNA using an
Applied Biosystems High-Capacity cDNA Reverse Transcription
kit. Each reaction (20 µL) was made up of the following: 1 µL
reverse transcriptase, 2 µL 10X RT buffer, 2 µL 10X random
primers, 0.8 µL 25X dNTP (100 mM), 4.2 µL nuclease-free
water and 10 µL RNA. A Bio-Rad T100 Thermal Cycler was
used to generate cDNA as follows: 25°C for 10 minutes (min),
37°C for 120 min; 85°C for 5 min with all resulting cDNA
stored at –20°C.

Wolbachia and Asaia detection

The detection of *Wolbachia* strains was initially carried out by
amplification of three conserved *Wolbachia* genes; 16S rRNA
gene using primers W-Spec-16S-F: 5'-CATACCTATTCGAAG-
GGATA-3' and W-Spec-16s-R: 5'-AGCTTCGAGTGAAAC-
CAATTC-3'⁴¹, *Wolbachia* surface protein (wsp) gene using
primers wsp81F: 5'-TGGTCCAATAAGTGATGAAGAAAC-
3' and wsp691R: 5'-AAAAATTAAACGCTACTCCA-3'⁴².
A Bio-Rad T100 Thermal Cycler using standard cycling
conditions was used to amplify 16S rRNA and wsp gene
products which were then separated and visualised using
an Invitrogen E-Gel iBase Real-Time Transilluminator
with 2% SYBR safe E-Gel EX agarose gels. Real-time
PCR reactions for the 16S rRNA gene were carried out with
5 µL of FastStart SYBR Green Master mix (Roche Diagnos-
tics), primers at a final concentration of 1 µM, 1 µL of PCR
grade water and 2 µL cDNA (10 µL final volume/reaction) as
previously described using no template controls (NTC) and a
limit of detection was previously established using a synthetic
oligonucleotide standard through ten-fold serial dilutions⁴.
A Roche LightCycler 96 System was used to amplify PCR
products using the following cycling conditions: 15 min at
95°C, 40 cycles of 95°C for 15 seconds (sec) and 58°C for
30 sec. *Asaia* detection was also carried out using Real time
PCR by amplifying the 16S rRNA gene³⁰ with the same
mastermix, reagent concentrations and cycling conditions as
for *Wolbachia* genes. PCR assays included a dissociation
curve (95°C for 10 sec, 65°C for 60 sec and 97°C for 1 sec) to
check that the correct amplicon was being amplified. Fluores-
cence was quantified using LightCycler 96 software (Roche
Diagnostics).

Molecular mosquito species identification

For *Aedes* mosquitoes collected from Central Cameroon sites,
a SYBR-green based assay that can distinguish *Ae. aegypti*
from *Ae. albopictus* based on the internal transcribed spacer 1
(ITS1)⁴³ was used. PCR cycling conditions for the ITS1 assay
were: 95°C for 15 min, 40 cycles of 95°C for 10 sec, 55°C
for 30 sec, 72°C for 20 sec and a dissociation curve (see above).
For *Culex* mosquitoes collected from Central Cameroon sites,
a multiplex PCR assay targeting the ACE1 gene⁴⁴ that can dis-
tinguish *Cx. pipiens pipiens* from *Cx. pipiens quinquefasciatus*
was also undertaken, given these sibling species are morpho-
logically indistinguishable. *PCR* cycling conditions for the
ACE1: 95°C for 10 min, 34 cycles of 95°C for 30 sec, 55°C
for 30 sec, 72°C for 1 min and 2°C for 5 min. To determine the
species for additional samples that were *Wolbachia*-positive,
Sanger sequencing and phylogenetic analysis of the cyto-
chrome c oxidase subunit 1 (*COI*) gene⁴⁵ was undertaken as
this provided the most available sequences for comparison to
ensure the optimal method for species identification.

Wolbachia MLST

Five conserved genes (gatB, coxA, hcpA, ftsZ and fbpA) were
amplified to determine any newly discovered *Wolbachia*
strains as previously described with the use of M13 adapt-
tors or degenerate primers⁴⁶. MLST PCRs consisted of 10 µL of
Phire Hot Start II PCR Master Mix (Thermo Scientific), prim-
ers with a final concentration of 1 µM, 1 µL of PCR-grade
water and 2 µL template cDNA (20 µL total). PCR cycling was
carried out in a Bio-Rad T100 Thermal Cycler using cycling
conditions that were optimised for different MLST genes
tested with the Phire Hot Start II PCR Master Mix. Three
genes (gatB, hcpA and fbpA genes) had the following cycling:
98°C for 30 sec, 34 cycles of 98°C for 5 sec, 65°C for 5 sec,
72°C for 10 sec, 72°C for 1 min. For two genes (coxA and
ftsZ) cycling was: 98°C for 30 sec, 34 cycles of 98°C for
5 sec, 55°C for 5 sec and 72°C for 30 sec, 72°C for 1 min.

Sanger sequencing

PCR products were deemed worthy of sequencing when
producing a strong, clear band at the correct product size
when visualised using an Invitrogen E-Gel iBase Real-Time
Transilluminator with 2% SYBR safe E-Gel EX agarose gels
run for 10 mins. Products were sent to Source BioScience
(Nottingham, UK) for cleanup prior to forward and reverse
sanger sequencing. The MLST primers used were gene-specific
and in the case of MLST genes just the M13 primers
(M13_adaptor_F: 5'-TGTAAAACGACGGCCAGT-3' and M13_
adaptor_R: 5'-CAGGAAACAGCTATGACC-3') were used
if these adaptors were included in the initial PCR to generate
the product. MEGAX⁴⁷ was used for all analysis of sequences
with manual checking of both forward and reverse chroma-
tograms. Editing of sequences included trimming and then
alignment to produce consensus sequences was undertaken
using ClustalW. Nucleotide BLAST (NCBI) database que-
ries and searches against the *Wolbachia* MLST database were
combined to determine if new alleles and strain types were
to species level, using morphological identification keys, then preserved in RNAlater (Invitrogen) and kept at below -20°C, prior to being sent for molecular analysis at LSHTM.

## RNA extraction and generation of complementary DNA (cDNA)

From West Cameroon collections, mosquitoes of the same species, from the same location and season, were pooled prior to RNA extraction (650 mosquitoes, 192 pools, pool size range was 3–5 mosquitoes). From Central Cameroon collections, a sub-sample (n=576) was selected for molecular analysis based on diversity of genera and collection sites. Mosquito RNA was extracted from either pools or individuals using Qiagen 96 RNeasy Kits and a Qiagen Tissue Lyser II (Hilden, Germany) with 5 mm stainless steel beads (Qiagen) to homogenize mosquitoes. Resulting RNA was eluted in 45 µL of RNase-free water and stored at –70°C. cDNA was generated from RNA using an Applied Biosystems High-Capacity cDNA Reverse Transcription kit. Each reaction (20 µL) was made up of the following: 1 µL reverse transcriptase, 2 µL 10X RT buffer, 2 µL 10X random primers, 0.8 µL 25X dNTP (100 mM), 4.2 µL nuclease-free water and 10 µL RNA. A Bio-Rad T100 Thermal Cycler was used to generate cDNA as follows: 25°C for 10 minutes (min), 37°C for 120 min; 85°C for 5 min with all resulting cDNA stored at –20°C.

## *Wolbachia* and *Asaia* detection

The detection of *Wolbachia* strains was initially carried out by amplification of three conserved *Wolbachia* genes; 16S rRNA gene using primers W-Spec-16S-F: 5'-CATACCTATTCGAAGGGATA-3' and W-Spec-16s-R: 5'-AGCTTCGAGTGAAACCAATTC-3'⁴¹, *Wolbachia* surface protein (wsp) gene using primers wsp81F: 5'-TGGTCCAATAAGTGATGAAGAAAC-3' and wsp691R: 5'-AAAAATTAAACGCTACTCCA-3'⁴². A Bio-Rad T100 Thermal Cycler using standard cycling conditions was used to amplify 16S rRNA and wsp gene products which were then separated and visualised using an Invitrogen E-Gel iBase Real-Time Transilluminator with 2% SYBR safe E-Gel EX agarose gels. Real-time PCR reactions for the 16S rRNA gene were carried out with 5 µL of FastStart SYBR Green Master mix (Roche Diagnostics), primers at a final concentration of 1 µM, 1 µL of PCR grade water and 2 µL cDNA (10 µL final volume/reaction) as previously described using no template controls (NTC) and a limit of detection was previously established using a synthetic oligonucleotide standard through ten-fold serial dilutions⁴³. A Roche LightCycler 96 System was used to amplify PCR products using the following cycling conditions: 15 min at 95°C, 40 cycles of 95°C for 15 seconds (sec) and 58°C for 30 sec. *Asaia* detection was also carried out using Real time PCR by amplifying the 16S rRNA gene³⁰ with the same mastermix, reagent concentrations and cycling conditions as for *Wolbachia* genes. PCR assays included a dissociation curve (95°C for 10 sec, 65°C for 60 sec and 97°C for 1 sec) to check that the correct amplicon was being amplified. Fluorescence was quantified using LightCycler 96 software (Roche Diagnostics).

## Molecular mosquito species identification

For *Aedes* mosquitoes collected from Central Cameroon sites, a SYBR-green based assay that can distinguish *Ae. aegypti* from *Ae. albopictus* based on the internal transcribed spacer 1 (ITS1)⁴³ was used. PCR cycling conditions for the ITS1 assay were: 95°C for 15 min, 40 cycles of 95°C for 10 sec, 55°C for 30 sec, 72°C for 20 sec and a dissociation curve (see above). For *Culex* mosquitoes collected from Central Cameroon sites, a multiplex PCR assay targeting the ACE1 gene⁴⁴ that can distinguish *Cx. pipiens pipiens* from *Cx. pipiens quinquefasciatus* was also undertaken, given these sibling species are morphologically indistinguishable. PCR cycling conditions for the ACE1: 95°C for 10 min, 34 cycles of 95°C for 30 sec, 55°C for 30 sec, 72°C for 1 min and 2°C for 5 min. To determine the species for additional samples that were *Wolbachia*-positive, Sanger sequencing and phylogenetic analysis of the cytochrome c oxidase subunit 1 (*COI*) gene⁴⁵ was undertaken as this provided the most available sequences for comparison to ensure the optimal method for species identification.

## *Wolbachia* MLST

Five conserved genes (*gatB*, *coxA*, *hcpA*, *ftsZ* and *fbpA*) were amplified to determine any newly discovered *Wolbachia* strains as previously described with the use of M13 adaptors or degenerate primers⁴⁶. MLST PCRs consisted of 10 µL of Phire Hot Start II PCR Master Mix (Thermo Scientific), primers with a final concentration of 1 µM, 1 µL of PCR-grade water and 2 µL template cDNA (20 µL total). PCR cycling was carried out in a Bio-Rad T100 Thermal Cycler using cycling conditions that were optimised for different MLST genes tested with the Phire Hot Start II PCR Master Mix. Three genes (*gatB*, *hcpA* and *fbpA* genes) had the following cycling: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 65°C for 5 sec, 72°C for 10 sec, 72°C for 1 min. For two genes (*coxA* and *ftsZ*) cycling was: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 55°C for 5 sec and 72°C for 30 sec, 72°C for 1 min.

## Sanger sequencing

PCR products were deemed worthy of sequencing when producing a strong, clear band at the correct product size when visualised using an Invitrogen E-Gel iBase Real-Time Transilluminator with 2% SYBR safe E-Gel EX agarose gels run for 10 mins. Products were sent to Source BioScience (Nottingham, UK) for cleanup prior to forward and reverse sanger sequencing. The MLST primers used were gene-specific and in the case of MLST genes just the M13 primers (M13_adaptor_F: 5'-TGTAAAACGACGGCCAGT-3' and M13_adaptor_R: 5'-CAGGAAACAGCTATGACC-3') were used if these adaptors were included in the initial PCR to generate the product. MEGAX⁴⁷ was used for all analysis of sequences with manual checking of both forward and reverse chromatograms. Editing of sequences included trimming and then alignment to produce consensus sequences was undertaken using ClustalW. Nucleotide BLAST (NCBI) database queries and searches against the *Wolbachia* MLST database were combined to determine if new alleles and strain types were
present in our collection. We also submitted our sequences to GenBank and obtained accession numbers.

## Phylogenetic analysis

Alignments were constructed in MEGAX and ClustalW was also used to align our sequences alongside additional sequences obtained from NCBI BLAST and *Wolbachia* MLST database searches. Maximum Likelihood (ML) phylogenetic trees were generated after initially determining the optimal nucleotide substitution model using the “Find Best-Fit Substitution Model (ML)” option within MEGAX. The lowest Bayesian Information Criterion (BIC) score was one of the criteria used and this resulted in two models: the Jukes-Cantor model<sup>48</sup> and the General Time Reversible model<sup>49</sup>. For our phylogenetic analysis, we used the highest log likelihood and included next to the branches the percentage of trees in which the associated taxa clustered together. In all phylogenetic analyses we used a Bootstrap method with 1000 replications and Neighbor-Join and BioNJ algorithms using tMaximum Composite Likelihood (MCL). Our phylogenetic trees were then generated to scale, with branch lengths measured in the number of substitutions per site and we removed all gaps and missing data.

## Statistical analysis

Fisher's exact post hoc tests in GraphPad prism version 9 (P<0.05 significance threshold) were used to determine any association between prevalence rates of *Wolbachia* and *Asaia*

for each mosquito genus from the different regions (West and Central). Samples were categorised as *Wolbachia*-infected, *Asaia*-infected, co-infected or uninfected.

## Ethical approval

We previously obtained permission and ethical approval for mosquito sampling<sup>4,40</sup>. Ethical approval for undertaking Human landing catches was obtained from the LSHTM ethics committee (reference no. 16684) in addition to local ethical approval (clearance no. 2016/01/685/CE/CNERSH/SP) delivered by the Cameroon National Ethics (CNE) Committee for Research on Human Health).

## Results

### *Wolbachia* and *Asaia* prevalence rates

We compared the prevalence rates of *Wolbachia* and *Asaia* rates using the 16S rRNA gene from the three major Culicine genera collected from both the West and Central Regions, with the caveat that the West Region samples were monospecific pools from the same species at the same location (Figure 2, Table 1). In the Central Region, 97.96% (n=115) of *Aedes* genera mosquitoes were infected only with *Wolbachia*, and *Asaia* was only detected in a single mosquito as a coinfection. In contrast, the majority of *Culex* genera mosquitoes collected from the Central Region were uninfected (85.83%, n=103), with *Asaia* detected in 13.45% (n=16) of individuals and only a single individual infected with *Wolbachia*. A similar infection prevalence was observed in *Mansonia* collected from the

<figure><img src="image_14.png" /><figcaption>**Figure 2.** *Wolbachia*, *Asaia* and co-infection prevalence rates from major Culicine mosquito genera collected from the West and Central regions of Cameroon. Mosquitoes from the Central region were individually extracted and analysed. Mosquitoes from the West region were extracted from monospecific pools (same species from same collection location) and prevalence analysis represents pooled samples. No *Mansonia* mosquitoes were collected from the West region.</figcaption></figure>
present in our collection. We also submitted our sequences to GenBank and obtained accession numbers.

## Phylogenetic analysis

Alignments were constructed in MEGAX and ClustalW was also used to align our sequences alongside additional sequences obtained from NCBI BLAST and *Wolbachia* MLST database searches. Maximum Likelihood (ML) phylogenetic trees were generated after initially determining the optimal nucleotide substitution model using the “Find Best-Fit Substitution Model (ML)” option within MEGAX. The lowest Bayesian Information Criterion (BIC) score was one of the criteria used and this resulted in two models: the Jukes-Cantor model⁴⁸ and the General Time Reversible model¹⁹. For our phylogenetic analysis, we used the highest log likelihood and included next to the branches the percentage of trees in which the associated taxa clustered together. In all phylogenetic analyses we used a Bootstrap method with 1000 replications and Neighbor-Join and BioNJ algorithms using tMaximum Composite Likelihood (MCL). Our phylogenetic trees were then generated to scale, with branch lengths measured in the number of substitutions per site and we removed all gaps and missing data.

## Statistical analysis

Fisher's exact post hoc tests in GraphPad prism version 9 (P<0.05 significance threshold) were used to determine any association between prevalence rates of *Wolbachia* and *Asaia*

for each mosquito genus from the different regions (West and Central). Samples were categorised as *Wolbachia*-infected, *Asaia*-infected, co-infected or uninfected.

## Ethical approval

We previously obtained permission and ethical approval for mosquito sampling⁴,⁴⁰. Ethical approval for undertaking Human landing catches was obtained from the LSHTM ethics committee (reference no. 16684) in addition to local ethical approval (clearance no. 2016/01/685/CE/CNERSH/SP) delivered by the Cameroon National Ethics (CNE) Committee for Research on Human Health).

## Results

### *Wolbachia* and *Asaia* prevalence rates

We compared the prevalence rates of *Wolbachia* and *Asaia* rates using the 16S rRNA gene from the three major Culicine genera collected from both the West and Central Regions, with the caveat that the West Region samples were monospecific pools from the same species at the same location (Figure 2, Table 1). In the Central Region, 97.96% (n=115) of *Aedes* genera mosquitoes were infected only with *Wolbachia*, and *Asaia* was only detected in a single mosquito as a coinfection. In contrast, the majority of *Culex* genera mosquitoes collected from the Central Region were uninfected (85.83%, n=103), with *Asaia* detected in 13.45% (n=16) of individuals and only a single individual infected with *Wolbachia*. A similar infection prevalence was observed in *Mansonia* collected from the

<figure><img src="image_15.png" /><figcaption>**Figure 2.** *Wolbachia*, *Asaia* and co-infection prevalence rates from major Culicine mosquito genera collected from the West and Central regions of Cameroon. Mosquitoes from the Central region were individually extracted and analysed. Mosquitoes from the West region were extracted from monospecific pools (same species from same collection location) and prevalence analysis represents pooled samples. No *Mansonia* mosquitoes were collected from the West region.</figcaption></figure>
<table><caption>**Table 1.** Prevalence rates of *Wolbachia* (16S rRNA gene), *Asaia* and coinfection from mosquito genera collected from the West and Central regions of Cameroon. Mosquitoes from the Central region were individually extracted and analysed. Mosquitoes from the West region were extracted from monospecific pools (same species from same collection location) and prevalence analysis represents pooled samples.</caption><thead><tr><th>Region</th><th>Genera</th><th>Wolbachia (%)</th><th>Asaia (%)</th><th>Co-infected (%)</th><th>Uninfected (%)</th><th>Totals</th><th>Fisher's P-value</th></tr></thead><tbody><tr><td>West</td><td>Aedes</td><td>32 (24.81)</td><td>0 (0.00)</td><td>97 (75.19)</td><td>0 (0.00)</td><td>129</td><td>&gt;0.99</td></tr><tr><td>West</td><td>Culex</td><td>48 (97.96)</td><td>0 (0.00)</td><td>0 (0.00)</td><td>1 (2.04)</td><td>49</td><td>&gt;0.99</td></tr><tr><td>Central</td><td>Aedes</td><td>115 (97.46)</td><td>0 (0.00)</td><td>1 (0.85)</td><td>2 (1.69)</td><td>118</td><td>&gt;0.99</td></tr><tr><td>Central</td><td>Culex</td><td>1 (0.83)</td><td>16 (13.45)</td><td>0 (0.00)</td><td>103 (85.83)</td><td>120</td><td>&gt;0.99</td></tr><tr><td>Central</td><td>Mansonia</td><td>0 (0.00)</td><td>39 (40.63)</td><td>0 (0.00)</td><td>57 (59.38)</td><td>96</td><td>&gt;0.99</td></tr></tbody></table>

Central Region, but a higher prevalence of *Asaia* was detected (40.63%, n=39) and there was no *Wolbachia* detected (59.38%, 59 individuals were uninfected for both bacterial species). In the West Region, *Aedes* mosquitoes were either co-infected (75.19%, n=97 pools) or singly infected with *Wolbachia* (24.81%, n=32 pools). For *Culex* genera mosquitoes, the vast majority (97.96%, n=48 pools) were infected with *Wolbachia* only. Results of Fisher's exact *post hoc* tests (P<0.05 significance threshold) indicated no significant associations were present in our data. As *Aedes* collections in the West Region were heavily dominated by *Ae. africanus* - a vector of YFV in forest and rainforest regions in Sub-Saharan Africa⁵⁰, we compared *Wolbachia* and *Asaia* prevalence rates for pooled mono-specific RNA pools (n=97) to look for any potential co-infections within this species. *Wolbachia* was detected in 100.00% of pools (97/97) and a high *Asaia* prevalence rate of 96.91% (94/97) pools was also seen, demonstrating a high likelihood of co-infections occurring in this species. However, as mono-specific pools consisting of an average of five female mosquitoes were used for analysis no statistical association analysis can be carried out.

## Confirmation of *Wolbachia* prevalence rates through amplification of the *wsp* gene

Our preliminary assessment of *Wolbachia* prevalence rates was generated from 16S rRNA gene amplification. However, using the 16S rRNA gene alone is insufficient because it can also be possible that prokaryotic 16S rRNA genes can be amplified from dead bacterial cells⁵¹ and *Wolbachia* 16S rRNA sequences has previously been detected in water containers that contained larvae of mosquitoes⁵². We undertook further wsp gene amplification on a wide variety of samples collected from the West Region in which morphological identification was done to species level and individuals, of the same species were pooled into groups of up to five individuals prior to RNA extraction (monospecific pools). Screening with the wsp gene revealed variable estimates of *Wolbachia* prevalence rates (Table 2). Of particular interest was the high prevalence in the most abundant species *Ae. africanus* collected in both locations in West Cameroon. A total of 341 *Ae. africanus* females from Fonakeukeu were grouped into 72 pools and 65/72 pools (90.3%) were *Wolbachia*-positive based on strong amplification

of the wsp gene. Similarly, 34/46 pools (73.9%) of the pools, representing a total of 228 *Ae. africanus* females from Toutsang amplified the wsp gene. Variable prevalence rates were also seen in other morphologically identified species, including wsp amplification in species within the *Culex*, *Aedes*, *Mansonia*, *Uranotaenia* and *Eretmapodites* genera.

## Molecular species identification of selected *Wolbachia*-infected mosquito samples

After using the 16S rRNA and wsp genes to provide a preliminary indication of infection status, COI gene barcoding⁴⁵ was done to molecularly identify the species of a sub-sample of mosquitoes, given the difficulties associated with morphological identification of less well-studied species (Table 3–Table 4, Figure 3). Our results showed that within these 13 sub-selected *Wolbachia*-infected samples, there were seven *Culex* species, three *Aedes* species and one species each of five additional genera, confirmed to species level using Sanger sequencing of the COI gene: *Cx. quinquefasciatus*, *Cx. watti*, *Cx. cinereus*, *Cx. nigripalpus*, *Cx. perexiguus*, *Cx. rima*, *Cx. cinctellus*, *Ae. africanus*, *Ae. denderensis*, *Ma. uniformis*, *Ca. argenteopunctata*, *Lu. tigripes*, *Er. chrysogaster* and *Ur. bilineata* (Table 2). To differentiate between sibling species within the *Cx. pipiens* complex, we amplified the ACE1 gene and gel electrophoresis indicated a band size of 274 base pairs, which is diagnostic for *Cx. quinquefasciatus*. To avoid potentially mis-labelling species without sufficient sequence similarity, samples with species identity below 94% were designated 'cf' as this would be more indicative of a species that is closely related. However, the lack of sequences available for many of these species could result in genetic variation within the same species accounting for lower-than-expected sequence similarities.

## *Wolbachia* genetic diversity and MLST gene allelic profiling

We used 16S rRNA phylogeny to put the strains detected in this study into context with existing strains (Figure 4). Our results showed eight strains are clustering closely together. In addition, there is sequence diversity among strains found infecting *Ae. africanus* (samples 8–10). An in-depth analysis was undertaken through MLST gene allelic profiling (Table 5) from
<table><caption>**Table 1.** Prevalence rates of *Wolbachia* (16S rRNA gene), *Asaia* and coinfection from mosquito genera collected from the West and Central regions of Cameroon. Mosquitoes from the Central region were individually extracted and analysed. Mosquitoes from the West region were extracted from monospecific pools (same species from same collection location) and prevalence analysis represents pooled samples.</caption><thead><tr><th>Region</th><th>Genera</th><th>Wolbachia (%)</th><th>Asaia (%)</th><th>Co-infected (%)</th><th>Uninfected (%)</th><th>Totals</th><th>Fisher's P-value</th></tr></thead><tbody><tr><td>West</td><td>Aedes</td><td>32 (24.81)</td><td>0 (0.00)</td><td>97 (75.19)</td><td>0 (0.00)</td><td>129</td><td>&gt;0.99</td></tr><tr><td>West</td><td>Culex</td><td>48 (97.96)</td><td>0 (0.00)</td><td>0 (0.00)</td><td>1 (2.04)</td><td>49</td><td>&gt;0.99</td></tr><tr><td>Central</td><td>Aedes</td><td>115 (97.46)</td><td>0 (0.00)</td><td>1 (0.85)</td><td>2 (1.69)</td><td>118</td><td>&gt;0.99</td></tr><tr><td>Central</td><td>Culex</td><td>1 (0.83)</td><td>16 (13.45)</td><td>0 (0.00)</td><td>103 (85.83)</td><td>120</td><td>&gt;0.99</td></tr><tr><td>Central</td><td>Mansonia</td><td>0 (0.00)</td><td>39 (40.63)</td><td>0 (0.00)</td><td>57 (59.38)</td><td>96</td><td>&gt;0.99</td></tr></tbody></table>

Central Region, but a higher prevalence of *Asaia* was detected (40.63%, n=39) and there was no *Wolbachia* detected (59.38%, 59 individuals were uninfected for both bacterial species). In the West Region, *Aedes* mosquitoes were either co-infected (75.19%, n=97 pools) or singly infected with *Wolbachia* (24.81%, n=32 pools). For *Culex* genera mosquitoes, the vast majority (97.96%, n=48 pools) were infected with *Wolbachia* only. Results of Fisher's exact *post hoc* tests (P<0.05 significance threshold) indicated no significant associations were present in our data. As *Aedes* collections in the West Region were heavily dominated by *Ae. africanus* - a vector of YFV in forest and rainforest regions in Sub-Saharan Africa⁵⁰, we compared *Wolbachia* and *Asaia* prevalence rates for pooled mono-specific RNA pools (n=97) to look for any potential co-infections within this species. *Wolbachia* was detected in 100.00% of pools (97/97) and a high *Asaia* prevalence rate of 96.91% (94/97) pools was also seen, demonstrating a high likelihood of co-infections occurring in this species. However, as mono-specific pools consisting of an average of five female mosquitoes were used for analysis no statistical association analysis can be carried out.

### Confirmation of *Wolbachia* prevalence rates through amplification of the *wsp* gene

Our preliminary assessment of *Wolbachia* prevalence rates was generated from 16S rRNA gene amplification. However, using the 16S rRNA gene alone is insufficient because it can also be possible that prokaryotic 16S rRNA genes can be amplified from dead bacterial cells⁵¹ and *Wolbachia* 16S rRNA sequences has previously been detected in water containers that contained larvae of mosquitoes⁵². We undertook further wsp gene amplification on a wide variety of samples collected from the West Region in which morphological identification was done to species level and individuals, of the same species were pooled into groups of up to five individuals prior to RNA extraction (monospecific pools). Screening with the wsp gene revealed variable estimates of *Wolbachia* prevalence rates (Table 2). Of particular interest was the high prevalence in the most abundant species *Ae. africanus* collected in both locations in West Cameroon. A total of 341 *Ae. africanus* females from Fonakeukeu were grouped into 72 pools and 65/72 pools (90.3%) were *Wolbachia*-positive based on strong amplification

of the wsp gene. Similarly, 34/46 pools (73.9%) of the pools, representing a total of 228 *Ae. africanus* females from Toutsang amplified the wsp gene. Variable prevalence rates were also seen in other morphologically identified species, including wsp amplification in species within the *Culex*, *Aedes*, *Mansonia*, *Uranotaenia* and *Eretmapodites* genera.

### Molecular species identification of selected *Wolbachia*-infected mosquito samples

After using the 16S rRNA and wsp genes to provide a preliminary indication of infection status, COI gene barcoding⁴⁵ was done to molecularly identify the species of a sub-sample of mosquitoes, given the difficulties associated with morphological identification of less well-studied species (Table 3–Table 4, Figure 3). Our results showed that within these 13 sub-selected *Wolbachia*-infected samples, there were seven *Culex* species, three *Aedes* species and one species each of five additional genera, confirmed to species level using Sanger sequencing of the COI gene: *Cx. quinquefasciatus*, *Cx. watti*, *Cx. cinereus*, *Cx. nigripalpus*, *Cx. perexiguus*, *Cx. rima*, *Cx. cinctellus*, *Ae. africanus*, *Ae. denderensis*, *Ma. uniformis*, *Ca. argenteopunctata*, *Lu. tigripes*, *Er. chrysogaster* and *Ur. bilineata* (Table 2). To differentiate between sibling species within the *Cx. pipiens* complex, we amplified the ACE1 gene and gel electrophoresis indicated a band size of 274 base pairs, which is diagnostic for *Cx. quinquefasciatus*. To avoid potentially mis-labelling species without sufficient sequence similarity, samples with species identity below 94% were designated 'cf' as this would be more indicative of a species that is closely related. However, the lack of sequences available for many of these species could result in genetic variation within the same species accounting for lower-than-expected sequence similarities.

### *Wolbachia* genetic diversity and MLST gene allelic profiling

We used 16S rRNA phylogeny to put the strains detected in this study into context with existing strains (Figure 4). Our results showed eight strains are clustering closely together. In addition, there is sequence diversity among strains found infecting *Ae. africanus* (samples 8–10). An in-depth analysis was undertaken through MLST gene allelic profiling (Table 5) from
<table><caption>**Table 2.** Wolbachia infection prevalence using the wsp gene. Based on morphological identification to genera/species and wsp gene amplification in mosquitoes collected from the West Region of Cameroon.</caption><thead><tr><th>Collection site</th><th>Genera</th><th>Species(number)</th><th>wsp+/totalpools (%)</th></tr></thead><tbody><tr><td>Fonakeukeu</td><td>Aedes</td><td>africanus (341)</td><td>65/72 (90.3%)</td></tr><tr><td>Fonakeukeu</td><td>Aedes</td><td>argenteopunctatus (1)</td><td>1/1 (100%)</td></tr><tr><td>Fonakeukeu</td><td>Aedes</td><td>tarsalis (3)</td><td>1/3 (33.3%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>unknown (5)</td><td>0/5 (0.0%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>moucheti (25)</td><td>5/8 (62.5%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>ornathotoracis (4)</td><td>0/4 (0.0%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>tigripes (2)</td><td>1/1 (100%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>univitattus (4)</td><td>1/3 (33.3%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>wigglesworthi (7)</td><td>1/3 (33.3%)</td></tr><tr><td>Fonakeukeu</td><td>Mansonia</td><td>maculipennis (1)</td><td>0/1 (0.0%)</td></tr><tr><td>Fonakeukeu</td><td>Mansonia</td><td>annetii (1)</td><td>0/1 (0.0%)</td></tr><tr><td>Fonakeukeu</td><td>Eretmapodites</td><td>chrysogaster var (2)</td><td>1/3 (33.3%)</td></tr><tr><td>Fonakeukeu</td><td>Uranotaenia</td><td>billineata connali (6)</td><td>2/4 (50%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>africanus (228)</td><td>34/46 (73.9%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>tarsalis (2)</td><td>0/1 (0.0%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>unknown (1)</td><td>1/1 (100%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>circumluteolus (1)</td><td>1/1 (100%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>fraseri (1)</td><td>1/1 (100%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>gibbinsi (3)</td><td>1/1 (100%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>unknown (14)</td><td>2/5 (40.0%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>moucheti (28)</td><td>2/5 (40.0%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>tigripes (7)</td><td>0/4 (0.0%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>univitattus (7)</td><td>1/2 (50.0%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>duttoni (4)</td><td>1/3 (33.3%)</td></tr><tr><td>Toutsang</td><td>Mansonia</td><td>maculipennis (2)</td><td>0/1 (0.0%)</td></tr><tr><td>Toutsang</td><td>Mansonia</td><td>annetii (1)</td><td>0/1 (0.0%)</td></tr><tr><td>Toutsang</td><td>Eretmapodites</td><td>chrysogaster var (4)</td><td>2/3 (66.7%)</td></tr></tbody></table>

representatives of each mosquito species from wsp-positive individuals (Central Region) or monospecific pools (West Region) after species identification was confirmed. Complete MLST sequences are present for *Cx. quinquefasciatus*, *Cx. watti*, *Ae. africanus* (Sample 8) and *Ca. argenteopunctata*. The remaining samples had sequences of sufficient quality from 2–4 genes. For example, we were only able to obtain MLST gene sequences for two genes for *Ma. uniformis* (*gatB* and *coxA*). All sequences of sufficient quality were submitted to Genbank to obtain accession numbers Table 6.

As expected, the MLST allelic profile for *Cx. quinquefasciatus* mostly matched with strain type (ST) 9 for the wPip strain which infects *Cx. pipiens* and *Cx. quinquefasciatus*, although our sample had a match to ftsZ allele number 241, whereas existing profiles for ST 9 had an ftsZ allele number of 22. However, this represents only four nucleotide differences across the 435 base pairs for ftsZ alleles 241 and 22 resulting in 99.1% sequence identity. In *Cx. watti*, the *Wolbachia* strain allelic profile is most similar to Supergroup B *Wolbachia* strains found in the *Coenonympha hero* (the scarce heath
<table><caption>**Table 2.** Wolbachia infection prevalence using the wsp gene. Based on morphological identification to genera/species and wsp gene amplification in mosquitoes collected from the West Region of Cameroon.</caption><thead><tr><th>Collection site</th><th>Genera</th><th>Species(number)</th><th>wsp+/totalpools (%)</th></tr></thead><tbody><tr><td>Fonakeukeu</td><td>Aedes</td><td>africanus (341)</td><td>65/72 (90.3%)</td></tr><tr><td>Fonakeukeu</td><td>Aedes</td><td>argenteopunctatus (1)</td><td>1/1 (100%)</td></tr><tr><td>Fonakeukeu</td><td>Aedes</td><td>tarsalis (3)</td><td>1/3 (33.3%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>unknown (5)</td><td>0/5 (0.0%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>moucheti (25)</td><td>5/8 (62.5%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>ornathotoracis (4)</td><td>0/4 (0.0%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>tigripes (2)</td><td>1/1 (100%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>univitattus (4)</td><td>1/3 (33.3%)</td></tr><tr><td>Fonakeukeu</td><td>Culex</td><td>wigglesworthi (7)</td><td>1/3 (33.3%)</td></tr><tr><td>Fonakeukeu</td><td>Mansonia</td><td>maculipennis (1)</td><td>0/1 (0.0%)</td></tr><tr><td>Fonakeukeu</td><td>Mansonia</td><td>annetii (1)</td><td>0/1 (0.0%)</td></tr><tr><td>Fonakeukeu</td><td>Eretmapodites</td><td>chrysogaster var (2)</td><td>1/3 (33.3%)</td></tr><tr><td>Fonakeukeu</td><td>Uranotaenia</td><td>billineata connali (6)</td><td>2/4 (50%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>africanus (228)</td><td>34/46 (73.9%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>tarsalis (2)</td><td>0/1 (0.0%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>unknown (1)</td><td>1/1 (100%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>circumluteolus (1)</td><td>1/1 (100%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>fraseri (1)</td><td>1/1 (100%)</td></tr><tr><td>Toutsang</td><td>Aedes</td><td>gibbinsi (3)</td><td>1/1 (100%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>unknown (14)</td><td>2/5 (40.0%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>moucheti (28)</td><td>2/5 (40.0%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>tigripes (7)</td><td>0/4 (0.0%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>univitattus (7)</td><td>1/2 (50.0%)</td></tr><tr><td>Toutsang</td><td>Culex</td><td>duttoni (4)</td><td>1/3 (33.3%)</td></tr><tr><td>Toutsang</td><td>Mansonia</td><td>maculipennis (2)</td><td>0/1 (0.0%)</td></tr><tr><td>Toutsang</td><td>Mansonia</td><td>annetii (1)</td><td>0/1 (0.0%)</td></tr><tr><td>Toutsang</td><td>Eretmapodites</td><td>chrysogaster var (4)</td><td>2/3 (66.7%)</td></tr></tbody></table>

representatives of each mosquito species from wsp-positive individuals (Central Region) or monospecific pools (West Region) after species identification was confirmed. Complete MLST sequences are present for *Cx. quinquefasciatus*, *Cx. watti*, *Ae. africanus* (Sample 8) and *Ca. argenteopunctata*. The remaining samples had sequences of sufficient quality from 2-4 genes. For example, we were only able to obtain MLST gene sequences for two genes for *Ma. uniformis* (*gatB* and *coxA*). All sequences of sufficient quality were submitted to Genbank to obtain accession numbers Table 6.

As expected, the MLST allelic profile for *Cx. quinquefasciatus* mostly matched with strain type (ST) 9 for the wPip strain which infects *Cx. pipiens* and *Cx. quinquefasciatus*, although our sample had a match to ftsZ allele number 241, whereas existing profiles for ST 9 had an ftsZ allele number of 22. However, this represents only four nucleotide differences across the 435 base pairs for ftsZ alleles 241 and 22 resulting in 99.1% sequence identity. In *Cx. watti*, the *Wolbachia* strain allelic profile is most similar to Supergroup B *Wolbachia* strains found in the *Coenonympha hero* (the scarce heath
<caption>**Table 3.** CO1 gene sanger sequencing for molecular confirmation of mosquito species.</caption>

The NCBI BLAST percentage (%) identity and coverage are shown alongside the closest NCBI accession number (no.) and associated species. For identity 94% and under 'cf' has been added given the uncertainty of species identification.

<table><caption>**Table 4.** CO1 and Wolbachia 16S rRNA GenBank accession numbers. Location, species and sample code are shown alongside Genbank accession numbers. Sample sequences without accession numbers were of insufficient quality to obtain GenBank accession numbers.</caption><thead><tr><th>Sample</th><th>Collection site</th><th>identity (%)</th><th>coverage (%)</th><th>NCBI accession number</th><th>Species</th></tr></thead><tbody><tr><td>S1</td><td>Yaoundé</td><td>99</td><td>99</td><td>MK300247.1</td><td>Cx. quinquefasciatus</td></tr><tr><td>S2</td><td>Olama village</td><td>98</td><td>91</td><td>KU187063.1</td><td>Cx. watti</td></tr><tr><td>S3</td><td>Toutsang</td><td>97</td><td>100</td><td>LC473616.1</td><td>Cx. cinereus</td></tr><tr><td>S4</td><td>Toutsang</td><td>94</td><td>100</td><td>MT999280.1</td><td>Cx. cf nigripalpus</td></tr><tr><td>S5</td><td>Fonakeukeu</td><td>98</td><td>99</td><td>KU380382.1</td><td>Cx. perexiguus</td></tr><tr><td>S6</td><td>Fonakeukeu</td><td>94</td><td>99</td><td>LC473614.1</td><td>Cx. cf rima</td></tr><tr><td>S7</td><td>Olama</td><td>95</td><td>89</td><td>AB738190.1</td><td>Cx. cinctellus</td></tr><tr><td>S8</td><td>Toutsang</td><td>95</td><td>100</td><td>GQ165786.1</td><td>Ae. africanus</td></tr><tr><td>S9</td><td>Toutsang</td><td>94</td><td>100</td><td>GQ165786.1</td><td>Ae. cf. africanus</td></tr><tr><td>S10</td><td>Toutsang</td><td>94</td><td>100</td><td>GQ165786.1</td><td>Ae. cf. africanus</td></tr><tr><td>S11</td><td>Fonakeukeu</td><td>99</td><td>97</td><td>GQ165787.1</td><td>Ae. denderensis</td></tr><tr><td>S12</td><td>Olama village</td><td>99</td><td>93</td><td>KU380420.1</td><td>Ma. uniformis</td></tr><tr><td>S13</td><td>Fonakeukeu</td><td>94</td><td>100</td><td>MN552301.1</td><td>Ca. cf. argenteopunctata</td></tr><tr><td>S14</td><td>Toutsang</td><td>100</td><td>99</td><td>LC507833.1</td><td>Lu. tigripes</td></tr><tr><td>S15</td><td>Toutsang</td><td>90</td><td>99</td><td>MK533645.1</td><td>Er. cf. chrysogaster</td></tr><tr><td>S16</td><td>Toutsang</td><td>99</td><td>99</td><td>LC473729.1</td><td>Ur. bilineata</td></tr></tbody></table>

<table><thead><tr><th>Location</th><th>Sample ID</th><th>Species</th><th>CO1 accession number</th><th>16S rRNA accession number</th></tr></thead><tbody><tr><td>Yaoundé</td><td>S1</td><td>Cx. quinquefasciatus</td><td></td><td>OP745953</td></tr><tr><td>Olama village</td><td>S2</td><td>Cx. watti</td><td>OP744462</td><td></td></tr><tr><td>Toutsang</td><td>S3</td><td>Cx. cinereus</td><td>OP744463</td><td></td></tr><tr><td>Toutsang</td><td>S4</td><td>Cx. cf nigripalpus</td><td>OP744465</td><td>OP746031</td></tr><tr><td>Fonakeukeu</td><td>S5</td><td>Cx. perexiguus</td><td>OP744466</td><td>OP746061</td></tr><tr><td>Fonakeukeu</td><td>S6</td><td>Cx. cf rima</td><td>OP744493</td><td>OP746056</td></tr><tr><td>Olama</td><td>S7</td><td>Cx. cinctellus</td><td></td><td>OP746069</td></tr><tr><td>Toutsang</td><td>S8</td><td>Ae. africanus</td><td>OP744519</td><td>OP746071</td></tr><tr><td>Toutsang</td><td>S9</td><td>Ae. cf. africanus</td><td>OP744523</td><td>OP747286</td></tr><tr><td>Toutsang</td><td>S10</td><td>Ae. cf. africanus</td><td></td><td>OP750996</td></tr><tr><td>Fonakeukeu</td><td>S11</td><td>Ae. denderensis</td><td>OP744531</td><td>OP747294</td></tr><tr><td>Olama village</td><td>S12</td><td>Ma. uniformis</td><td>OP744580</td><td>OP747304</td></tr><tr><td>Fonakeukeu</td><td>S13</td><td>Ca. cf. argenteopunctata</td><td>OP744988</td><td>OP747416</td></tr><tr><td>Toutsang</td><td>S14</td><td>Lu. tigripes</td><td>OP745009</td><td>OP747419</td></tr><tr><td>Toutsang</td><td>S15</td><td>Er. cf. chrysogaster</td><td>OP745018</td><td>OP747455</td></tr><tr><td>Toutsang</td><td>S16</td><td>Ur. bilineata</td><td>OP745056</td><td>OP747456</td></tr></tbody></table>
<caption>**Table 3.** CO1 gene sanger sequencing for molecular confirmation of mosquito species.</caption>

The NCBI BLAST percentage (%) identity and coverage are shown alongside the closest NCBI accession number (no.) and associated species. For identity 94% and under 'cf' has been added given the uncertainty of species identification.

<table><caption>**Table 4.** CO1 and Wolbachia 16S rRNA GenBank accession numbers. Location, species and sample code are shown alongside Genbank accession numbers. Sample sequences without accession numbers were of insufficient quality to obtain GenBank accession numbers.</caption><thead><tr><th>Sample</th><th>Collection site</th><th>identity (%)</th><th>coverage (%)</th><th>NCBI accession number</th><th>Species</th></tr></thead><tbody><tr><td>S1</td><td>Yaoundé</td><td>99</td><td>99</td><td>MK300247.1</td><td>Cx. quinquefasciatus</td></tr><tr><td>S2</td><td>Olama village</td><td>98</td><td>91</td><td>KU187063.1</td><td>Cx. watti</td></tr><tr><td>S3</td><td>Toutsang</td><td>97</td><td>100</td><td>LC473616.1</td><td>Cx. cinereus</td></tr><tr><td>S4</td><td>Toutsang</td><td>94</td><td>100</td><td>MT999280.1</td><td>Cx. cf nigripalpus</td></tr><tr><td>S5</td><td>Fonakeukeu</td><td>98</td><td>99</td><td>KU380382.1</td><td>Cx. perexiguus</td></tr><tr><td>S6</td><td>Fonakeukeu</td><td>94</td><td>99</td><td>LC473614.1</td><td>Cx. cf rima</td></tr><tr><td>S7</td><td>Olama</td><td>95</td><td>89</td><td>AB738190.1</td><td>Cx. cinctellus</td></tr><tr><td>S8</td><td>Toutsang</td><td>95</td><td>100</td><td>GQ165786.1</td><td>Ae. africanus</td></tr><tr><td>S9</td><td>Toutsang</td><td>94</td><td>100</td><td>GQ165786.1</td><td>Ae. cf. africanus</td></tr><tr><td>S10</td><td>Toutsang</td><td>94</td><td>100</td><td>GQ165786.1</td><td>Ae. cf. africanus</td></tr><tr><td>S11</td><td>Fonakeukeu</td><td>99</td><td>97</td><td>GQ165787.1</td><td>Ae. denderensis</td></tr><tr><td>S12</td><td>Olama village</td><td>99</td><td>93</td><td>KU380420.1</td><td>Ma. uniformis</td></tr><tr><td>S13</td><td>Fonakeukeu</td><td>94</td><td>100</td><td>MN552301.1</td><td>Ca. cf. argenteopunctata</td></tr><tr><td>S14</td><td>Toutsang</td><td>100</td><td>99</td><td>LC507833.1</td><td>Lu. tigripes</td></tr><tr><td>S15</td><td>Toutsang</td><td>90</td><td>99</td><td>MK533645.1</td><td>Er. cf. chrysogaster</td></tr><tr><td>S16</td><td>Toutsang</td><td>99</td><td>99</td><td>LC473729.1</td><td>Ur. bilineata</td></tr></tbody></table>

<table><thead><tr><th>Location</th><th>Sample ID</th><th>Species</th><th>CO1 accession number</th><th>16S rRNA accession number</th></tr></thead><tbody><tr><td>Yaoundé</td><td>S1</td><td>Cx. quinquefasciatus</td><td></td><td>OP745953</td></tr><tr><td>Olama village</td><td>S2</td><td>Cx. watti</td><td>OP744462</td><td></td></tr><tr><td>Toutsang</td><td>S3</td><td>Cx. cinereus</td><td>OP744463</td><td></td></tr><tr><td>Toutsang</td><td>S4</td><td>Cx. cf nigripalpus</td><td>OP744465</td><td>OP746031</td></tr><tr><td>Fonakeukeu</td><td>S5</td><td>Cx. perexiguus</td><td>OP744466</td><td>OP746061</td></tr><tr><td>Fonakeukeu</td><td>S6</td><td>Cx. cf rima</td><td>OP744493</td><td>OP746056</td></tr><tr><td>Olama</td><td>S7</td><td>Cx. cinctellus</td><td></td><td>OP746069</td></tr><tr><td>Toutsang</td><td>S8</td><td>Ae. africanus</td><td>OP744519</td><td>OP746071</td></tr><tr><td>Toutsang</td><td>S9</td><td>Ae. cf. africanus</td><td>OP744523</td><td>OP747286</td></tr><tr><td>Toutsang</td><td>S10</td><td>Ae. cf. africanus</td><td></td><td>OP750996</td></tr><tr><td>Fonakeukeu</td><td>S11</td><td>Ae. denderensis</td><td>OP744531</td><td>OP747294</td></tr><tr><td>Olama village</td><td>S12</td><td>Ma. uniformis</td><td>OP744580</td><td>OP747304</td></tr><tr><td>Fonakeukeu</td><td>S13</td><td>Ca. cf. argenteopunctata</td><td>OP744988</td><td>OP747416</td></tr><tr><td>Toutsang</td><td>S14</td><td>Lu. tigripes</td><td>OP745009</td><td>OP747419</td></tr><tr><td>Toutsang</td><td>S15</td><td>Er. cf. chrysogaster</td><td>OP745018</td><td>OP747455</td></tr><tr><td>Toutsang</td><td>S16</td><td>Ur. bilineata</td><td>OP745056</td><td>OP747456</td></tr></tbody></table>
<figure><img src="image_15.png" /><figcaption>**Figure 3.** *CO1 gene phylogenetic analysis of mosquito species collected from Cameroon.* Maximum Likelihood phylogenetic analysis using the General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.6443)). The tree comprises 30 nucleotide sequences with 725 positions in the dataset. Drawn to a 0.05 scale.</figcaption></figure>

butterfly) – (ST 296), the moth *Amblyptilia punctidactyla*, *Coenonympha pamphilus* (the small heath butterfly), the Fischer's butterfly *Tongeia fischeri* (ST 300) and the thrrip *Pezo-thrips kellyanus* (ST 430). However, only *gatB* (allele 9) and *ftsZ* (allele 7) show exact matches to these three strain types with *hcpA* and *fbpA* alleles being variable both for the novel strain in *Cx. watti* and STs 296, 300 and 430. The allelic profile of the *Wolbachia* strain detected in *Cx. cinerus* was most similar to 11 different strain types (3,108,151,213,366, 382,387,461,462,472,492) but appears a novel strain as none of these typed strains contain the combination of the four alleles identified in *Cx. cinerus* (we were unable to sequence the

*ftsZ* gene). For example, ST 108 has the same *gatB* allele (71) but the remaining three genes for comparison (*coxA*, *hcpA* and *fbpA*) have variable allele numbers which are not the same as those sequenced from the *Wolbachia* strain in *Cx. cinerus*.

The *Wolbachia* strain detected in *Cx. cf. nigripalpus* produced an exact match to strain type 13 which is found in numerous insect species, including *Drosophila recens* and *Leucophenga maculosa* fruit flies and *Rhagoletis cerasi* (cherry fruit fly). The widespread occurrence of this *Wolbachia* strain type across multiple insect genera requires further investigation given 19 isolates are present in the database. We were only able
<figure><img src="image_16.png" /><figcaption>**Figure 3.** *CO1 gene phylogenetic analysis of mosquito species collected from Cameroon.* Maximum Likelihood phylogenetic analysis using the General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.6443)). The tree comprises 30 nucleotide sequences with 725 positions in the dataset. Drawn to a 0.05 scale.</figcaption></figure>

butterfly) – (ST 296), the moth *Amblyptilia punctidactyla*, *Coenonympha pamphilus* (the small heath butterfly), the Fischer’s butterfly *Tongeia fischeri* (ST 300) and the thrrip *Pezo-thrips kellyanus* (ST 430). However, only *gatB* (allele 9) and *ftsZ* (allele 7) show exact matches to these three strain types with *hcpA* and *fbpA* alleles being variable both for the novel strain in *Cx. watti* and STs 296, 300 and 430. The allelic profile of the *Wolbachia* strain detected in *Cx. cinerus* was most similar to 11 different strain types (3,108,151,213,366, 382,387,461,462,472,492) but appears a novel strain as none of these typed strains contain the combination of the four alleles identified in *Cx. cinerus* (we were unable to sequence the

*ftsZ* gene). For example, ST 108 has the same *gatB* allele (71) but the remaining three genes for comparison (*coxA*, *hcpA* and *fbpA*) have variable allele numbers which are not the same as those sequenced from the *Wolbachia* strain in *Cx. cinerus*.

The *Wolbachia* strain detected in *Cx. cf. nigripalpus* produced an exact match to strain type 13 which is found in numerous insect species, including *Drosophila recens* and *Leucophenga maculosa* fruit flies and *Rhagoletis cerasi* (cherry fruit fly). The widespread occurrence of this *Wolbachia* strain type across multiple insect genera requires further investigation given 19 isolates are present in the database. We were only able
<figure><img src="image_16.png" /><figcaption>**Figure 4.** 16S rRNA gene phylogenetic analysis of *Wolbachia* strains. The tree was produced using the Maximum Likelihood method and Jukes-Cantor model. The tree contains 31 nucleotide sequences and 1610 positions in the dataset. *Wolbachia* strains detected in this study are highlighted in red, sequences from additional strains obtained from Genbank with accession numbers are shown in black. Drawn to a 0.05 scale.</figcaption></figure>

to sequence three genes for the *Wolbachia* strain detected in *Cx. perexiguus* (*coxA*, *fbpA* and *hcpA*) resulting in three nearest strain types (108, 187,467) of which none had allele number 11 for *coxA*. However, a complete MLST profile would help confirm what appears to be a novel strain in *Cx. perexiguus* given its unique combination of three sequenced MLST genes. Likewise, the *Wolbachia* strain detected in *Cx. rima* would appear novel given only two of four allelic loci (*gatB* and *coxA*) matched the closest ST (52) previously reported in *Anoplolepis gracillipes* (the yellow crazy ant).

Given that we collected large numbers of *Ae. africanus* from the West Region of Cameroon, we included three monospecific pools (A-C) for MLST allelic profiling (Table 5–Table 6). Our results provide evidence for multiple *Wolbachia* strains through analysis indicating allelic matches for four of five MLST genes (and divergence seen in the 16S rRNA phylogeny between samples 8–10). Our Sanger sequencing indicated no evidence for the presence of multiple strains within the same pool, but further analysis is required to further determine if superinfections can be present within individual
<figure><img src="image_17.png" /><figcaption>**Figure 4.** 16S rRNA gene phylogenetic analysis of *Wolbachia* strains. The tree was produced using the Maximum Likelihood method and Jukes-Cantor model. The tree contains 31 nucleotide sequences and 1610 positions in the dataset. *Wolbachia* strains detected in this study are highlighted in red, sequences from additional strains obtained from Genbank with accession numbers are shown in black. Drawn to a 0.05 scale.</figcaption></figure>

to sequence three genes for the *Wolbachia* strain detected in *Cx. perexiguus* (*coxA*, *fbpA* and *hcpA*) resulting in three nearest strain types (108, 187,467) of which none had allele number 11 for *coxA*. However, a complete MLST profile would help confirm what appears to be a novel strain in *Cx. perexiguus* given its unique combination of three sequenced MLST genes. Likewise, the *Wolbachia* strain detected in *Cx. rima* would appear novel given only two of four allelic loci (*gatB* and *coxA*) matched the closest ST (52) previously reported in *Anoplolepis gracillipes* (the yellow crazy ant).

Given that we collected large numbers of *Ae. africanus* from the West Region of Cameroon, we included three monospecific pools (A-C) for MLST allelic profiling (Table 5–Table 6). Our results provide evidence for multiple *Wolbachia* strains through analysis indicating allelic matches for four of five MLST genes (and divergence seen in the 16S rRNA phylogeny between samples 8–10). Our Sanger sequencing indicated no evidence for the presence of multiple strains within the same pool, but further analysis is required to further determine if superinfections can be present within individual
<table><caption>**Table 5.** MLST gene allelic profiling. Assigned allele numbers matching those present in the Wolbachia MLST database (https://pubmlst.org/organisms/wolbachia-spp/) indicating nucleotide differences in brackets.</caption><thead><tr><th>Sample ID</th><th>Mosquito species</th><th>gatB</th><th>coxA</th><th>hcpA</th><th>ftsZ</th><th>fbpA</th></tr></thead><tbody><tr><td>S1</td><td>Cx. quinquefasciatus</td><td>4</td><td>3</td><td>3 (1)</td><td>241</td><td>4</td></tr><tr><td>S2</td><td>Cx. watti</td><td>9</td><td>14 (2)</td><td>12</td><td>7</td><td>203 (1)</td></tr><tr><td>S3</td><td>Cx. cinereus</td><td>71</td><td>11</td><td>303</td><td>-</td><td>90</td></tr><tr><td>S4</td><td>Cx. cf nigripalpus</td><td>1</td><td>1</td><td>1</td><td>3</td><td>1</td></tr><tr><td>S5</td><td>Cx. perexiguus</td><td>-</td><td>11</td><td>74</td><td>-</td><td>6</td></tr><tr><td>S6</td><td>Cx. cf rima</td><td>22</td><td>2</td><td>74</td><td>258</td><td>-</td></tr><tr><td>S7</td><td>Cx. cinctellus</td><td>71</td><td>11</td><td>303</td><td>-</td><td>90</td></tr><tr><td>S8</td><td>Ae. africanus</td><td>9</td><td>8</td><td>74</td><td>106</td><td>6</td></tr><tr><td>S9</td><td>Ae. cf. africanus</td><td>9</td><td>8</td><td>74</td><td>117</td><td>203</td></tr><tr><td>S10</td><td>Ae. cf. africanus</td><td>9</td><td>8</td><td>12</td><td>117</td><td>203</td></tr><tr><td>S11</td><td>Ae. denderensis</td><td>9</td><td>11</td><td>74</td><td>106</td><td>6</td></tr><tr><td>S12</td><td>Ma. uniformis</td><td>9</td><td>14</td><td>-</td><td>-</td><td>43 (12)</td></tr><tr><td>S13</td><td>Ca. cf. argenteopunctata</td><td>9</td><td>11</td><td>303</td><td>7</td><td>446</td></tr><tr><td>S14</td><td>Lu. tigripes</td><td>71</td><td>-</td><td>74</td><td>117</td><td>6</td></tr><tr><td>S15</td><td>Er. cf. chrysogaster</td><td>-</td><td>275</td><td>-</td><td>106</td><td>6</td></tr><tr><td>S16</td><td>Ur. bilineata</td><td>9</td><td>11</td><td>74</td><td>-</td><td>90</td></tr></tbody></table>

<table><caption>**Table 6.** GenBank accession numbers for MLST gene sequences. Sample sequences without accession numbers were of insufficient quality to obtain GenBank accession numbers.</caption><thead><tr><th>Sample ID</th><th>Mosquito species</th><th>gatB</th><th>coxA</th><th>hcpA</th><th>ftsZ</th><th>fbpA</th></tr></thead><tbody><tr><td>S1</td><td>Cx. quinquefasciatus</td><td>OQ236162</td><td>OQ236174</td><td>OQ236185</td><td>OQ236197</td><td>OQ236208</td></tr><tr><td>S2</td><td>Cx. watti</td><td>OQ236163</td><td>OQ236175</td><td>OQ236186</td><td>OQ236198</td><td>OQ236209</td></tr><tr><td>S3</td><td>Cx. cinereus</td><td>OQ236164</td><td>OQ236176</td><td>OQ236187</td><td></td><td>OQ236210</td></tr><tr><td>S4</td><td>Cx. cf nigripalpus</td><td>OQ236165</td><td></td><td>OQ236188</td><td>OQ236199</td><td></td></tr><tr><td>S5</td><td>Cx. perexiguus</td><td></td><td>OQ236177</td><td>OQ236189</td><td></td><td></td></tr><tr><td>S6</td><td>Cx. cf rima</td><td>OQ236166</td><td>OQ236178</td><td>OQ236190</td><td>OQ236200</td><td></td></tr><tr><td>S8</td><td>Cx. cinctellus</td><td>OQ236167</td><td>OQ236179</td><td>OQ236191</td><td>OQ236201</td><td></td></tr><tr><td>S9</td><td>Ae. africanus</td><td>OQ236168</td><td></td><td>OQ236192</td><td>OQ236202</td><td>OQ236211</td></tr><tr><td>S10</td><td>Ae. cf. africanus</td><td>OQ236169</td><td>OQ236180</td><td>OQ236193</td><td>OQ236203</td><td>OQ236212</td></tr><tr><td>S11</td><td>Ae. cf. africanus</td><td></td><td></td><td></td><td>OQ236204</td><td>OQ236213</td></tr><tr><td>S12</td><td>Ae. denderensis</td><td>OQ236170</td><td>OQ236181</td><td></td><td></td><td></td></tr><tr><td>S13</td><td>Ma. uniformis</td><td>OQ236171</td><td>OQ236182</td><td>OQ236194</td><td>OQ236205</td><td>OQ236214</td></tr><tr><td>S14</td><td>Ca. cf. argenteopunctata</td><td>OQ236172</td><td></td><td>OQ236195</td><td>OQ236206</td><td></td></tr><tr><td>S15</td><td>Lu. tigripes</td><td></td><td>OQ236183</td><td></td><td>OQ236207</td><td>OQ236215</td></tr><tr><td>S16</td><td>Er. cf. chrysogaster</td><td>OQ236173</td><td>OQ236184</td><td>OQ236196</td><td></td><td>OQ236216</td></tr></tbody></table>
<table><caption>**Table 5.** MLST gene allelic profiling. Assigned allele numbers matching those present in the Wolbachia MLST database (https://pubmlst.org/organisms/wolbachia-spp/) indicating nucleotide differences in brackets.</caption><thead><tr><th>Sample ID</th><th>Mosquito species</th><th>gatB</th><th>coxA</th><th>hcpA</th><th>ftsZ</th><th>fbpA</th></tr></thead><tbody><tr><td>S1</td><td>Cx. quinquefasciatus</td><td>4</td><td>3</td><td>3 (1)</td><td>241</td><td>4</td></tr><tr><td>S2</td><td>Cx. watti</td><td>9</td><td>14 (2)</td><td>12</td><td>7</td><td>203 (1)</td></tr><tr><td>S3</td><td>Cx. cinereus</td><td>71</td><td>11</td><td>303</td><td>-</td><td>90</td></tr><tr><td>S4</td><td>Cx. cf nigripalpus</td><td>1</td><td>1</td><td>1</td><td>3</td><td>1</td></tr><tr><td>S5</td><td>Cx. perexiguus</td><td>-</td><td>11</td><td>74</td><td>-</td><td>6</td></tr><tr><td>S6</td><td>Cx. cf rima</td><td>22</td><td>2</td><td>74</td><td>258</td><td>-</td></tr><tr><td>S7</td><td>Cx. cinctellus</td><td>71</td><td>11</td><td>303</td><td>-</td><td>90</td></tr><tr><td>S8</td><td>Ae. africanus</td><td>9</td><td>8</td><td>74</td><td>106</td><td>6</td></tr><tr><td>S9</td><td>Ae. cf. africanus</td><td>9</td><td>8</td><td>74</td><td>117</td><td>203</td></tr><tr><td>S10</td><td>Ae. cf. africanus</td><td>9</td><td>8</td><td>12</td><td>117</td><td>203</td></tr><tr><td>S11</td><td>Ae. denderensis</td><td>9</td><td>11</td><td>74</td><td>106</td><td>6</td></tr><tr><td>S12</td><td>Ma. uniformis</td><td>9</td><td>14</td><td>-</td><td>-</td><td>43 (12)</td></tr><tr><td>S13</td><td>Ca. cf. argenteopunctata</td><td>9</td><td>11</td><td>303</td><td>7</td><td>446</td></tr><tr><td>S14</td><td>Lu. tigripes</td><td>71</td><td>-</td><td>74</td><td>117</td><td>6</td></tr><tr><td>S15</td><td>Er. cf. chrysogaster</td><td>-</td><td>275</td><td>-</td><td>106</td><td>6</td></tr><tr><td>S16</td><td>Ur. bilineata</td><td>9</td><td>11</td><td>74</td><td>-</td><td>90</td></tr></tbody></table>

<table><caption>**Table 6.** GenBank accession numbers for MLST gene sequences. Sample sequences without accession numbers were of insufficient quality to obtain GenBank accession numbers.</caption><thead><tr><th>Sample ID</th><th>Mosquito species</th><th>gatB</th><th>coxA</th><th>hcpA</th><th>ftsZ</th><th>fbpA</th></tr></thead><tbody><tr><td>S1</td><td>Cx. quinquefasciatus</td><td>OQ236162</td><td>OQ236174</td><td>OQ236185</td><td>OQ236197</td><td>OQ236208</td></tr><tr><td>S2</td><td>Cx. watti</td><td>OQ236163</td><td>OQ236175</td><td>OQ236186</td><td>OQ236198</td><td>OQ236209</td></tr><tr><td>S3</td><td>Cx. cinereus</td><td>OQ236164</td><td>OQ236176</td><td>OQ236187</td><td></td><td>OQ236210</td></tr><tr><td>S4</td><td>Cx. cf nigripalpus</td><td>OQ236165</td><td></td><td>OQ236188</td><td>OQ236199</td><td></td></tr><tr><td>S5</td><td>Cx. perexiguus</td><td></td><td>OQ236177</td><td>OQ236189</td><td></td><td></td></tr><tr><td>S6</td><td>Cx. cf rima</td><td>OQ236166</td><td>OQ236178</td><td>OQ236190</td><td>OQ236200</td><td></td></tr><tr><td>S8</td><td>Cx. cinctellus</td><td>OQ236167</td><td>OQ236179</td><td>OQ236191</td><td>OQ236201</td><td></td></tr><tr><td>S9</td><td>Ae. africanus</td><td>OQ236168</td><td></td><td>OQ236192</td><td>OQ236202</td><td>OQ236211</td></tr><tr><td>S10</td><td>Ae. cf. africanus</td><td>OQ236169</td><td>OQ236180</td><td>OQ236193</td><td>OQ236203</td><td>OQ236212</td></tr><tr><td>S11</td><td>Ae. cf. africanus</td><td></td><td></td><td></td><td>OQ236204</td><td>OQ236213</td></tr><tr><td>S12</td><td>Ae. denderensis</td><td>OQ236170</td><td>OQ236181</td><td></td><td></td><td></td></tr><tr><td>S13</td><td>Ma. uniformis</td><td>OQ236171</td><td>OQ236182</td><td>OQ236194</td><td>OQ236205</td><td>OQ236214</td></tr><tr><td>S14</td><td>Ca. cf. argenteopunctata</td><td>OQ236172</td><td></td><td>OQ236195</td><td>OQ236206</td><td></td></tr><tr><td>S15</td><td>Lu. tigripes</td><td></td><td>OQ236183</td><td></td><td>OQ236207</td><td>OQ236215</td></tr><tr><td>S16</td><td>Er. cf. chrysogaster</td><td>OQ236173</td><td>OQ236184</td><td>OQ236196</td><td></td><td>OQ236216</td></tr></tbody></table>
*Ae. africanus*. A novel strain was detected in *Ae. denderensis* as only three loci (*gatB*, *hcpA* and *fbpA*) matched ST 467 of a *Wolbachia* strain found in *Cabera pusaria* (Common white wave moth). A complete MLST profile was generated for the *Wolbachia* strain in *Ca. argenteopunctata* which appears novel given only two loci in combination match existing strains. For *Lutzia tigripes*, we could only produce sequences for three MLST genes, but this strain also appears novel with only three of four loci matching ST 108 – a strain found in the butterfly *Brangas felderi*. Likewise, novel strains appear to be present in both *Eretmapodites chrysogaster* and *Uranotaenia bilineata* as their partial allelic profiles did not match any other strain types in the database.

Discussion

In Cameroon, we previously showed that the richness of
mosquito species was dependent on both habitat type and
seasonality⁴⁰. Therefore, in this current study we analysed
mosquitoes from diverse environmental settings to capture as
much potential diversity in both mosquito species and corre-
sponding resident *Wolbachia* strains. We identified what appears
to be either novel strains or variants of existing characterised
*Wolbachia* strains in 13 different mosquito species. A natural
*Wolbachia* strain in *Cx. quinquefasciatus* mostly matching
ST 9 is to be expected given the wPip strain is widespread in
species of the *Cx. pipiens* complex⁵³⁻⁵⁵. Our allelic profiling
indicated evidence of some genetic diversity in the ftsZ allele
although this was only four of 435 nucleotides (99.1% sequence
similarity). This also highlights the requirement of using MLST
allelic profiling given the 16S analysis provided little sequence
similarity to existing sequences from *Wolbachia* strains detected
in the *Cx. pipiens* complex (Figure 4). Interestingly, there was no
evidence of *Wolbachia* in *Cx. pipiens* collected from Madagascar⁶
despite the prevailing assumption that the wPip strain widely
infects both *Cx. pipiens* and *Cx. quinquefasciatus* populations.
Further studies across sub-Saharan Africa are needed to deter-
mine variability in both the prevalence rates and genetic strain
diversity of the wPip strain in members of the *Cx. pipiens*
complex, given their important role as vectors of multiple
human pathogens such as West Nile virus and filarial nematodes
such as *Wuchereria bancrofti*.

Another vector of human pathogens analysed in our study was
*Ae. africanus* – a major vector of YFV. Although *Ae. africanus*
is considered a sylvatic vector in rural areas, recent studies
have suggested it has the capacity to colonise peri-domestic
and domestic habitats⁵⁰. Our MLST analysis suggests there are
multiple *Wolbachia* strain variants present in *Ae. africanus*
with variation in gene sequences in three of the five MLST
genes (Table 5). It could also be possible that resident
*Wolbachia* strain superinfections occur in *Ae. africanus* as
have been seen in *Ae. albopictus*⁵⁶. *Mansonia uniformis* has
been shown to transmit numerous arboviruses, such as Murray
Valley encephalitis and Ross River virus, and has been shown
to be a vector of Bancroftian lymphatic filariasis in Ghana⁵⁷.
Interestingly, although *Wolbachia* has been previously identi-
fied in this species, no complete allelic profile is present. Our
results match *gatB* and *coxA* allelic loci from *Ma. uniformis*

collected in Kenya⁸ but not fbpA, suggesting the possibility
of a different strain variant present in populations from
Cameroon. The remaining novel *Wolbachia* strains that we
have identified were in mosquito species that are considered
either minor vectors of human disease or implicated in trans-
mission of WNV, such as *Cx. perexiguus*, *Cx. watti*, and
*Cx. rima*. With the exception of *Cx. quinquefasciatus* and
*Ma. uniformis*, no *Wolbachia* MLST sequences are avail-
able (pubmlst.org/organisms/wolbachia-spp) for the remaining
mosquito species.

Although we undertook molecular barcoding by sequencing
the mosquito CO1 gene to try and provide as much confidence
in species identification, caution must be taken with any
results as this is dependent on the availability of sequences for
comparison. For example, we identified *Wolbachia* strains
in multiple individuals in which sequence identity was only
94%, suggesting these may be closely related species to the
closest match sequence available on GenBank. Sample 7
was identified as *Cx. cinctellus* but with only 95% identity
and 89% coverage, indicating this could also be another
closely-related species. The inability to accurately identify
mosquito species using molecular barcoding is common for
species in which few sequences have previously been made
available in databases such as GenBank. However, providing
the CO1 sequences will inform future studies looking at
closely related species.

Our results comparing *Wolbachia* and *Asaia* prevalence rates
across major Culicine genera indicated a significant association
only in *Mansonia* mosquitoes. These results for *Mansonia* are
consistent with other mosquito species such as *Ae. koreicus* in
which studies from field collected mosquitoes indicate a mutual
exclusion between these two symbionts⁵⁸. In contrast, high lev-
els of co-infections (particularly within *Ae. africanus*, which
dominated our collections from the West Region) add to grow-
ing evidence that *Wolbachia* and *Asaia* can co-exist in wild
mosquitoes³ despite studies clearly demonstrating an antago-
nistic association in lab colonies²⁷,²⁸. As *Asaia* can be acquired
from the environment throughout the mosquito life cycle, the
collection location becomes a significant factor that complicates
this tripartite association and therefore our results are limited
to both our collection locations and species collected. Another
major limitation of our study is that we were unable to pro-
vide comparative data to the species level for the Central region
due to the high levels of misidentification of Culicine species²⁹
and missing or damaged morphological features during
mosquito collections. Furthermore, as tissue-specific detection
was not feasible for the large number of diverse field-collected
mosquitoes in our study, it could be possible that *Wolbachia*
and *Asaia* co-exist within a given individual mosquito but are
located in different tissues⁶⁰. Likewise, the detection of both
*Wolbachia* and *Asaia* in samples from the West region (mono-
specific pools) needs further investigation given the limitations
of using pooled samples. The possibility of morphological
misidentification resulting in the addition of an ‘incorrect’
species to the pool or results reflecting single infections
(ie. one *Wolbachia*-infected individual, one *Asaia*-infected
*Ae. africanus*. A novel strain was detected in *Ae. denderensis* as only three loci (*gatB*, *hcpA* and *fbpA*) matched ST 467 of a *Wolbachia* strain found in *Cabera pusaria* (Common white wave moth). A complete MLST profile was generated for the *Wolbachia* strain in *Ca. argenteopunctata* which appears novel given only two loci in combination match existing strains. For *Lutzia tigripes*, we could only produce sequences for three MLST genes, but this strain also appears novel with only three of four loci matching ST 108 – a strain found in the butterfly *Brangas felderi*. Likewise, novel strains appear to be present in both *Eretmapodites chrysogaster* and *Uranotaenia bilineata* as their partial allelic profiles did not match any other strain types in the database.

Discussion

In Cameroon, we previously showed that the richness of
mosquito species was dependent on both habitat type and
seasonality⁴⁰. Therefore, in this current study we analysed
mosquitoes from diverse environmental settings to capture as
much potential diversity in both mosquito species and corre-
sponding resident *Wolbachia* strains. We identified what appears
to be either novel strains or variants of existing characterised
*Wolbachia* strains in 13 different mosquito species. A natural
*Wolbachia* strain in *Cx. quinquefasciatus* mostly matching
ST 9 is to be expected given the wPip strain is widespread in
species of the *Cx. pipiens* complex⁵³⁻⁵⁵. Our allelic profiling
indicated evidence of some genetic diversity in the ftsZ allele
although this was only four of 435 nucleotides (99.1% sequence
similarity). This also highlights the requirement of using MLST
allelic profiling given the 16S analysis provided little sequence
similarity to existing sequences from *Wolbachia* strains detected
in the *Cx. pipiens* complex (Figure 4). Interestingly, there was no
evidence of *Wolbachia* in *Cx. pipiens* collected from Madagascar⁶
despite the prevailing assumption that the wPip strain widely
infects both *Cx. pipiens* and *Cx. quinquefasciatus* populations.
Further studies across sub-Saharan Africa are needed to deter-
mine variability in both the prevalence rates and genetic strain
diversity of the wPip strain in members of the *Cx. pipiens*
complex, given their important role as vectors of multiple
human pathogens such as West Nile virus and filarial nematodes
such as *Wuchereria bancrofti*.

Another vector of human pathogens analysed in our study was
*Ae. africanus* – a major vector of YFV. Although *Ae. africanus*
is considered a sylvatic vector in rural areas, recent studies
have suggested it has the capacity to colonise peri-domestic
and domestic habitats⁵⁰. Our MLST analysis suggests there are
multiple *Wolbachia* strain variants present in *Ae. africanus*
with variation in gene sequences in three of the five MLST
genes (Table 5). It could also be possible that resident
*Wolbachia* strain superinfections occur in *Ae. africanus* as
have been seen in *Ae. albopictus*⁵⁶. *Mansonia uniformis* has
been shown to transmit numerous arboviruses, such as Murray
Valley encephalitis and Ross River virus, and has been shown
to be a vector of Bancroftian lymphatic filariasis in Ghana⁵⁷.
Interestingly, although *Wolbachia* has been previously identi-
fied in this species, no complete allelic profile is present. Our
results match *gatB* and *coxA* allelic loci from *Ma. uniformis*

collected in Kenya⁸ but not *fbpA*, suggesting the possibility
of a different strain variant present in populations from
Cameroon. The remaining novel *Wolbachia* strains that we
have identified were in mosquito species that are considered
either minor vectors of human disease or implicated in trans-
mission of WNV, such as *Cx. perexiguus*, *Cx. watti*, and
*Cx. rima*. With the exception of *Cx. quinquefasciatus* and
*Ma. uniformis*, no *Wolbachia* MLST sequences are avail-
able (pubmlst.org/organisms/wolbachia-spp) for the remaining
mosquito species.

Although we undertook molecular barcoding by sequencing
the mosquito CO1 gene to try and provide as much confidence
in species identification, caution must be taken with any
results as this is dependent on the availability of sequences for
comparison. For example, we identified *Wolbachia* strains
in multiple individuals in which sequence identity was only
94%, suggesting these may be closely related species to the
closest match sequence available on GenBank. Sample 7
was identified as *Cx. cinctellus* but with only 95% identity
and 89% coverage, indicating this could also be another
closely-related species. The inability to accurately identify
mosquito species using molecular barcoding is common for
species in which few sequences have previously been made
available in databases such as GenBank. However, providing
the CO1 sequences will inform future studies looking at
closely related species.

Our results comparing *Wolbachia* and *Asaia* prevalence rates
across major Culicine genera indicated a significant association
only in *Mansonia* mosquitoes. These results for *Mansonia* are
consistent with other mosquito species such as *Ae. koreicus* in
which studies from field collected mosquitoes indicate a mutual
exclusion between these two symbionts⁵⁸. In contrast, high lev-
els of co-infections (particularly within *Ae. africanus*, which
dominated our collections from the West Region) add to grow-
ing evidence that *Wolbachia* and *Asaia* can co-exist in wild
mosquitoes³ despite studies clearly demonstrating an antago-
nistic association in lab colonies²⁷,²⁸. As *Asaia* can be acquired
from the environment throughout the mosquito life cycle, the
collection location becomes a significant factor that complicates
this tripartite association and therefore our results are limited
to both our collection locations and species collected. Another
major limitation of our study is that we were unable to pro-
vide comparative data to the species level for the Central region
due to the high levels of misidentification of Culicine species²⁹
and missing or damaged morphological features during
mosquito collections. Furthermore, as tissue-specific detection
was not feasible for the large number of diverse field-collected
mosquitoes in our study, it could be possible that *Wolbachia*
and *Asaia* co-exist within a given individual mosquito but are
located in different tissues⁶⁰. Likewise, the detection of both
*Wolbachia* and *Asaia* in samples from the West region (mono-
specific pools) needs further investigation given the limitations
of using pooled samples. The possibility of morphological
misidentification resulting in the addition of an ‘incorrect’
species to the pool or results reflecting single infections
(ie. one *Wolbachia*-infected individual, one *Asaia*-infected
individual) cannot be ruled out. However, this would seem unlikely given *Aedes* monospecific pools mosquitoes were either co-infected (75%) or singly infected with *Wolbachia* (25%) and for *Culex* 98% were infected with *Wolbachia* only. Larger cohort collections of mosquitoes from diverse environmental settings will provide further insight into how these two widespread bacteria co-exist (or do not co-exist) in different mosquito species.

*Wolbachia* MLST gene allelic profiling was undertaken to provide more assurances on detection of genuine endosymbiotic strains found in wild mosquito populations. We defined a ‘novel’ strain based on MLST to contain either new MLST gene sequences not present in https://pubmlst.org/organisms/wolbachia-spp or a combination of MLST gene sequences that does match an existing strain in the database. Despite being widely used, MLST For *Wolbachia* strains has limitations and the five genes may not represent the optimal loci to capture strain variation⁶¹. Furthermore, defining whether a novel strain exists based only on PCR amplification of genes is problematic given the numerous examples of environmental contamination or host genome integration⁵¹,⁵². Caution must be taken when extrapolating PCR amplification to indicate the presence of a living endosymbiotic bacterium – particularly so when only a few gene targets such as 16S rRNA are amplified and sequenced. It has been shown that 16S rRNA prokaryotic DNA can be amplified from dead cells⁵¹ and *Wolbachia* 16S rRNA can be detected just from water that previously contained mosquito larvae⁵². If possible, extraction of mosquito RNA (as carried out in this study) to confirm expression of *Wolbachia* genes provides further evidence³. Once novel strains are detected using MLST profiling, further studies are needed to confirm a genuine stable endosymbiotic association is present with the mosquito host species. This is important when low prevalence rates are detected given this may otherwise suggest that the *Wolbachia* strain is not inducing CI. Furthermore, there are several additional experiments that can be undertaken to provide further confirmation of resident *Wolbachia* strains in mosquitoes. Methods that can visualise *Wolbachia* bacteria in mosquito tissues using microscopy, such as fluorescent *in situ* hybridization, and *Wolbachia* genome sequencing, to compare genome depth and coverage of novel strains to those of other known infections, should be carried out to fully characterise novel *Wolbachia* strains.

## Conclusions

Novel *Wolbachia* strains in Culicine mosquitoes collected from ecologically diverse settings in Cameroon add to the diversity of this highly prevalent endosymbiont in insect populations. Resident *Wolbachia* strains should be further characterised to determine the tissue tropism and density of newly

discovered strains. Our study also suggests that co-infection with environmentally acquired *Asaia* bacteria is widespread in wild mosquito populations (except the *Mansonia* genera) and the antagonistic relationship observed in lab colonies may not be present in some wild Culicine populations. Novel *Wolbachia* strains could be considered as candidate strains for biocontrol strategies given their ability to reside naturally within existing mosquito populations and co-exist with environmentally acquired *Asaia* bacteria.

## Data availability

### Underlying data

GenBank: *Wolbachia* endosymbiont of *Culex pipiens* isolate 1 16S ribosomal RNA gene, partial sequence. Accession number OP745953; https://identifiers.org/insdc:OP745953⁶²

GenBank: *Culex watti* isolate 1 cytochrome c oxidase subunit I (COX1) gene, partial cds; mitochondrial. Accession number OP744462; https://identifiers.org/insdc:OP744462⁶³

Additional COI, *Wolbachia* 16S gene GenBank accession numbers are listed in Table 4;

GenBank: *Wolbachia pipientis* isolate S1 glutamyl-tRNA(Gln)a midotransferase subunit B (gatB) mRNA, partial cds. Accession number OQ236162; https://identifiers.org/insdc:OQ236162⁶⁴

GenBank: *Wolbachia pipientis* isolate S1 cytochrome c oxidase subunit I (coxA) mRNA, partial cds. Accession number OQ236174; https://identifiers.org/insdc: OQ236174⁶⁵

Additional *Wolbachia* multi-locus sequence typing genes GenBank accession numbers are listed in Table 6.

## Extended data

Open Science Framework: Diverse Novel *Wolbachia* strains in Culicine mosquitoes from ecologically diverse regions of Cameroon, https://doi.org/10.17605/OSF.IO/V75DU

This project contains the raw PCR screening data.

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

## Acknowledgements

We thank staff and volunteers of OCEAC and VBID-RUBAE for their help with mosquito collections.
individual) cannot be ruled out. However, this would seem
unlikely given *Aedes* monospecific pools mosquitoes were either
co-infected (75%) or singly infected with *Wolbachia* (25%)
and for *Culex* 98% were infected with *Wolbachia* only. Larger
cohort collections of mosquitoes from diverse environmental
settings will provide further insight into how these two
widespread bacteria co-exist (or do not co-exist) in different
mosquito species.

Wolbachia MLST gene allelic profiling was undertaken to
provide more assurances on detection of genuine endosymbiotic
strains found in wild mosquito populations. We defined a ‘novel’
strain based on MLST to contain either new MLST gene sequences
not present in https://pubmlst.org/organisms/wolbachia-spp or a
combination of MLST gene sequences that does match an exist-
ing strain in the database. Despite being widely used, MLST
For *Wolbachia* strains has limitations and the five genes may
not represent the optimal loci to capture strain variation⁶¹. Fur-
thermore, defining whether a novel strain exists based only on
PCR amplification of genes is problematic given the numer-
ous examples of environmental contamination or host genome
integration⁵¹,⁵². Caution must be taken when extrapolating PCR
amplification to indicate the presence of a living endosymbiotic
bacterium – particularly so when only a few gene targets such
as 16S rRNA are amplified and sequenced. It has been shown
that 16S rRNA prokaryotic DNA can be amplified from dead
cells⁵¹ and *Wolbachia* 16S rRNA can be detected just from
water that previously contained mosquito larvae⁵². If possible,
extraction of mosquito RNA (as carried out in this study)
to confirm expression of *Wolbachia* genes provides further
evidence³. Once novel strains are detected using MLST pro-
filing, further studies are needed to confirm a genuine stable
endosymbiotic association is present with the mosquito host
species. This is important when low prevalence rates are
detected given this may otherwise suggest that the *Wolbachia*
strain is not inducing CI. Furthermore, there are several addi-
tional experiments that can be undertaken to provide further
confirmation of resident *Wolbachia* strains in mosquitoes.
Methods that can visualise *Wolbachia* bacteria in mosquito
tissues using microscopy, such as fluorescent *in situ* hybridi-
zation, and *Wolbachia* genome sequencing, to compare
genome depth and coverage of novel strains to those of other
known infections, should be carried out to fully characterise
novel *Wolbachia* strains.

Conclusions

Novel *Wolbachia* strains in Culicine mosquitoes collected
from ecologically diverse settings in Cameroon add to the
diversity of this highly prevalent endosymbiont in insect
populations. Resident *Wolbachia* strains should be further char-
acterised to determine the tissue tropism and density of newly

discovered strains. Our study also suggests that co-infection
with environmentally acquired *Asaia* bacteria is widespread in
wild mosquito populations (except the *Mansonia* genera) and
the antagonistic relationship observed in lab colonies may not
be present in some wild Culicine populations. Novel *Wolbachia*
strains could be considered as candidate strains for biocontrol
strategies given their ability to reside naturally within exist-
ing mosquito populations and co-exist with environmentally
acquired *Asaia* bacteria.

Data availability

Underlying data

GenBanK: Wolbachia endosymbiont of Culex pipiens isolate 1 16S ribosomal RNA gene, partial sequence. Accession number OP745953; https://identifiers.org/insdc:OP745953⁶²

GenBanK: Culex watti isolate 1 cytochrome c oxidase subunit I (COX1) gene, partial cds; mitochondrial. Accession number OP744462; https://identifiers.org/insdc:OP744462⁶³

Additional CO1, *Wolbachia* 16S gene GenBank accession numbers are listed in Table 4;

GenBank: Wolbachia pipientis isolate S1 glutamyl-tRNA(Gln)a midotransferase subunit B (gatB) mRNA, partial cds. Accession number OQ236162; https://identifiers.org/insdc:OQ236162⁶⁴

GenBank: Wolbachia pipientis isolate S1 cytochrome c oxi-
dase subunit I (coxA) mRNA, partial cds. Accession number
OQ236174; https://identifiers.org/insdc: OQ236174⁶⁵

Additional *Wolbachia* multi-locus sequence typing genes
GenBank accession numbers are listed in Table 6.

Extended data

Open Science Framework: Diverse Novel Wolbachia strains in Culicine mosquitoes from ecologically diverse regions of Cameroon, https://doi.org/10.17605/OSF.IO/V75DU

This project contains the raw PCR screening data.

Data are available under the terms of the [Creative Commons Zero "No rights reserved" data waiver](https://creativecommons.org/licenses/by/0.0) (CC0 1.0 Public domain dedication).

Acknowledgements

We thank staff and volunteers of OCEAC and VBID-RUBAE
for their help with mosquito collections.
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36. Braack L, de Almeida APG, Cornel AJ, et al.: Mosquito-borne arboviruses of African origin: review of key viruses and vectors. *Parasit Vectors*. 2018; 11(1): 29.

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37. Tchuandom SB, Tchouangue TF, Antonio-Nkondjio C, et al.: Seroprevalence of dengue virus among children presenting with febrile illness in some public health facilities in Cameroon. *Pan Afr Med J*. 2018; 31: 177.

PubMed Abstract | Publisher Full Text | Free Full Text

38. Mordecai EA, Ryan SJ, Caldwell JM, et al.: Climate change could shift disease burden from malaria to arboviruses in Africa. *Lancet Planet Health*. 2020; 4(9): e416–e423.

PubMed Abstract | Publisher Full Text | Free Full Text

39. Cornel AJ, Lee Y, Almeida APG, et al.: Mosquito community composition in South Africa and some neighboring countries. *Parasit Vectors*. 2018; 11(1): 331.

PubMed Abstract | Publisher Full Text | Free Full Text

40. Mayi MPA, Bamou R, Djiappi-Tchamen B, et al.: Habitat and Seasonality Affect *Mosquito Community Composition in the West Region of Cameroon*. *Insects*. 2020; 11(5): 312.

PubMed Abstract | Publisher Full Text | Free Full Text

41. Werren JH, Windsor DM: *Wolbachia* infection frequencies in insects: evidence of a global equilibrium? *Proc Biol Sci*. 2000; 267(1450): 1277–85.

PubMed Abstract | Publisher Full Text | Free Full Text

42. Zhou W, Rousset F, O'Neill S: Phylogeny and PCR-based classification of *Wolbachia* strains using wsp gene sequences. *Proc Biol Sci*. 1998; 265(1395):
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33. Hegde S, Khanipov K, Albayrak L, et al.: Microbiome Interaction Networks and Community Structure From Laboratory-Reared and Field-Collected *Aedes aegypti*, *Aedes albopictus*, and *Culex quinquefasciatus* Mosquito Vectors. *Front Microbiol*. 2018; 9: 2160.

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36. Braack L, de Almeida APG, Cornel AJ, et al.: Mosquito-borne arboviruses of African origin: review of key viruses and vectors. *Parasit Vectors*. 2018; 11(1): 29.

PubMed Abstract | Publisher Full Text | Free Full Text

37. Tchuandom SB, Tchouangue TF, Antonio-Nkondjio C, et al.: Seroprevalence of dengue virus among children presenting with febrile illness in some public health facilities in Cameroon. *Pan Afr Med J*. 2018; 31: 177.

PubMed Abstract | Publisher Full Text | Free Full Text

38. Mordecai EA, Ryan SJ, Caldwell JM, et al.: Climate change could shift disease burden from malaria to arboviruses in Africa. *Lancet Planet Health*. 2020; 4(9): e416–e423.

PubMed Abstract | Publisher Full Text | Free Full Text

39. Cornel AJ, Lee Y, Almeida APG, et al.: Mosquito community composition in South Africa and some neighboring countries. *Parasit Vectors*. 2018; 11(1): 331.

PubMed Abstract | Publisher Full Text | Free Full Text

40. Mayi MPA, Bamou R, Djiappi-Tchamen B, et al.: Habitat and Seasonality Affect *Mosquito Community Composition in the West Region of Cameroon*. *Insects*. 2020; 11(5): 312.

PubMed Abstract | Publisher Full Text | Free Full Text

41. Werren JH, Windsor DM: *Wolbachia* infection frequencies in insects: evidence of a global equilibrium? *Proc Biol Sci*. 2000; 267(1450): 1277–85.

PubMed Abstract | Publisher Full Text | Free Full Text

42. Zhou W, Rousset F, O'Neill S: Phylogeny and PCR-based classification of *Wolbachia* strains using wsp gene sequences. *Proc Biol Sci*. 1998; 265(1395):
509-15.

PubMed Abstract | Publisher Full Text | Free Full Text

43. Hill LA, Davis JB, Hapgood G, et al.: Rapid identification of *Aedes albopictus*, *Aedes scutellaris*, and *Aedes aegypti* life stages using real-time polymerase chain reaction assays. *Am J Trop Med Hyg.* 2008; **79**(6): 866-75.
PubMed Abstract | Publisher Full Text

44. Smith JL, Fonseca DM: Rapid assays for identification of members of the *Culex* (*Culex pipiens* complex, their hybrids, and other sibling species (*Diptera: culicidae*). *Am J Trop Med Hyg.* 2004; **70**(4): 339-45.
PubMed Abstract | Publisher Full Text

45. Folmer O, Black M, Hoeh W, et al.: DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. *Mol Mar Biol Biotechnol.* 1994; **3**(5): 294-9.
PubMed Abstract

46. Baldo L, Dunning Hotopp JC, Jolley KA, et al.: Multilocus sequence typing system for the endosymbiont *Wolbachia pipientis*. *Appl Environ Microbiol.* 2006; **72**(11): 7098-110.
PubMed Abstract | Publisher Full Text | Free Full Text

47. Kumar S, Stecher G, Li M, et al.: MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. *Mol Biol Evol.* 2018; **35**(6): 1547-1549.
PubMed Abstract | Publisher Full Text | Free Full Text

48. Jukes TH, Cantor CR: CHAPTER 24 - Evolution of Protein Molecules. In: Munro HN editor. *Mammalian Protein Metabolism*. Academic Press; 1969; 21-132.
Publisher Full Text

49. Nei M, Kumar S: Molecular Evolution and Phylogenetics. Oxford University Press ed, 2000.
Reference Source

50. Guindo-Coulibaly N, Adja AM, Coulibaly JT, et al.: Expansion of *Aedes africanus* (*Diptera: Culicidae*), a sylvatic vector of arboviruses, into an urban environment of Abidjan, Cote d'Ivoire. *J Vector Ecol.* 2019; **44**(2): 248-255.
PubMed Abstract | Publisher Full Text

51. Carini P, Marsden PJ, Leff JW, et al.: Relic DNA is abundant in soil and obscures estimates of soil microbial diversity. *Nat Microbiol.* 2016; **2**: 16242.
PubMed Abstract | Publisher Full Text

52. Chrostek E, Gerth M: *Is Anopheles gambiae* a Natural Host of *Wolbachia?*
*mBio*. 2019; **10**(3): e00784-19.
PubMed Abstract | Publisher Full Text | Free Full Text

53. Klasson L, Walker T, Sebaihia M, et al.: Genome evolution of *Wolbachia* strain *wPip* from the *Culex pipiens* group. *Mol Biol Evol.* 2008; **25**(9): 1877-87.
PubMed Abstract | Publisher Full Text | Free Full Text

54. Walker T, Klasson L, Sebaihia M, et al.: Ankyrin repeat domain-encoding

genes in the wPip strain of *Wolbachia* from the *Culex pipiens* group. *BMC
Biol.* 2007; 5: 39.
PubMed Abstract | Publisher Full Text | Free Full Text

55. Walker T, Song S, Sinkins SP: *Wolbachia* in the *Culex pipiens* group mosquitoes: introgression and superinfection. *J Hered.* 2009; **100**(2): 192-6.
PubMed Abstract | Publisher Full Text | Free Full Text

56. Dutton TJ, Sinkins SP: Strain-specific quantification of *Wolbachia* density in *Aedes albopictus* and effects of larval rearing conditions. *Insect Mol Biol.* 2004; **13**(3): 317-22.
PubMed Abstract | Publisher Full Text

57. Ughasi J, Bekard HE, Coulibaly M, et al.: *Mansonia africana* and *Mansonia uniformis* are vectors in the transmission of *Wuchereria bancrofti* lymphatic filariasis in Ghana. *Parasit Vectors.* 2012; **5**: 89.
PubMed Abstract | Publisher Full Text | Free Full Text

58. Damiani C, Cappelli A, Comandatore F, et al.: *Wolbachia* in *Aedes koreicus*: Rare Detections and Possible Implications. *Insects.* 2022; **13**(2): 216.
PubMed Abstract | Publisher Full Text | Free Full Text

59. Jourdain F, Picard M, Sulesco T, et al.: Identification of mosquitoes (Diptera: Culicidae): an external quality assessment of medical entomology laboratories in the MediLabSecure Network. *Parasit Vectors.* 2018; **11**(1): 553.
PubMed Abstract | Publisher Full Text | Free Full Text

60. Hegde S, Khanipov K, Albayrak L, et al.: Microbiome Interaction Networks and Community Structure From Laboratory-Reared and Field-Collected *Aedes aegypti*, *Aedes albopictus*, and *Culex quinquefasciatus* Mosquito Vectors. *Front Microbiol.* 2018; **9**: 2160.
PubMed Abstract | Publisher Full Text | Free Full Text

61. Bleidorn C, Gerth M: A critical re-evaluation of multilocus sequence typing (MLST) efforts in *Wolbachia*. *FEMS Microbiol Ecol.* 2018; **94**(1).
PubMed Abstract | Publisher Full Text

62. Walker, et al.: Wolbachia endosymbiont of *Culex pipiens* isolate 1 16S ribosomal RNA gene, partial sequence. GenBank [Dataset], Accession number OP745953, 2023.

63. Walker, et al.: *Culex watti* isolate 1 cytochrome c oxidase subunit I (COX1) gene, partial cds; mitochondrial. GenBank [Dataset], Accession number OP744462, 2023.

64. Walker, et al.: *Wolbachia pipientis* isolate S1 glutamyl-tRNA(Gln)amidotran sferase subunit B (gatB) mRNA, partial cds. GenBank [Dataset], Accession number OQ236162, 2023.

65. Walker, et al.: *Wolbachia pipientis* isolate S1 cytochrome c oxidase subunit I (*coxA*) mRNA, partial cds. GenBank [Dataset], Accession number OQ236174, 2023.
509-15.

PubMed Abstract | Publisher Full Text | Free Full Text

43. Hill LA, Davis JB, Hapgood G, et al.: Rapid identification of *Aedes albopictus*, *Aedes scutellaris*, and *Aedes aegypti* life stages using real-time polymerase chain reaction assays. *Am J Trop Med Hyg.* 2008; **79**(6): 866-75.
PubMed Abstract | Publisher Full Text

44. Smith JL, Fonseca DM: Rapid assays for identification of members of the *Culex* (*Culex pipiens* complex, their hybrids, and other sibling species (*Diptera: culicidae*). *Am J Trop Med Hyg.* 2004; **70**(4): 339-45.
PubMed Abstract | Publisher Full Text

45. Folmer O, Black M, Hoeh W, et al.: DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. *Mol Mar Biol Biotechnol.* 1994; **3**(5): 294-9.
PubMed Abstract

46. Baldo L, Dunning Hotopp JC, Jolley KA, et al.: Multilocus sequence typing system for the endosymbiont *Wolbachia pipientis*. *Appl Environ Microbiol.* 2006; **72**(11): 7098-110.
PubMed Abstract | Publisher Full Text | Free Full Text

47. Kumar S, Stecher G, Li M, et al.: MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. *Mol Biol Evol.* 2018; **35**(6): 1547-1549.
PubMed Abstract | Publisher Full Text | Free Full Text

48. Jukes TH, Cantor CR: CHAPTER 24 - Evolution of Protein Molecules. In: Munro HN editor. *Mammalian Protein Metabolism*. Academic Press; 1969; 21-132.
Publisher Full Text

49. Nei M, Kumar S: Molecular Evolution and Phylogenetics. Oxford University Press ed, 2000.
Reference Source

50. Guindo-Coulibaly N, Adja AM, Coulibaly JT, et al.: Expansion of *Aedes africanus* (*Diptera: Culicidae*), a sylvatic vector of arboviruses, into an urban environment of Abidjan, Cote d'Ivoire. *J Vector Ecol.* 2019; **44**(2): 248-255.
PubMed Abstract | Publisher Full Text

51. Carini P, Marsden PJ, Leff JW, et al.: Relic DNA is abundant in soil and obscures estimates of soil microbial diversity. *Nat Microbiol.* 2016; **2**: 16242.
PubMed Abstract | Publisher Full Text

52. Chrostek E, Gerth M: *Is Anopheles gambiae* a Natural Host of *Wolbachia?*
*mBio*. 2019; **10**(3): e00784-19.
PubMed Abstract | Publisher Full Text | Free Full Text

53. Klasson L, Walker T, Sebaihia M, et al.: Genome evolution of *Wolbachia* strain *wPip* from the *Culex pipiens* group. *Mol Biol Evol.* 2008; **25**(9): 1877-87.
PubMed Abstract | Publisher Full Text | Free Full Text

54. Walker T, Klasson L, Sebaihia M, et al.: Ankyrin repeat domain-encoding

genes in the wPip strain of *Wolbachia* from the *Culex pipiens* group. *BMC
Biol.* 2007; 5: 39.
PubMed Abstract | Publisher Full Text | Free Full Text

55. Walker T, Song S, Sinkins SP: *Wolbachia* in the *Culex pipiens* group mosquitoes: introgression and superinfection. *J Hered.* 2009; **100**(2): 192-6.
PubMed Abstract | Publisher Full Text | Free Full Text

56. Dutton TJ, Sinkins SP: Strain-specific quantification of *Wolbachia* density in *Aedes albopictus* and effects of larval rearing conditions. *Insect Mol Biol.* 2004; **13**(3): 317-22.
PubMed Abstract | Publisher Full Text

57. Ughasi J, Bekard HE, Coulibaly M, et al.: *Mansonia africana* and *Mansonia uniformis* are vectors in the transmission of *Wuchereria bancrofti* lymphatic filariasis in Ghana. *Parasit Vectors.* 2012; **5**: 89.
PubMed Abstract | Publisher Full Text | Free Full Text

58. Damiani C, Cappelli A, Comandatore F, et al.: *Wolbachia* in *Aedes koreicus*: Rare Detections and Possible Implications. *Insects.* 2022; **13**(2): 216.
PubMed Abstract | Publisher Full Text | Free Full Text

59. Jourdain F, Picard M, Sulesco T, et al.: Identification of mosquitoes (Diptera: Culicidae): an external quality assessment of medical entomology laboratories in the MediLabSecure Network. *Parasit Vectors.* 2018; **11**(1): 553.
PubMed Abstract | Publisher Full Text | Free Full Text

60. Hegde S, Khanipov K, Albayrak L, et al.: Microbiome Interaction Networks and Community Structure From Laboratory-Reared and Field-Collected *Aedes aegypti*, *Aedes albopictus*, and *Culex quinquefasciatus* Mosquito Vectors. *Front Microbiol.* 2018; **9**: 2160.
PubMed Abstract | Publisher Full Text | Free Full Text

61. Bleidorn C, Gerth M: A critical re-evaluation of multilocus sequence typing (MLST) efforts in *Wolbachia*. *FEMS Microbiol Ecol.* 2018; **94**(1).
PubMed Abstract | Publisher Full Text

62. Walker, et al.: Wolbachia endosymbiont of *Culex pipiens* isolate 1 16S ribosomal RNA gene, partial sequence. GenBank [Dataset], Accession number OP745953, 2023.

63. Walker, et al.: *Culex watti* isolate 1 cytochrome c oxidase subunit I (COX1) gene, partial cds; mitochondrial. GenBank [Dataset], Accession number OP744462, 2023.

64. Walker, et al.: *Wolbachia pipientis* isolate S1 glutamyl-tRNA(Gln)amidotran sferase subunit B (gatB) mRNA, partial cds. GenBank [Dataset], Accession number OQ236162, 2023.

65. Walker, et al.: *Wolbachia pipientis* isolate S1 cytochrome c oxidase subunit I (*coxA*) mRNA, partial cds. GenBank [Dataset], Accession number OQ236174, 2023.
# Open Peer Review

Current Peer Review Status: ✓ ✓ ✓

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

## Version 2

Reviewer Report 10 October 2023

https://doi.org/10.21956/wellcomeopenres.22165.r67662

© 2023 Caragata E. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_20.png" /></figure>

### ✓ Eric P. Caragata

<figure><img src="image_21.png" /></figure>

Florida Medical Entomology Laboratory, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA

All of my previous comments have been addressed to my satisfaction. The only other note I want to give is that it could be beneficial to explicitly delineate methodological differences in the handling of Western and Central region mosquitoes in the methods section.

**Competing Interests:** No competing interests were disclosed.

**Reviewer Expertise:** Mosquito microbiology, molecular biology, immunity, and metabolism. Wolbachia. Mosquito-microbe-pathogen interactions.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Reviewer Report 06 October 2023

https://doi.org/10.21956/wellcomeopenres.22165.r67661

© 2023 Favia G. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_22.png" /></figure>

### ✓ Guido Favia

School of Bioscience and Veterinary Medicine, University of Camerino, Camerino, Italy

I have read the revised version of the manuscript and in my opinion, the authors improved the quality well addressing the concerns raised at the first evaluation. Consequently the manuscript is worthy for indexing.
# Open Peer Review

Current Peer Review Status: ✓ ✓ ✓

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

## Version 2

Reviewer Report 10 October 2023

https://doi.org/10.21956/wellcomeopenres.22165.r67662

© 2023 **Caragata** E. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_21.png" /></figure>

### ✓ Eric P. Caragata

<figure><img src="image_22.png" /></figure>

Florida Medical Entomology Laboratory, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA

All of my previous comments have been addressed to my satisfaction. The only other note I want to give is that it could be beneficial to explicitly delineate methodological differences in the handling of Western and Central region mosquitoes in the methods section.

**Competing Interests:** No competing interests were disclosed.

**Reviewer Expertise:** Mosquito microbiology, molecular biology, immunity, and metabolism. Wolbachia. Mosquito-microbe-pathogen interactions.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Reviewer Report 06 October 2023

https://doi.org/10.21956/wellcomeopenres.22165.r67661

© 2023 **Favia** G. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_23.png" /></figure>

### ✓ Guido Favia

School of Bioscience and Veterinary Medicine, University of Camerino, Camerino, Italy

I have read the revised version of the manuscript and in my opinion, the authors improved the quality well addressing the concerns raised at the first evaluation. Consequently the manuscript is worthy for indexing.
**Competing Interests:** No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Reviewer Report 03 October 2023

https://doi.org/10.21956/wellcomeopenres.22165.r67660

© 2023 Ross P. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_23.png" /></figure>

Perran Ross

<figure><img src="image_24.png" /></figure>

The University of Melbourne, Melbourne, Victoria, Australia

The authors have substantially revised the manuscript and all issues raised have been sufficiently addressed. I have no further suggestions.

**Competing Interests:** I am currently collaborating on a project with a lab from the Liverpool School of Tropical Medicine of which a coauthor of this paper is currently a member. The project is unrelated to the current manuscript and I have not worked with any of the other authors, so I don't believe this will affect the objectivity of my review.

**Reviewer Expertise:** Wolbachia infections in Aedes mosquitoes and their use in control programs

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Version 1

Reviewer Report 01 August 2023

https://doi.org/10.21956/wellcomeopenres.20603.r61513

© 2023 Ross P. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_25.png" /></figure>

Perran Ross

<figure><img src="image_26.png" /></figure>

The University of Melbourne, Melbourne, Victoria, Australia

**General comments**
**Competing Interests:** No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Reviewer Report 03 October 2023

https://doi.org/10.21956/wellcomeopenres.22165.r67660

© 2023 Ross P. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_24.png" /></figure>

Perran Ross

<figure><img src="image_25.png" /></figure>

The University of Melbourne, Melbourne, Victoria, Australia

The authors have substantially revised the manuscript and all issues raised have been sufficiently addressed. I have no further suggestions.

**Competing Interests:** I am currently collaborating on a project with a lab from the Liverpool School of Tropical Medicine of which a coauthor of this paper is currently a member. The project is unrelated to the current manuscript and I have not worked with any of the other authors, so I don't believe this will affect the objectivity of my review.

**Reviewer Expertise:** Wolbachia infections in Aedes mosquitoes and their use in control programs

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Version 1

Reviewer Report 01 August 2023

https://doi.org/10.21956/wellcomeopenres.20603.r61513

© 2023 Ross P. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_26.png" /></figure>

Perran Ross

<figure><img src="image_27.png" /></figure>

The University of Melbourne, Melbourne, Victoria, Australia

**General comments**
Wolbachia endosymbionts are prevalent in insect populations and can enact dramatic effects on their hosts. Understanding the prevalence and diversity of Wolbachia in natural mosquito populations has important implications for the control of mosquito-borne disease because Wolbachia infections can influence mosquito vectorial capacity and modulate the success of vector control programs. Other microbes such as Asaia can also reside in mosquito tissues where they can influence host phenotypes and interact with Wolbachia. Here, the authors performed field surveys of Culicine mosquitoes in two regions of Cameroon and screen either individuals or pools of mosquitoes for Wolbachia and Asaia using PCR detection and sequencing of 16S rRNA, wsp and MLST markers. They detect Wolbachia and Asaia at variable frequencies which depend on the mosquito genus and region. While these results provide important data for vector control programs and may lead to candidate strains for Wolbachia release programs, there are a few key limitations with their study design and their conclusions are overstated.

The authors claim to detect co-infections of Wolbachia and Asaia in field-collected mosquitoes, contradicting evidence from laboratory studies that the two microbes have an antagonistic relationship with each other. However, these two observations are not necessarily in conflict. While there may be mutual exclusion of Asaia and Wolbachia in the reproductive tissues of some mosquitoes, this does not preclude the two microbes from residing in the same mosquito but within different tissues. There are several cases where Wolbachia and Asaia have been detected in the same mosquito (e.g. Schrieke et al. 2022 Computational and Structural Biotechnology ¹, Hegde et al. 2018 Frontiers in Microbiology ², da Silva et al. 2022 genes ³, Chen et al. 2020 Frontiers in Microbiology ⁴) but they may not be occupying the same tissues, especially given that Asaia can be environmentally acquired and often resides in the gut. This study also does not provide sufficient evidence for a Wolbachia-Asaia coinfection. Firstly, all samples where both Wolbachia and Asaia were detected are from pools of mosquitoes, so it is possible that these samples contain a mix of singly infected individuals. Secondly, molecular detection is not sufficient evidence of an infection as pointed out by previous studies (Chrostek and Gerth 2019 mBio ⁵, Ross et al. 2020 Ecol Evol ⁶). This is acknowledged at the end of the discussion but the rest of the paper is written as if all these detections represent true infections.

There are also some limitations and apparent gaps/inconsistencies in their study design. Mosquitoes from the West region were pooled while mosquitoes from the Central region were tested individually, but mosquitoes were only identified to the species level in the West region. The use of pools is likely to overestimate the prevalence of Wolbachia and Asaia in a sample given that not all individuals need to test positive for the pool to be considered positive. There is also the risk that any errors with morphological species identification could lead to an incorrect assignment of Wolbachia or Asaia status of a pool if one species is positive and the other is negative. The authors also use a mix of 16S rRNA and wsp detection for Wolbachia, but the data for 16S are combined across species within a genus while the wsp data are only presented for individual species. Furthermore, the results for 16S include only Aedes, Mansonia and Culex while there are other genera included for the wsp marker. The justification for this is unclear, and it would be useful to know how concordant the two markers are with each other, especially when the authors note that the use of the 16S rRNA gene has limitations for Wolbachia detection. The authors also mention in the methods that they also used the FtsZ marker for initial Wolbachia detection but these results are not presented.

The authors then select a sample of mosquito species testing positive for Wolbachia for further
Wolbachia endosymbionts are prevalent in insect populations and can enact dramatic effects on their hosts. Understanding the prevalence and diversity of Wolbachia in natural mosquito populations has important implications for the control of mosquito-borne disease because Wolbachia infections can influence mosquito vectorial capacity and modulate the success of vector control programs. Other microbes such as Asaia can also reside in mosquito tissues where they can influence host phenotypes and interact with Wolbachia. Here, the authors performed field surveys of Culicine mosquitoes in two regions of Cameroon and screen either individuals or pools of mosquitoes for Wolbachia and Asaia using PCR detection and sequencing of 16S rRNA, wsp and MLST markers. They detect Wolbachia and Asaia at variable frequencies which depend on the mosquito genus and region. While these results provide important data for vector control programs and may lead to candidate strains for Wolbachia release programs, there are a few key limitations with their study design and their conclusions are overstated.

The authors claim to detect co-infections of Wolbachia and Asaia in field-collected mosquitoes, contradicting evidence from laboratory studies that the two microbes have an antagonistic relationship with each other. However, these two observations are not necessarily in conflict. While there may be mutual exclusion of Asaia and Wolbachia in the reproductive tissues of some mosquitoes, this does not preclude the two microbes from residing in the same mosquito but within different tissues. There are several cases where Wolbachia and Asaia have been detected in the same mosquito (e.g. Schrieke et al. 2022 Computational and Structural Biotechnology ¹, Hegde et al. 2018 Frontiers in Microbiology ², da Silva et al. 2022 genes ³, Chen et al. 2020 Frontiers in Microbiology ⁴) but they may not be occupying the same tissues, especially given that Asaia can be environmentally acquired and often resides in the gut. This study also does not provide sufficient evidence for a Wolbachia-Asaia coinfection. Firstly, all samples where both Wolbachia and Asaia were detected are from pools of mosquitoes, so it is possible that these samples contain a mix of singly infected individuals. Secondly, molecular detection is not sufficient evidence of an infection as pointed out by previous studies (Chrostek and Gerth 2019 mBio ⁵, Ross et al. 2020 Ecol Evol ⁶). This is acknowledged at the end of the discussion but the rest of the paper is written as if all these detections represent true infections.

There are also some limitations and apparent gaps/inconsistencies in their study design. Mosquitoes from the West region were pooled while mosquitoes from the Central region were tested individually, but mosquitoes were only identified to the species level in the West region. The use of pools is likely to overestimate the prevalence of Wolbachia and Asaia in a sample given that not all individuals need to test positive for the pool to be considered positive. There is also the risk that any errors with morphological species identification could lead to an incorrect assignment of Wolbachia or Asaia status of a pool if one species is positive and the other is negative. The authors also use a mix of 16S rRNA and wsp detection for Wolbachia, but the data for 16S are combined across species within a genus while the wsp data are only presented for individual species. Furthermore, the results for 16S include only Aedes, Mansonia and Culex while there are other genera included for the wsp marker. The justification for this is unclear, and it would be useful to know how concordant the two markers are with each other, especially when the authors note that the use of the 16S rRNA gene has limitations for Wolbachia detection. The authors also mention in the methods that they also used the FtsZ marker for initial Wolbachia detection but these results are not presented.

The authors then select a sample of mosquito species testing positive for Wolbachia for further
analysis of the Wolbachia strains with 16S rRNA and MLST markers. The authors place an emphasis on the fact that the Wolbachia strains that they detect are novel and diverse but these terms are not well defined. Has Wolbachia been detected in any of these species besides Cx. quinquefasciatus before, and how different do Wolbachia sequences have to be before they are considered novel and diverse? The authors compared their sequences to the MLST database but this may not represent the full diversity of Wolbachia strains that have been identified given that Wolbachia sequences have been obtained through other approaches (e.g. Scholz et al. Nature Communications). There are also some issues with the use of the MLST system for strain typing and diversity as outlined by Bleidorn and Gerth 2018 (FEMS Microbiol Ecol) ⁷ and these should be acknowledged.

**Specific comments**

**Title** – The title is misleading – the authors have detected Wolbachia and Asaia sequences in a range of field-collected mosquito species, but there is insufficient evidence that these represent true infections. The terms “diverse” and “novel” are not well defined and it is unclear how many of these strains have been detected for the first time.

**Introduction** – the authors mention that identifying Wolbachia strains can provide important comparative data but they don't elaborate on how this information would be useful for biocontrol strategies. For instance, natural infections could interfere with releases of transinfections because they may change patterns of cytoplasmic incompatibility between the released and resident mosquitoes. The authors also note that novel strains could be considered candidates for biocontrol strategies, but what properties are you looking for in a strain for it to be considered a candidate, and what steps would need to be taken for the strain to be used (e.g. introgression or microinjection into a different mosquito line, virus blocking, cytoplasmic incompatibility and so on)?

**Introduction paragraph 1** – References 11 and 12 refer to Ae. polynesiensis, not Ae. albopictus.

**Introduction paragraph 1** – There is an emphasis on wMel here specifically, but also consider including the wAlbB strain which was the first transinfected strain in Ae. aegypti (Xi et al. 2005, Science) and which has also been released in wild mosquito populations to inhibit virus transmission (Nazni et al. 2019 Current Biology).

**Introduction paragraph 2** – “However, a more targeted approach amplifying *Wolbachia*-specific genes is required to confirm a resident strain is present” – This really requires additional evidence such as microscopy, removal of the infection, demonstration of maternal transmission and so on.

**Introduction paragraph 2** – provide an example of a superinfection comprising strains from the same supergroup

**Introduction paragraph 3** – “outbreaks of arboviral diseases including DENV, YFV, CHIKV and Rift Valley fever virus (RVFV) are a possibility.” is a bit vague – do they occur and how frequently?

**Methods** - How many mosquitoes per pool? In the results you mention an average of 5 female mosquitoes, but how much did this vary?
analysis of the Wolbachia strains with 16S rRNA and MLST markers. The authors place an
emphasis on the fact that the Wolbachia strains that they detect are novel and diverse but these
terms are not well defined. Has Wolbachia been detected in any of these species besides Cx.
quinquefasciatus before, and how different do Wolbachia sequences have to be before they are
considered novel and diverse? The authors compared their sequences to the MLST database but
this may not represent the full diversity of Wolbachia strains that have been identified given that
Wolbachia sequences have been obtained through other approaches (e.g. Scholz et al. Nature
Communications). There are also some issues with the use of the MLST system for strain typing
and diversity as outlined by Bleidorn and Gerth 2018 (FEMS Microbiol Ecol) ⁷ and these should be
acknowledged.

**<u>Specific comments</u>**

**Title** – The title is misleading – the authors have detected Wolbachia and Asaia sequences in a range of field-collected mosquito species, but there is insufficient evidence that these represent true infections. The terms “diverse” and “novel” are not well defined and it is unclear how many of these strains have been detected for the first time.

**Introduction** – the authors mention that identifying Wolbachia strains can provide important comparative data but they don't elaborate on how this information would be useful for biocontrol strategies. For instance, natural infections could interfere with releases of transinfections because they may change patterns of cytoplasmic incompatibility between the released and resident mosquitoes. The authors also note that novel strains could be considered candidates for biocontrol strategies, but what properties are you looking for in a strain for it to be considered a candidate, and what steps would need to be taken for the strain to be used (e.g. introgression or microinjection into a different mosquito line, virus blocking, cytoplasmic incompatibility and so on)?

**Introduction paragraph 1** – References 11 and 12 refer to Ae. polynesiensis, not Ae. albopictus.

**Introduction paragraph 1** – There is an emphasis on wMel here specifically, but also consider
including the wAlbB strain which was the first transinfected strain in Ae. aegypti (Xi et al. 2005,
Science) and which has also been released in wild mosquito populations to inhibit virus
transmission (Nazni et al. 2019 Current Biology).

**Introduction paragraph 2** – “However, a more targeted approach amplifying *Wolbachia*-specific genes is required to confirm a resident strain is present” – This really requires additional evidence such as microscopy, removal of the infection, demonstration of maternal transmission and so on.

**Introduction paragraph 2** – provide an example of a superinfection comprising strains from the
same supergroup

**Introduction paragraph 3** – “outbreaks of arboviral diseases including DENV, YFV, CHIKV and Rift Valley fever virus (RVFV) are a possibility.” is a bit vague – do they occur and how frequently?

**Methods** - How many mosquitoes per pool? In the results you mention an average of 5 female mosquitoes, but how much did this vary?
**Methods** – sanger sequencing – which PCR products were considered to be worthy of sequencing and why?

**Results paragraph 1** – When presenting West region results, do these n values represent the number of pools? Or were these actually individuals?

**Results paragraph 1** – What is the hypothesis being tested with the Fisher's exact tests? Please elaborate on what associations are being tested and include these statistical tests in the methods section

**Results** – There appear to be some inconsistencies when presenting frequency data- sometimes samples from the West region are specifically referred to as pools while at other times there is no mention of them being individuals or pools (e.g. in the first paragraph of the results).

**Table 1** – results for Wolbachia 16S are reported, but what about other genes? Were they concordant with the 16S results?

**Table 1** – What is the Fisher test comparing?

**Table 1** – Although mentioned as a caveat in the text, it is misleading to put "co-infection" for the West samples when these consist of pools of mosquitoes – it is plausible that pools could consist of singly infected Wolbachia and Asaia individuals

**Table 2** – Only the last three rows indicate that pools were tested, but aren't all samples in this table pools?

**Figure 1** – Please provide the 16S data by species to allow for a comparison with the wsp data

**Results** - "Wolbachia 16S rRNA sequences has previously been detected in water containers that contained larvae of mosquitoes" - Is this not also possible for the other genes?

**Discussion** – What makes a strain a candidate? Is any newly detected strain a candidate strain, or do they need to display a certain phenotype such as cytoplasmic incompatibility or virus blocking? In my opinion a candidate strain must possess some desirable traits and should be able to be maintained in the laboratory.

**References**

1. Schrieke H, Maignien L, Constancias F, Trigodet F, et al.: The mosquito microbiome includes habitat-specific but rare symbionts. *Comput Struct Biotechnol J.* 2022; **20**: 410-420 PubMed Abstract | **Publisher Full Text**

2. Hegde S, Khanipov K, Albayrak L, Golovko G, et al.: Microbiome Interaction Networks and Community Structure From Laboratory-Reared and Field-Collected Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus Mosquito Vectors. *Front Microbiol.* 2018; **9**: 2160 PubMed Abstract | **Publisher Full Text**

3. da Silva H, Oliveira TMP, Sallum MAM: Bacterial Community Diversity and Bacterial Interaction Network in Eight Mosquito Species. *Genes (Basel)*. 2022; **13** (11). PubMed Abstract | **Publisher Full Text**

4. Chen S, Zhang D, Augustinos A, Doudoumis V, et al.: Multiple Factors Determine the Structure of
**Methods** – sanger sequencing – which PCR products were considered to be worthy of sequencing and why?

**Results paragraph 1** – When presenting West region results, do these n values represent the number of pools? Or were these actually individuals?

**Results paragraph 1** – What is the hypothesis being tested with the Fisher's exact tests? Please elaborate on what associations are being tested and include these statistical tests in the methods section

**Results** – There appear to be some inconsistencies when presenting frequency data- sometimes samples from the West region are specifically referred to as pools while at other times there is no mention of them being individuals or pools (e.g. in the first paragraph of the results).

**Table 1** – results for Wolbachia 16S are reported, but what about other genes? Were they concordant with the 16S results?

**Table 1** – What is the Fisher test comparing?

**Table 1** – Although mentioned as a caveat in the text, it is misleading to put "co-infection" for the West samples when these consist of pools of mosquitoes – it is plausible that pools could consist of singly infected Wolbachia and Asaia individuals

**Table 2** – Only the last three rows indicate that pools were tested, but aren't all samples in this table pools?

**Figure 1** – Please provide the 16S data by species to allow for a comparison with the wsp data

**Results** - "Wolbachia 16S rRNA sequences has previously been detected in water containers that contained larvae of mosquitoes" - Is this not also possible for the other genes?

**Discussion** – What makes a strain a candidate? Is any newly detected strain a candidate strain, or do they need to display a certain phenotype such as cytoplasmic incompatibility or virus blocking? In my opinion a candidate strain must possess some desirable traits and should be able to be maintained in the laboratory.

**References**

1. Schrieke H, Maignien L, Constancias F, Trigodet F, et al.: The mosquito microbiome includes habitat-specific but rare symbionts. *Comput Struct Biotechnol J.* 2022; **20**: 410-420 PubMed Abstract | **Publisher Full Text**

2. Hegde S, Khanipov K, Albayrak L, Golovko G, et al.: Microbiome Interaction Networks and Community Structure From Laboratory-Reared and Field-Collected Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus Mosquito Vectors. *Front Microbiol.* 2018; **9**: 2160 PubMed Abstract | **Publisher Full Text**

3. da Silva H, Oliveira TMP, Sallum MAM: Bacterial Community Diversity and Bacterial Interaction Network in Eight Mosquito Species. *Genes (Basel)*. 2022; **13** (11). PubMed Abstract | **Publisher Full Text**

4. Chen S, Zhang D, Augustinos A, Doudoumis V, et al.: Multiple Factors Determine the Structure of
Bacterial Communities Associated With Aedes albopictus Under Artificial Rearing Conditions. *Front Microbiol.* 2020; **11**: 605 PubMed Abstract | Publisher Full Text

5. Chrostek E, Gerth M: Is Anopheles gambiae a Natural Host of Wolbachia?. *mBio*. 2019; **10** (3). PubMed Abstract | Publisher Full Text

6. Ross PA, Callahan AG, Yang Q, Jasper M, et al.: An elusive endosymbiont: Does Wolbachia occur naturally in Aedes aegypti?. *Ecol Evol.* 2020; **10** (3): 1581-1591 PubMed Abstract | Publisher Full Text

7. Bleidorn C, Gerth M: A critical re-evaluation of multilocus sequence typing (MLST) efforts in Wolbachia. *FEMS Microbiol Ecol.* 2018; **94** (1). PubMed Abstract | Publisher Full Text

**Is the work clearly and accurately presented and does it cite the current literature?**

Partly

**Is the study design appropriate and is the work technically sound?**

Partly

**Are sufficient details of methods and analysis provided to allow replication by others?**

Partly

**If applicable, is the statistical analysis and its interpretation appropriate?**

Partly

**Are all the source data underlying the results available to ensure full reproducibility?**

Partly

**Are the conclusions drawn adequately supported by the results?**

Partly

**Competing Interests:** I am currently collaborating on a project with a lab from the Liverpool School of Tropical Medicine of which a coauthor of this paper is currently a member. The project is unrelated to the current manuscript and I have not worked with any of the other authors, so I don't believe this will affect the objectivity of my review.

**Reviewer Expertise:** Wolbachia infections in Aedes mosquitoes and their use in control programs

**I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.**

Author Response 04 Sep 2023

**Thomas Walker**

Dear Perran, **Thank you so much for your very comprehensive review. We have addressed your comments as follows with responses in bold: Wolbachia endosymbionts are prevalent in insect populations and can enact dramatic effects on their hosts. Understanding the prevalence and diversity of Wolbachia in natural mosquito populations**
Bacterial Communities Associated With Aedes albopictus Under Artificial Rearing Conditions. *Front Microbiol.* 2020; **11**: 605 PubMed Abstract | Publisher Full Text

5. Chrostek E, Gerth M: Is Anopheles gambiae a Natural Host of Wolbachia?. *mBio*. 2019; **10** (3). PubMed Abstract | Publisher Full Text

6. Ross PA, Callahan AG, Yang Q, Jasper M, et al.: An elusive endosymbiont: Does Wolbachia occur naturally in Aedes aegypti?. *Ecol Evol.* 2020; **10** (3): 1581-1591 PubMed Abstract | Publisher Full Text

7. Bleidorn C, Gerth M: A critical re-evaluation of multilocus sequence typing (MLST) efforts in Wolbachia. *FEMS Microbiol Ecol.* 2018; **94** (1). PubMed Abstract | Publisher Full Text

**Is the work clearly and accurately presented and does it cite the current literature?**

Partly

**Is the study design appropriate and is the work technically sound?**

Partly

**Are sufficient details of methods and analysis provided to allow replication by others?**

Partly

**If applicable, is the statistical analysis and its interpretation appropriate?**

Partly

**Are all the source data underlying the results available to ensure full reproducibility?**

Partly

**Are the conclusions drawn adequately supported by the results?**

Partly

**Competing Interests:** I am currently collaborating on a project with a lab from the Liverpool School of Tropical Medicine of which a coauthor of this paper is currently a member. The project is unrelated to the current manuscript and I have not worked with any of the other authors, so I don't believe this will affect the objectivity of my review.

**Reviewer Expertise:** Wolbachia infections in Aedes mosquitoes and their use in control programs

**I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.**

Author Response 04 Sep 2023

**Thomas Walker**

Dear Perran, **Thank you so much for your very comprehensive review. We have addressed your comments as follows with responses in bold: Wolbachia endosymbionts are prevalent in insect populations and can enact dramatic effects on their hosts. Understanding the prevalence and diversity of Wolbachia in natural mosquito populations**
has important implications for the control of mosquito-borne disease because Wolbachia infections can influence mosquito vectorial capacity and modulate the success of vector control programs. Other microbes such as Asaia can also reside in mosquito tissues where they can influence host phenotypes and interact with Wolbachia. Here, the authors performed field surveys of Culicine mosquitoes in two regions of Cameroon and screen either individuals or pools of mosquitoes for Wolbachia and Asaia using PCR detection and sequencing of 16S rRNA, wsp and MLST markers. They detect Wolbachia and Asaia at variable frequencies which depend on the mosquito genus and region. While these results provide important data for vector control programs and may lead to candidate strains for Wolbachia release programs, there are a few key limitations with their study design and their conclusions are overstated.

The authors claim to detect co-infections of Wolbachia and Asaia in field-collected mosquitoes, contradicting evidence from laboratory studies that the two microbes have an antagonistic relationship with each other. However, these two observations are not necessarily in conflict. While there may be mutual exclusion of Asaia and Wolbachia in the reproductive tissues of some mosquitoes, this does not preclude the two microbes from residing in the same mosquito but within different tissues. There are several cases where Wolbachia and Asaia have been detected in the same mosquito (e.g. Schrieke et al. 2022 Computational and Structural Biotechnology ¹, Hegde et al. 2018 Frontiers in Microbiology ², da Silva et al. 2022 genes ³, Chen et al. 2020 Frontiers in Microbiology ⁴) but they may not be occupying the same tissues, especially given that Asaia can be environmentally acquired and often resides in the gut. **Thank you for these points. We have expanded paragraph 4 of our discussion to provide a more balanced view on the relationship between Wolbachia and Asaia.** This study also does not provide sufficient evidence for a Wolbachia-Asaia coinfection. Firstly, all samples where both Wolbachia and Asaia were detected are from pools of mosquitoes, so it is possible that these samples contain a mix of singly infected individuals. **We have added a caveat to our discussion on 'pooling' mosquitoes (paragraph 4) but given that you have pointed out that 'there are several cases where Wolbachia and Asaia have been detected in the same mosquito' it would be contradictory to those studies to assume a coinfection is not possible. Unfortunately these collections were undertaken during the covid pandemic so a pooling approach was taken to minimise time in labs under severe travel restrictions. Our data from the West region (Aedes monospecific pools mosquitoes were either co-infected (75%) or singly infected with Wolbachia (25%) and for Culex 98% were infected with Wolbachia only) would also indicate a mix of singly infected individuals is not likely to represent most of our data.** Secondly, molecular detection is not sufficient evidence of an infection as pointed out by previous studies (Chrostek and Gerth 2019 mBio ⁵, Ross et al. 2020 Ecol Evol ⁶). This is acknowledged at the end of the discussion but the rest of the paper is written as if all these detections represent true infections. **We agree there have been many recent papers that have just presented molecular data without providing further evidence but we feel our manuscript provides sufficient caveats to justify our results as more than likely coming from genuine infections (paragraphs 4 and 5 of the discussion). We acknowledge that reliance on just one Wolbachia gene (eg. 16S rRNA or wsp) would not be sufficient as has been the case for numerous recent publications. However, having multiple MLST gene sequences from Wolbachia housekeeping genes that match (or are very close in sequence) to existing sequencing in
has important implications for the control of mosquito-borne disease because Wolbachia infections can influence mosquito vectorial capacity and modulate the success of vector control programs. Other microbes such as Asaia can also reside in mosquito tissues where they can influence host phenotypes and interact with Wolbachia. Here, the authors performed field surveys of Culicine mosquitoes in two regions of Cameroon and screen either individuals or pools of mosquitoes for Wolbachia and Asaia using PCR detection and sequencing of 16S rRNA, wsp and MLST markers. They detect Wolbachia and Asaia at variable frequencies which depend on the mosquito genus and region. While these results provide important data for vector control programs and may lead to candidate strains for Wolbachia release programs, there are a few key limitations with their study design and their conclusions are overstated.

The authors claim to detect co-infections of Wolbachia and Asaia in field-collected mosquitoes, contradicting evidence from laboratory studies that the two microbes have an antagonistic relationship with each other. However, these two observations are not necessarily in conflict. While there may be mutual exclusion of Asaia and Wolbachia in the reproductive tissues of some mosquitoes, this does not preclude the two microbes from residing in the same mosquito but within different tissues. There are several cases where Wolbachia and Asaia have been detected in the same mosquito (e.g. Schrieke et al. 2022 Computational and Structural Biotechnology ¹, Hegde et al. 2018 Frontiers in Microbiology ², da Silva et al. 2022 genes ³, Chen et al. 2020 Frontiers in Microbiology ⁴) but they may not be occupying the same tissues, especially given that Asaia can be environmentally acquired and often resides in the gut. **Thank you for these points. We have expanded paragraph 4 of our discussion to provide a more balanced view on the relationship between Wolbachia and Asaia.** This study also does not provide sufficient evidence for a Wolbachia-Asaia coinfection. Firstly, all samples where both Wolbachia and Asaia were detected are from pools of mosquitoes, so it is possible that these samples contain a mix of singly infected individuals. **We have added a caveat to our discussion on 'pooling' mosquitoes (paragraph 4) but given that you have pointed out that 'there are several cases where Wolbachia and Asaia have been detected in the same mosquito' it would be contradictory to those studies to assume a coinfection is not possible. Unfortunately these collections were undertaken during the covid pandemic so a pooling approach was taken to minimise time in labs under severe travel restrictions. Our data from the West region (Aedes monospecific pools mosquitoes were either co-infected (75%) or singly infected with Wolbachia (25%) and for Culex 98% were infected with Wolbachia only) would also indicate a mix of singly infected individuals is not likely to represent most of our data.** Secondly, molecular detection is not sufficient evidence of an infection as pointed out by previous studies (Chrostek and Gerth 2019 mBio ⁵, Ross et al. 2020 Ecol Evol ⁶). This is acknowledged at the end of the discussion but the rest of the paper is written as if all these detections represent true infections. **We agree there have been many recent papers that have just presented molecular data without providing further evidence but we feel our manuscript provides sufficient caveats to justify our results as more than likely coming from genuine infections (paragraphs 4 and 5 of the discussion). We acknowledge that reliance on just one Wolbachia gene (eg. 16S rRNA or wsp) would not be sufficient as has been the case for numerous recent publications. However, having multiple MLST gene sequences from Wolbachia housekeeping genes that match (or are very close in sequence) to existing sequencing in