# Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

Claire L. Jeffries, Gena G. Lawrence, George Golovko, Mojca Kristan, James Orsborne, Kirstin Spence, Eliot Hurn, Janvier Bandibabone, Luciano M Tantely, Fara Nantenaina Raharimalala, et al.

► To cite this version:

Claire L. Jeffries, Gena G. Lawrence, George Golovko, Mojca Kristan, James Orsborne, et al. Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa. Wellcome Open Research, 2018, 3, pp.113. 10.12688/wellcomeopenres.14765.2 . pasteur-01967418

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RESEARCH ARTICLE

**REVISED** **Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa [version 2; referees: 3 approved]**

Claire L. Jeffries<fnref n="1" />, Gena G. Lawrence<fnref n="2" />, George Golovko<fnref n="3" />, Mojca Kristan<fnref n="1" />, James Orsborne<fnref n="1" />, Kirstin Spence<fnref n="1" />, Eliot Hurn<fnref n="1" />, Janvier Bandibabone<fnref n="1" />, Luciano M. Tantely<fnref n="1" />, Fara N. Raharimalala<fnref n="1" />, Kalil Keita<fnref n="6" />, Denka Camara<fnref n="6" />, Yaya Barry<fnref n="6" />, Francis Wat'senga<fnref n="7" />, Emile Z. Manzambi<fnref n="7" />, Yaw A. Afrane<fnref n="1" />, Abdul R. Mohammed<fnref n="1" />, Tarekegn A. Abeku<fnref n="9" />, Shivanand Hegde<fnref n="10" />, Kamil Khanipov<fnref n="1" />, Maria Pimenova<fnref n="3" />, Yuriy Fofanov<fnref n="3" />, Sebastien Boyer<fnref n="1" />, Seth R. Irish<fnref n="11" />, Grant L. Hughes<fnref n="12" />, Thomas Walker<fnref n="1" />

**v2** First published: 12 Sep 2018, 3:113 (
https://doi.org/10.12688/wellcomeopenres.14765.1)

Latest published: 27 Nov 2018, 3:113 (
https://doi.org/10.12688/wellcomeopenres.14765.2)

**Abstract**

**Background:** Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the Anopheles (An.) genera, but has recently been found in An. gambiae s.l. populations in West Africa. As there are numerous Anopheles species that have the capacity to transmit malaria, we analysed a range of species across five malaria endemic countries to determine Wolbachia prevalence rates, characterise novel Wolbachia strains and determine any correlation between the presence of Plasmodium, Wolbachia and the competing bacterium *Asaia*.

**Methods:** Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC),

**Open Peer Review**

Referee Status:

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Invited Referees

1

2

3

REVISED

report

version 2

published
27 Nov 2018

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

version 1
published
12 Sep 2018

report

report

report

<footnote n="1">¹Department of Disease Control, London School of Hygiene & Tropical Medicine, London, WC1E 7HT, UK</footnote>

<footnote n="2">²Entomology Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, 30033, USA</footnote>

<footnote n="3">³Department of Pharmacology and Toxicology, University of Texas Medical Branch at Galveston, Galveston, Texas, USA</footnote>

<footnote n="4">⁴Laboratoire d'entomologie médicale et parasitologie, Centre de Recherche en Sciences Naturelles (CRSN/LWIRO), Sud-Kivu, Congo, Democratic Republic</footnote>

<footnote n="5">⁵Unité d'Entomologie Médicale, Institut Pasteur de Madagascar, Antananarivo, Madagascar</footnote>

<footnote n="6">⁶Nationale de Lutte contre le Paludisme, Ministère de la Santé, Conakry, Guinée</footnote>

<footnote n="7">⁷National Institute of Biomedical Research, Kinshasa, Congo, Democratic Republic</footnote>

<footnote n="8">⁸Department of Medical Microbiology, University of Ghana, Accra, Ghana</footnote>

<footnote n="9">⁹Malaria Consortium, London, EC2A 4LT, UK</footnote>

<footnote n="10">¹⁰Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA</footnote>

<footnote n="11">¹¹The US President's Malaria Initiative and Entomology Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, 30329-4027, USA</footnote>

<footnote n="12">¹²Department of Pathology, Institute for Human Infections and Immunity, Center for Tropical Diseases, Center for Biodefense and Emerging Infectious Disease, University of Texas Medical Branch, Galveston, Texas, USA</footnote>
Ghana, Uganda and Madagascar, between 2013 and 2017. Molecular analysis was undertaken using quantitative PCR, Sanger sequencing, *Wolbachia* multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial 16S rRNA gene.

**Results:** Novel *Wolbachia* strains were discovered in five species: *An. coluzzii*, *An. gambiae* s.s., *An. arabiensis*, *An. moucheti* and *An. coluzzii* species A, increasing the number of *Anopheles* species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with *Wolbachia* supergroup B strains. We also provide evidence for resident strain variants within *An. coluzzii* species A. *Wolbachia* is the dominant member of the microbiome in *An. moucheti* and *An. coluzzii* species A but present at lower densities in *An. coluzzii*. Interestingly, no evidence of *Wolbachia/Asaia* co-infections was seen and *Asaia* infection densities were shown to be variable and location dependent.

**Conclusions:** The important discovery of novel *Wolbachia* strains in *Anopheles* provides greater insight into the prevalence of resident *Wolbachia* strains in diverse malaria vectors. Novel *Wolbachia* strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other *Anopheles* mosquito species, which could be used for population replacement or suppression control strategies.

## Keywords

Wolbachia, mosquitoes, malaria, Anopheles, Asaia, endosymbionts

1. **Ottavia Romoli** ID, Institut Pasteur de la Guyane, French Guiana

Mathilde Gendrin ID, Institut Pasteur de la Guyane, French Guiana
Institut Pasteur, France

2. **Anne Duplouy** ID, Lund University, Sweden

3. **Christophe Antonio-Nkondjio** ID, Organisation for Coordination in the Fight Against Endemic Diseases in Central Africa, Cameroon

## Discuss this article

## Comments (0)

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

**Author roles:** Jeffries CL: Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; Lawrence GG: Methodology; Golovko G: Investigation, Methodology; Kristan M: Investigation, Methodology; Orsborne J: Investigation, Methodology; Spence K: Investigation, Methodology; Hurn E: Investigation, Methodology; Bandibabone J: Investigation, Methodology; Tantely LM: Investigation, Methodology; Raharimalala FN: Investigation, Methodology; Keita K: Investigation, Methodology; Camara D: Investigation, Methodology; Barry Y: Investigation, Methodology; Wat'senga F: Investigation, Methodology; Manzambi EZ: Investigation, Methodology; Afrane YA: Investigation, Methodology; Mohammed AR: Investigation, Methodology; Abeku TA: Investigation, Methodology; Hegde S: Investigation, Methodology; Khanipov K: Investigation, Methodology; Pimenova M: Investigation, Methodology; Fofanov Y: Investigation, Methodology; Boyer S: Investigation, Methodology; Irish SR: Investigation, Methodology; Hughes GL: Funding Acquisition, Investigation, Methodology, Supervision; Walker T: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

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

**Grant information:** CLJ and TW were supported by a Wellcome Trust /Royal Society grant awarded to TW (101285): http://www.wellcome.ac.uk; https://royalsociety.org. GLH is supported by NIH grants (R21AI124452 and R21AI129507), a University of Texas Rising Star award, the John S. Dunn Foundation Collaborative Research Award, the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation, and the Centers for Disease Control and Prevention (CDC) (Cooperative Agreement Number U01CK000512). The papers contents are solely the responsibility of the authors and do not necessarily represent the official views of the CDC or the Department of Health and Human Services. This work was also supported by a James W. McLaughlin postdoctoral fellowship at the University of Texas Medical Branch to SH. Field work in Uganda was funded by UK aid (through the Programme Partnership Arrangement grant to Malaria Consortium). YAA and ARM were supported by a NIH grant R01AI123074. SRI was funded by the U.S. President's Malaria Initiative.

*The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.*

**Copyright:** © 2018 Jeffries CL et al. This is an open access article distributed under the terms of the [Creative Commons Attribution Licence](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**How to cite this article:** Jeffries CL, Lawrence GG, Golovko G et al. Novel *Wolbachia* strains in *Anopheles* malaria vectors from Sub-Saharan Africa [version 2; referees: 3 approved] Wellcome Open Research 2018, 3:113 (https://doi.org/10.12688/wellcomeopenres.14765.2)

**First published:** 12 Sep 2018, 3:113 (https://doi.org/10.12688/wellcomeopenres.14765.1)
<figure><img src="image_5.png" /></figure>

Amendments from Version 1

This revised version contains modifications to Table 1 & Table 2 and Figure 1 & Figure 7 to provide greater clarity on these data sets. We have highlighted how our study was undertaken across diverse malaria endemic countries beyond West Africa and the revised manuscript contains minor editing (including the addition of primer sequences) that was suggested by the reviewers. In addition, we have modified our discussion on the correlation between *Plasmodium* and *Wolbachia* prevalence in *An. gambiae* s.s. to provide a more balanced viewpoint on our data.

See referee reports

## Background

Malaria is a mosquito-borne disease caused by infection with *Plasmodium* (P.) parasites, with transmission to humans occurring through the inoculation of *Plasmodium* sporozoites during blood-feeding of an infectious female *Anopheles* (An.) mosquito. The genus *Anopheles* consists of 475 formally recognised species with ~40 vector species/species complexes responsible for the transmission of malaria at a level of public health concern¹. During the mosquito infection cycle, *Plasmodium* parasites encounter a variety of resident microbiota both in the mosquito midgut and other tissues. Numerous studies have shown that certain species of bacteria can inhibit *Plasmodium* development²⁻⁴. For example, *Enterobacter* bacteria that reside in the *Anopheles* midgut can inhibit the development of *Plasmodium* parasites prior to their invasion of the midgut epithelium⁵,⁶. *Wolbachia* endosymbiotic bacteria are estimated to naturally infect ~40% of insect species⁷ including mosquito vector species that are responsible for transmission of human diseases, such as *Culex* (*Cx.*) *quinquefasciatus*⁸⁻¹⁰ and *Aedes* (*Ae.*) *albopictus*¹¹,¹². Although *Wolbachia* strains have been shown to have variable effects on arboviral infections in their native mosquito hosts¹³⁻¹⁵, transinfected *Wolbachia* strains have been considered for mosquito biocontrol strategies, due to observed arbovirus transmission blocking abilities and a variety of synergistic phenotypic effects. Transinfected strains in *Ae. aegypti* and *Ae. albopictus* provide strong inhibitory effects on arboviruses, with maternal transmission and cytoplasmic incompatibility enabling introduced strains to spread through populations¹⁶⁻²². Open releases of *Wolbachia*-transinfected *Ae. aegypti* populations have demonstrated the ability of the *wMel* *Wolbachia* strain to invade wild populations²³ and provide strong inhibitory effects on viruses from field populations²⁴, with releases currently occurring in arbovirus endemic countries such as Indonesia, Vietnam, Brazil and Colombia (https://www.worldmosquitoprogram.org).

The prevalence of *Wolbachia* in *Anopheles* species has not been extensively studied, with most studies focused in Asia using classical PCR-based screening; up until 2014 there was no evidence of resident strains in mosquitoes from this genus²⁵⁻²⁹. Furthermore, significant efforts to establish artificially infected lines were, up until recently, also unsuccessful³⁰. Somatic, transient infections of the *Wolbachia* strains *wMelPop* and *wAlbB* in *An. gambiae* were shown to significantly inhibit *P. falciparum*³¹, but the interference phenotype is variable with other *Wolbachia* strain-parasite combinations³²⁻³⁴. A stable line was established

in *An. stephensi*, a vector of malaria in southern Asia, using the *wAlbB* strain and this was also shown to confer resistance to *P. falciparum* infection³⁵. One potential reason postulated for the absence of *Wolbachia* in *Anopheles* species was thought to be the presence of other bacteria, particularly from the genus *Asaia*³⁶. This acetic acid bacterium is stably associated with several *Anopheles* species and is often the dominant species in the mosquito microbiota³⁷. In laboratory studies, *Asaia* has been shown to impede the vertical transmission of *Wolbachia* in *Anopheles*³⁶ and was shown to have a negative correlation with *Wolbachia* in mosquito reproductive tissues³⁸.

Recently, resident *Wolbachia* strains (those naturally present in wild insect populations) have been discovered in the *An. gambiae* s.l. complex, which consists of multiple morphologically indistinguishable species including several major malaria vector species. *Wolbachia* strains (collectively named *wAnga*) were found in *An. gambiae* s.l. populations in Burkina Faso³⁹ and Mali⁴⁰, suggesting that *Wolbachia* may be more abundant in the *An. gambiae* complex across Sub-Saharan Africa. Globally, there is a large variety of *Anopheles* vector species (~70) that have the capacity to transmit malaria⁴¹ and could potentially contain resident *Wolbachia* strains. Additionally, this number of malaria vector species may be an underestimate given that recent studies using molecular barcoding have also revealed a larger diversity of *Anopheles* species than would be identified using morphological identification alone⁴²,⁴³.

Investigating the prevalence and diversity of *Wolbachia* strains naturally present in *Anopheles* populations across diverse malaria endemic countries would allow a greater understanding of how this bacterium could be influencing malaria transmission in field populations and identify candidate strains for transmission. In this study, we collected *Anopheles* mosquitoes from five malaria-endemic countries; Ghana, Democratic Republic of the Congo (DRC), Guinea, Uganda and Madagascar, from 2013–2017. Wild-caught adult female *Anopheles* were screened for *P. falciparum* malaria parasites, *Wolbachia* and *Asaia* bacteria. In total, we analysed mosquitoes from 17 *Anopheles* species that are known malaria vectors or implicated in transmission, and some unidentified species, discovering five species of *Anopheles* with resident *Wolbachia* strains; *An. coluzzii* from Ghana, *An. gambiae* s.s., *An. arabiensis*, *An. moucheti* and *An. spe*cies A from DRC. Using *Wolbachia* gene sequencing, including multilocus sequence typing (MLST), we show that the resident strains in these malaria vectors are diverse, novel strains and quantitative PCR (qPCR) and 16S rRNA amplicon sequencing data suggests that the strains in *An. moucheti* and *An. species A* are higher density infections, compared to the strains found in the *An. gambiae* s.l. complex. We found no evidence for either *Wolbachia-Asaia* co-infections, or for either bacteria having any significant effect on the prevalence of *Plasmodium* in wild mosquito populations.

## Methods

### Study sites & collection methods

*Anopheles* adult mosquitoes were collected from five malaria-endemic countries in Sub-Saharan Africa (Guinea, Democratic
Republic of the Congo (DRC), Ghana, Uganda and Madagascar) between 2013 and 2017 (Figure 1). Human landing catches, Centers for Disease Control (CDC) light traps and pyrethrum spray catches were undertaken between April 2014 and February 2015 in 10 villages near four cities in Guinea; Foulayah (10.144633, -10.749717) and Balayani (10.1325, -10.7443) near Faranah; Djoumaya (10.836317, -14.2481) and Kaboye Amaraya (10.93435, -14.36995) near Boke; Tongbekoro (9.294295, -10.147953), Keredou (9.208919, -10.069525), and Gbangbadou (9.274363, -9.998639) near Kissidougou; and Makonon (10.291124, -9.363358), Balandou (10.407669, -9.219096), and Dalabani (10.463692, -9.451904) near Kankan. Human landing catches and pyrethrum spray catches were undertaken between January and September 2015 in seven sites of the DRC; Kinshasa (-4.415881,

15.412188), Mikalayi (-6.024184, 22.318251), Kisangani (0.516350, 25.221176), Katana (-2.225129, 28.831604), Kalemie (-5.919054, 29.186572), and Kapolowe (-10.939802, 26.952970). We also analysed a subset from collections obtained from Lwiro (-2.244097, 28.815232), a village near Katana, collected between September and October 2015. A combination of CDC light traps, pyrethrum spray catches and human landing catches were undertaken in Butemba, Kyankwanzi District in mid-western Uganda (1.1068444, 31.5910085) in August and September 2013, and June 2014. CDC light trap catches were undertaken in May 2017 in Dogo in Ada, Greater Accra, Ghana (5.874861111, 0.560611111). In Madagascar, sampling was undertaken in June 2016 at four sites: Anivorano Nord, located in the Northern domain, (-12.7645000, 49.2386944); Ambomiharina, Western domain,

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

**Figure 1.** Locations of *Anopheles* species collections (including *Wolbachia*-infected species) and *P. falciparum* malaria prevalence rates in mosquitoes (across all species for each location). (A) Overall map showing the five malaria-endemic countries where mosquito collections were undertaken. (B) High *P. falciparum* prevalence rates in mosquitoes from Guinea, and *Wolbachia*-infected *An. coluzzii* from Ghana (no *P. falciparum* detected). (C) *Wolbachia* strains in *An. gambiae* s.s., *An. arabiensis*, *An.** species A and *An. moucheti* from the Democratic Republic of the Congo (DRC) and variable *P. falciparum* prevalence rates in mosquitoes from DRC and Uganda. (D) Low *P. falciparum* infection rates in mosquitoes from Madagascar and no evidence of resident *Wolbachia* strains. (*W*+; *Wolbachia* detected in this species). Maps were generated using ArcMap™ within the ArcGIS 10.5 software package (Esri®, Redlands CA, USA, http://www.esri.com). Maps were constructed using country outline (level 0) data downloaded from the Database of Global Administrative Areas (GADM) (http://www.gadm.org) (release number 2.8) for both the world, and each country of interest. The coloured mosquito icons were generated by the authors themselves (CLJ).
(-16.3672778, 46.9928889); Antafia, Western domain, (-17.0271667, 46.7671389); and Ambohimarina, Central domain, (-18.3329444, 47.1092500). Trapping consisted of CDC light traps and a net trap baited with Zebu (local species of cattle) to attract zoophilic species<sup>44</sup>. Coordinate values for all locations are latitude and longitude respectively, in decimal degrees.

## DNA extraction and mosquito species identification

DNA was extracted from individual whole mosquitoes or abdo-
mens using QIAGEN DNeasy Blood and Tissue Kits accord-
ing to manufacturer's instructions. DNA extracts were eluted in
a final volume of 100 µl and stored at -20°C. Mosquito species
identification was initially undertaken using morphological keys
followed by diagnostic species-specific PCR assays to distinguish
between the morphologically indistinguishable sibling mosquito
species of the *An. gambiae*<sup>45-47</sup> and *An. funestus* complexes<sup>48</sup>.
To determine species identification for samples of interest and
for samples that could not be identified by species-specific
PCR, Sanger sequences were generated from *ITS2* PCR products<sup>49</sup>.

Detection of *P. falciparum* and *Asaia*

Detection of *P. falciparum* malaria was undertaken using qPCR
targeting an 120-bp sequence of the *P. falciparum* cytochrome
c oxidase subunit 1 (*Cox1*) mitochondrial gene using primers
5'-TTACATCAGGAATGTTATTGC-3' and 5'-ATATTGGATCT
CCTGCAAAT-3'⁵⁰. Positive controls from gDNA extracted
from a cultured *P. falciparum*-infected blood sample (parasitae-
mia of ~10%) were serially diluted to determine the threshold
limit of detection, in addition to the inclusion of no template con-
trols (NTCs). *Asaia* detection was undertaken targeting the 16S
rRNA gene using primers Asafor: 5'-GCGCGTAGGCGGTTT
ACAC-3' and Asarev: 5'-AGCGTCAGTAATGAGCCAGGT
T-3'³⁷,⁵¹. Ct values for both *P. falciparum* and *Asaia* assays in
selected *An. gambiae* extracts were normalized to Ct values
for a single copy *An. gambiae* rps17 housekeeping gene using
primers 5'-GACGAAACCACTGCGTAACA-3' and 5'-TGCT
CCAGTGCTGAAACATC-3' (accession no. AGAP004887 on
www vectorbase.org)⁵²,⁵³. As Ct values are inversely related to
the amount of amplified DNA, a higher target gene Ct: host
gene Ct ratio represented a lower estimated infection level. qPCR
reactions were prepared using 5 µl of FastStart SYBR Green Mas-
ter mix (Roche Diagnostics), a final concentration of 1 µM of
each primer, 1 µl of PCR grade water and 2 µl template DNA, to
a final reaction volume of 10 µl. Prepared reactions were run on a
Roche LightCycler® 96 System and amplification was followed
by a dissociation curve (95°C for 10 seconds, 65°C for 60 sec-
onds and 97°C for 1 second) to ensure the correct target sequence
was being amplified. PCR results were analysed using the
LightCycler® 96 software (Roche Diagnostics). A sub-selection
of PCR products from each assay was sequenced to confirm
correct amplification of the target gene fragment.

Wolbachia detection

Wolbachia detection was first undertaken targeting three
conserved *Wolbachia* genes previously shown to amplify a wide
diversity of strains; 16S rRNA gene using primers W-Spec-
16S-F: 5'-CATACCTATTCGAAGGGATA-3' and W-Spec-16s-
R: 5'-AGCTTCGAGTGAAACCAATTC-3'⁴⁰,⁵⁴, *Wolbachia*
surface protein (wsp) gene using primers wsp81F: 5'-TGGT

CCAATAAGTGATGAAGAAAC-3' and wsp691R: 5'-AAAAA
TTAAACGCTACTCCA-3'⁵⁵ and FtsZ cell cycle gene using
primers ftsZqPCR F: 5'-GCATTGCAGAGCTTGGACTT-3'
and ftsZqPCR R: 5'-TCTTCTCCTTCTGCCTCTCC-3'⁵⁶. DNA
extracted from a *Drosophila melanogaster* fly (infected with the
wMel strain of *Wolbachia*) was used as a positive control, in addi-
tion to no template controls (NTCs). The 16S rRNA⁵⁴ and wsp⁵⁵
gene PCR reactions were carried out in a Bio-Rad T100 Thermal
Cycler using standard cycling conditions and PCR products
were separated and visualised using 2% E-Gel EX agarose gels
(Invitrogen) with SYBR safe and an Invitrogen E-Gel iBase Real-
Time Transilluminator. FtsZ⁵⁶ and 16S rRNA⁴⁰ gene real time
PCR reactions were prepared using 5 µl of FastStart SYBR Green
Master mix (Roche Diagnostics), a final concentration of 1 µM
of each primer, 1 µl of PCR grade water and 2 µl template DNA,
to a final reaction volume of 10 µl. Prepared reactions were run
on a Roche LightCycler® 96 System for 15 minutes at 95°C,
followed by 40 cycles of 95°C for 15 seconds and 58°C for
30 seconds. Amplification was followed by a dissociation
curve (95°C for 10 seconds, 65°C for 60 seconds and 97°C for
1 second) to ensure the correct target sequence was being ampli-
fied. PCR results were analysed using the LightCycler® 96 soft-
ware (Roche Diagnostics). To estimate *Wolbachia* densities across
multiple *Anopheles* mosquito species, ftsZ and 16S qPCR Ct
values were compared to total dsDNA extracted, measured using
an Invitrogen Qubit 4 fluorometer. A serial dilution series of
a known *Wolbachia*-infected mosquito DNA extract was used
to correlate Ct values and amount of amplified target product.

Wolbachia multilocus strain typing (MLST)

MLST was undertaken to characterize *Wolbachia* strains using
the sequences of five conserved genes as molecular markers
to genotype each strain. In brief, 450–500 base pair fragments
of the *gatB*, *coxA*, *hcpA*, *ftsZ* and *fbpA* *Wolbachia* genes were
amplified from individual *Wolbachia*-infected mosquitoes using
previously optimised protocols⁵⁷. Primers used were as
follows: *gatB_F1*: 5'-GAKTTAAAYCGYGCAGGBGTT-3',
*gatB_R1*: 5'-TGGYAAYTCRGGYAAAGATGA-3', *coxA_F1*: 5'-
TTGGRGCRATYAACTTTATAG-3', *coxA_R1*: 5'-CTAAAGACT
TTKACRCCAGT-3', *hcpA_F1*: 5'-GAAATARCAGTTGCTGC
AAA-3', *hcpA_R1*: 5'-GAAAGTYRAGCAAGYTCTG-3',
*ftsZ_F1*: 5'-ATYATGGARCATATAAARGATAG-3', *ftsZ_R1*: 5'-
TCRAGYAATGGATTRGATAT-3', *fbpA_F1*: 5'-GCTGC
TCCRCTTGGYWTGAT-3' and *fbpA_R1*: 5'-CCRCCAG
ARAAAAYYACTATTC-3'. A *Cx. pipiens* gDNA extraction (pre-
viously shown to be infected with the *wPip* strain of *Wolbachia*)
was used as a positive control for each PCR run, in addition to
no template controls (NTCs). If initial amplification with these
primers was unsuccessful, the PCR was repeated using the
standard primers but with the addition of M13 adaptors. If no
amplification was detected using standard primers, further PCR
analysis was undertaken using degenerate primer sets, with or
without M13 adaptors, which for the *hcpA* gene of *wAnga-Ghana*
allowed improved amplification (using *hcpA_F3*: 5'-ATTA
GAGAAATARCAGTTGCTGC-3', *hcpA_R3*: 5'-CATGAA
AGACGAGCAARYTCTGG-3' (no M13 adaptors))⁵⁷. PCR prod-
ucts were separated and visualised using 2% E-Gel EX agar-
ose gels (Invitrogen) with SYBR safe and an Invitrogen E-Gel
iBase Real-Time Transilluminator. PCR products were submitted
to Source BioScience (Source BioScience Plc, Nottingham, UK) for PCR reaction clean-up, followed by Sanger sequencing to generate both forward and reverse reads. Where PCR primers included M13 adaptors, just the M13 primers alone (M13_adaptor_F: 5'-TGTAAAACGACGGCCAGT-3' and M13_adaptor_R: 5'-CAGGAAACAGCTATGACC-3') were used for sequencing, otherwise the same primers as utilised for PCR were used. Sequencing analysis was carried out in MEGA7⁵⁸ as follows. Both chromatograms (forward and reverse traces) from each sample were manually checked, edited, and trimmed as required, followed by alignment with ClustalW and checking to produce consensus sequences. Consensus sequences were used to perform nucleotide BLAST (NCBI) database queries, and searches against the *Wolbachia* MLST database⁵⁹. If a sequence produced an exact match in the MLST database we assigned the appropriate allele number, otherwise we obtained a new allele number for each novel gene locus sequence through submission of the FASTA and raw trace files on the *Wolbachia* MLST website for new allele assignment and inclusion within the database. Full consensus sequences were also submitted to GenBank and assigned accession numbers. The Sanger sequencing traces from the wsp gene were also treated in the same way and analysed alongside the MLST gene locus scheme, as an additional marker for strain typing.

## Phylogenetic analysis

Alignments were constructed in MEGA7 by ClustalW to include all relevant and available sequences highlighted through searches on the BLAST and *Wolbachia* MLST databases. Maximum Likelihood phylogenetic trees were constructed from Sanger sequences as follows. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model⁶⁰. The tree with the highest log likelihood in each case is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. The phylogeny test was by Bootstrap method with 1000 replications. Evolutionary analyses were conducted in MEGA7⁵⁸.

## Microbiome analysis

The microbiomes of selected individual *Anopheles* were analysed using barcoded high-throughput amplicon sequencing of the bacterial 16S rRNA gene. Sequencing libraries for each isolate were generated using universal 16S rRNA V3-V4 region primers⁶¹ in accordance with Illumina 16S rRNA metagenomic sequencing library protocols. The samples were barcoded for multiplexing using Nextera XT Index Kit v2. Sequencing was performed on an Illumina MiSeq instrument using a MiSeq Reagent Kit v2 (500-cycles). Quality control and taxonomical assignment of the resultant reads were performed using CLC Genomics Workbench 8.0.1 Microbial Genomics Module. Low quality reads containing nucleotides with quality threshold below 0.05 (using

the modified Richard Mott algorithm), as well as reads with two or more unknown nucleotides were removed from analysis. Additionally, reads were trimmed to remove sequenced Nextera adapters. Reference-based operational taxonomic unit (OTU) picking was performed using the SILVA SSU v128 97% database⁶². Sequences present in more than one copy but not clustered to the database were then placed into *de novo* OTUs (97% similarity) and aligned against the reference database with 80% similarity threshold to assign the “closest” taxonomical name where possible. Chimeras were removed from the dataset if the absolute crossover cost was 3 using a k-mer size of 6. Alpha diversity was measured using Shannon entropy (OTU level).

## Statistical analysis

Fisher's exact post hoc test in Graphpad Prism 7 was used to compare infection rates. Normalised qPCR Ct ratios were compared using unpaired t-tests in GraphPad Prism 7.

## Results

### Mosquito species and resident *Wolbachia* strains

*Anopheles* species composition varied depending on country and mosquito collection sites (Table 1). We detected *Wolbachia* in *An. coluzzii* mosquitoes from Ghana (prevalence of 4% - termed *wAnga-Ghana*) and *An. gambiae* s.s. from all six collection sites in DRC (prevalence range of 8–24%) in addition to a single infected *An. arabiensis* from Kalemie in DRC (Figure 1 and Table 1). The molecular phylogeny of the *ITS2* gene of *Anopheles gambiae* s.l. complex individuals (including both *Wolbachia*-infected and uninfected individuals analysed in our study) confirmed molecular species identifications made using species-specific PCR assays (Figure 2). Novel resident *Wolbachia* infections were detected in two additional *Anopheles* species from DRC; *An. moucheti* (termed *wAnM*) from Mikalayi, and *An. species A* (termed *wAnSA*) from Katana. Additionally, we screened adult female mosquitoes of *An. species A* (collected as larvae and adults) from Lwiro, a village near Katana in DRC, and detected *Wolbachia* in 30/33 (91%), indicating this resident *wAnSA* strain has a high infection prevalence in populations in this region. The molecular phylogeny of the *ITS2* gene revealed *Wolbachia*-infected individuals from Lwiro and Katana are the same *An. species A* (Figure 3) previously collected in Eastern Zambia⁴³ and Western Kenya⁶³. All *ITS2* sequences were deposited in GenBank (accession numbers MH598414–MH598445; listed in Supplementary Table 1).

### *Wolbachia* strain typing

Phylogenetic analysis of the 16S rRNA gene demonstrated that the 16S sequences for these strains cluster with other Super-group B strains such as *wPip* (99–100% nucleotide identity) (Figure 4a). When compared to the resident *Wolbachia* strains in *An. gambiae* s.l. populations from Mali⁴⁰ and Burkina Faso³⁹, *wAnga-Ghana* is more closely related to the Super-group B strain of *wAnga* from Burkina Faso. Although a resident strain was detected in *An. gambiae* s.s. and a single *An. arabiensis* from DRC through amplification of 16S rRNA fragments using two independent PCR assays⁴⁰,⁵⁴, we were unable to obtain 16S sequences of sufficient quality to allow further analysis. The *Wolbachia* wsp gene has been evolving at a faster rate and provides more
<table><caption>**Table 1.** *Anopheles* mosquito species collected from locations within five malaria-endemic countries, including the infection status of individuals from each location. Individuals were classified as having either single infections with *Plasmodium* (*Pla*), *Wolbachia* (*Wol*) or *Asaia* (*Asa*), co-infections, or uninfected. Species containing *Wolbachia*-infected individuals are shown in bold.</caption><thead><tr><th rowspan="2">Country</th><th rowspan="2">Location</th><th rowspan="2">Species</th><th colspan="3">Individuals with single infections</th><th colspan="3">Individuals with co-infections</th><th rowspan="2">Uninfected individuals</th><th rowspan="2">Total</th></tr><tr><th>Pla</th><th>Wol</th><th>Asa</th><th>Pla + Wol</th><th>Pla + Asa</th><th>Wol + Asa</th></tr></thead><tbody><tr><td rowspan="12">Guinea</td><td rowspan="3">Faranah</td><td>An. gambiae s.s.</td><td>9 (18.8)</td><td>0 (0)</td><td>13 (27.1)</td><td>0 (0)</td><td>11 (22.9)</td><td>0 (0)</td><td>15 (31.3)</td><td>48</td></tr><tr><td>An. arabiensis</td><td>0 (0)</td><td>0 (0)</td><td>7 (100.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>7</td></tr><tr><td>An. nili</td><td>0 (0)</td><td>0 (0)</td><td>6 (75.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>2 (25.0)</td><td>8</td></tr><tr><td rowspan="2">Kissidougou</td><td>An. gambiae s.s.</td><td>0 (0)</td><td>0 (0)</td><td>26 (74.3)</td><td>0 (0)</td><td>9 (25.7)</td><td>0 (0)</td><td>0 (0)</td><td>35</td></tr><tr><td>An. species O</td><td>0 (0)</td><td>0 (0)</td><td>1 (100.00)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1</td></tr><tr><td>Boke</td><td>An. gambiae s.s.</td><td>7 (33.3)</td><td>0 (0)</td><td>3 (14.3)</td><td>0 (0)</td><td>3 (14.3)</td><td>0 (0)</td><td>8 (38.1)</td><td>21</td></tr><tr><td rowspan="2">Kankan</td><td>An. gambiae s.s.</td><td>10 (21.7)</td><td>0 (0)</td><td>15 (32.6)</td><td>0 (0)</td><td>9 (19.6)</td><td>0 (0)</td><td>12 (26.1)</td><td>46</td></tr><tr><td>An. sp. unknown</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (100.0)</td><td>1</td></tr><tr><td rowspan="12">DRC</td><td rowspan="3">Mikalayi</td><td>An. gambiae s.s.</td><td>4 (25.0)</td><td>1 (6.3)</td><td>1 (6.3)</td><td>1 (6.3)</td><td>0 (0)</td><td>0 (0)</td><td>9 (56.3)</td><td>16</td></tr><tr><td>An. moucheti</td><td>0 (0)</td><td>1 (100.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1</td></tr><tr><td>An. funestus s.s.</td><td>3 (23.1)</td><td>0 (0)</td><td>1 (7.7)</td><td>0 (0)</td><td>1 (7.7)</td><td>0 (0)</td><td>8 (61.5)</td><td>13</td></tr><tr><td rowspan="2">Kisangani</td><td>An. gambiae s.s.</td><td>2 (8.0)</td><td>2 (8.0)</td><td>3 (12.0)</td><td>0 (0)</td><td>1 (4.0)</td><td>0 (0)</td><td>17 (68.0)</td><td>25</td></tr><tr><td>An. arabiensis</td><td>1 (25.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>3 (75.0)</td><td>4</td></tr><tr><td rowspan="3">Katana</td><td>An. gambiae s.s.</td><td>0 (0)</td><td>2 (8.7)</td><td>0 (0)</td><td>0 (0)</td><td>1 (4.3)</td><td>0 (0)</td><td>20 (87.0)</td><td>23</td></tr><tr><td>An. funestus s.s.</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>5 (100.0)</td><td>5</td></tr><tr><td>An. species A</td><td>0 (0)</td><td>1 (50.0)</td><td>0 (0)</td><td>1 (50.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>2</td></tr><tr><td>Lwiro (Katana)</td><td>An. species A*</td><td>NT</td><td>30 (91.0)</td><td>NT</td><td>NT</td><td>NT</td><td>NT</td><td>3 (9.0)</td><td>33</td></tr><tr><td rowspan="2">Kapolowe</td><td>An. gambiae s.s.</td><td>1 (11.0)</td><td>1 (11.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>7 (78.0)</td><td>9</td></tr><tr><td>An. funestus s.s.</td><td>1 (20.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>4 (80.0)</td><td>5</td></tr><tr><td rowspan="2">Kalemie</td><td>An. gambiae s.s.</td><td>2 (7.1)</td><td>6 (21.4)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>20 (71.4)</td><td>28</td></tr><tr><td>An. arabiensis</td><td>0 (0)</td><td>1 (50.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (50.0)</td><td>2</td></tr><tr><td rowspan="2">Kinshasa</td><td>An. gambiae s.s.</td><td>5 (19.2)</td><td>2 (7.7)</td><td>1 (3.8)</td><td>1 (3.8)</td><td>0 (0)</td><td>0 (0)</td><td>17 (65.4)</td><td>26</td></tr><tr><td>An. funestus s.s.</td><td>1 (50.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (50.0)</td><td>2</td></tr><tr><td rowspan="2">Ghana</td><td rowspan="2">Dogo</td><td>An. coluzzii</td><td>0 (0)</td><td>12 (4.2)</td><td>92 (32.1)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>183 (63.8)</td><td>287</td></tr><tr><td>An. melas</td><td>0 (0)</td><td>0 (0)</td><td>1 (100.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1</td></tr><tr><td rowspan="3">Uganda</td><td>Butemba (2013)</td><td>An. gambiae s.s.</td><td>2 (3.5)</td><td>0 (0)</td><td>41 (71.9)</td><td>0 (0)</td><td>9 (15.8)</td><td>0 (0)</td><td>5 (8.8)</td><td>57</td></tr><tr><td rowspan="2">Butemba (2014)</td><td>An. gambiae s.s.</td><td>23 (17.0)</td><td>0 (0)</td><td>38 (28.1)</td><td>0 (0)</td><td>27 (20.0)</td><td>0 (0)</td><td>47 (34.8)</td><td>135</td></tr><tr><td>An. arabiensis</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (100.00)</td><td>1</td></tr></tbody></table>
<table><thead><tr><th rowspan="2">Country</th><th rowspan="2">Location</th><th rowspan="2">Species</th><th colspan="3">Individuals with single infections</th><th colspan="3">Individuals with co-infections</th><th rowspan="2">Uninfected individuals</th><th rowspan="2">Total</th></tr><tr><th>Pla</th><th>Wol</th><th>Asa</th><th>Pla + Wol</th><th>Pla + Asa</th><th>Wol + Asa</th></tr></thead><tbody><tr><td rowspan="15">Madagascar</td><td rowspan="6">Anivorano Nord</td><td>An. funestus</td><td>0 (0)</td><td>0 (0)</td><td>3 (37.5)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>5 (62.5)</td><td>8</td></tr><tr><td>An. gambiae s.s.</td><td>0 (0)</td><td>0 (0)</td><td>1 (33.3)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>2 (66.6)</td><td>3</td></tr><tr><td>An. arabiensis</td><td>0 (0)</td><td>0 (0)</td><td>2 (100.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>2</td></tr><tr><td>An. mascarensis</td><td>0 (0)</td><td>0 (0)</td><td>15 (44.1)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>19 (55.9)</td><td>34</td></tr><tr><td>An. maculipalpis</td><td>0 (0)</td><td>0 (0)</td><td>2 (15.4)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>11 (84.6)</td><td>13</td></tr><tr><td>An. coustani</td><td>0 (0)</td><td>0 (0)</td><td>6 (28.6)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>15 (71.4)</td><td>21</td></tr><tr><td rowspan="5">Ambomiharina</td><td>An. rufipes</td><td>0 (0)</td><td>0 (0)</td><td>3 (27.3)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>8 (72.7)</td><td>11</td></tr><tr><td>An. funestus</td><td>0 (0)</td><td>0 (0)</td><td>9 (81.8)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>2 (18.2)</td><td>11</td></tr><tr><td>An. pharoensis</td><td>0 (0)</td><td>0 (0)</td><td>3 (42.9)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>4 (57.1)</td><td>7</td></tr><tr><td>An. rufipes</td><td>0 (0)</td><td>0 (0)</td><td>14 (66.7)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>7 (33.3)</td><td>21</td></tr><tr><td>An. maculipalpis</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>9 (100.0)</td><td>9</td></tr><tr><td rowspan="5">Antafia</td><td>An. gambiae s.s.</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>8 (100.0)</td><td>8</td></tr><tr><td>An. coustani</td><td>0 (0)</td><td>0 (0)</td><td>6 (25.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>18 (75.0)</td><td>24</td></tr><tr><td>An. squamosus</td><td>0 (0)</td><td>0 (0)</td><td>2 (20.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>8 (80.0)</td><td>10</td></tr><tr><td>An. mascarensis</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (100.0)</td><td>1</td></tr><tr><td>An. pauliani</td><td>0 (0)</td><td>0 (0)</td><td>3 (100.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>3</td></tr><tr><td rowspan="5">Ambohimarina</td><td>An. gambiae s.s.</td><td>1 (9.1)</td><td>0 (0)</td><td>3 (27.3)</td><td>0 (0)</td><td>2 (18.2)</td><td>0 (0)</td><td>5 (45.5)</td><td>11</td></tr><tr><td>An. pauliani</td><td>0 (0)</td><td>0 (0)</td><td>1 (50.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (50.0)</td><td>2</td></tr><tr><td>An. rufipes</td><td>0 (0)</td><td>0 (0)</td><td>1 (50.0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (50.0)</td><td>2</td></tr><tr><td>An. mascarensis</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>2 (100.0)</td><td>2</td></tr><tr><td>An. funestus</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (100.0)</td><td>1</td></tr><tr><td rowspan="6">Ambohimarina</td><td>An. gambiae s.s.</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (100.0)</td><td>1</td></tr><tr><td>An. arabiensis</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>2 (100.0)</td><td>2</td></tr><tr><td>An. rufipes</td><td>0 (0)</td><td>0 (0)</td><td>3 (42.9)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>4 (57.1)</td><td>7</td></tr><tr><td>An. coustani</td><td>0 (0)</td><td>0 (0)</td><td>2 (11.1)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>16 (88.9)</td><td>18</td></tr><tr><td>An. maculipalpis</td><td>0 (0)</td><td>0 (0)</td><td>1 (12.5)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>7 (87.5)</td><td>8</td></tr><tr><td>An. squamosus</td><td>0 (0)</td><td>0 (0)</td><td>2 (4.3)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>44 (95.7)</td><td>46</td></tr><tr><td>Ambohimarina</td><td>An. mascarensis</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>11 (100.0)</td><td>11</td></tr></tbody></table>

<footnote>*Adult individuals from Lwiro (Katana), DRC were collected as both larvae and adults so have been excluded from *P. falciparum* and *Asaia* prevalence analysis (NT; Not tested).</footnote>
<figure><img src="image_7.png" /><figcaption>**Figure 2.** Maximum Likelihood molecular phylogenetic analysis of *Anopheles gambiae* complex ITS2 sequences from field-collected mosquitoes. The tree with the highest log likelihood (-785.65) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 42 nucleotide sequences. There were a total of 475 positions in the final dataset. Symbols, colours and codes used for the sequences generated in this study are as follows: W+: individual was *Wolbachia* positive (solid coloured symbol), W-: individual was *Wolbachia* negative (empty coloured symbol). DRC, Democratic Republic of the Congo (red); KAL, Kalemie; MIK, Mikalayi; KIN, Kinshasa; KAT, Katana. GHA, Ghana (blue); DOG, Dogo. GUI, Guinea (green); KSK, Kissidougou. MAD, Madagascar (purple); ANT, Antafia. UGA, Uganda (maroon); BUT, Butemba. Different shape coloured symbols are used to differentiate between the different mosquito species. GenBank sequences included (for comparison with sequences generated in this study) are in black with their accession numbers provided. Where GenBank sequence subtrees have been compressed, this is denoted by a solid black diamond symbol. GenBank accession numbers for sequences included in compressed subtrees are: GQ870318.1 and GQ870320.1 for *Anopheles bwambae*, and GQ870315.1, JN664146.1 and KR014832.1 for *Anopheles quadriannulatus*.</figcaption></figure>
<figure><img src="image_8.png" /></figure>

**Figure 3.** Maximum Likelihood molecular phylogenetic analysis of *Anopheles ITS2* sequences from field-collected mosquitoes outside of the *An. gambiae* s.l. complex. The tree with the highest log likelihood (-3084.12) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 118 nucleotide sequences. There were a total of 156 positions in the final dataset. Symbols, colours and codes used for sequences generated in this study are as follows: W+; individual was *Wolbachia* positive (solid coloured symbol), W-; individual was *Wolbachia* negative (empty coloured symbol). DRC, Democratic Republic of the Congo (red); KAT, Katana; LWI, Lwiro; MIK, Mikalayi. GUI, Guinea (green); FAR, Faranah; KAN, Kankan; KSK, Kissidougou. MAD, Madagascar (purple); AMB, Ambomiharina. Different shape coloured symbols are used to differentiate between different mosquito species. GenBank sequences included (for comparison with sequences generated in this study) are in black with their accession numbers provided. Where GenBank sequence subtrees have been compressed, this is denoted by a solid black diamond symbol. GenBank accession numbers for sequences included in compressed subtrees are as follows: *Anopheles squamosus*; KJ522825.1 and KR014825.1. *Anopheles coustani*; JN994134.1, KJ522815.1 and KR014823.1. *Anopheles funestus*; AF062512.1, JN994135.1, JN994136.1, KJ522816.1 and KR014830.1. *Anopheles rivulorum*; JN994148.1, JN994149.1 and KR014822.1. *Anopheles lessoni*; JN994139.1, KJ522824.1 and KR014834.1. *Anopheles pretoriensis*; JN994145.1, KJ522820.1 and KR014829.1. *Anopheles maculipalpis*; JN994142.1, KJ522817.1 and KR014835.1. (The blue *Anopheles gambiae* complex compressed subtree is shown in Figure 2.)
<figure><img src="image_9.png" /></figure>

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

**Figure 4.** Resident *Wolbachia* strain phylogenetic analysis using 16S rRNA and wsp genes. (A) Maximum Likelihood molecular phylogenetic analysis of the 16S rRNA gene for resident strains in *An. coluzzii* (wAnga-Ghana; blue), *An. moucheti* (wAnM; green) and *An.* species A (wAnsA; red). The tree with the highest log likelihood (-660.03) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 17 nucleotide sequences. There were a total of 333 positions in the final dataset. Accession numbers of additional sequences obtained from GenBank are shown, including wPip (navy blue), wAnga-Mali (purple) and wAnga-Burkina Faso strains (maroon). (B) Maximum Likelihood molecular phylogenetic analysis of the wsp gene for wAnsA-infected representative individuals from the DRC (red). The tree with the highest log likelihood (-3663.41) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 83 nucleotide sequences. There were a total of 443 positions in the final dataset. Reference numbers of additional sequences obtained from the MLST database (IsoN; Isolate number) or GenBank (accession number) are shown. Strains isolated from mosquitoes are highlighted in navy blue. KAT, Katana; LWI, Lwiro.
informative strain phylogenies⁵⁵. As expected, however, and similar to *Wolbachia*-infected *An. gambiae* s.l. from Burkina Faso³⁹ and Mali⁴⁰, a fragment of the *wsp* gene was not amplified from *Wolbachia*-positive samples from *An. gambiae* s.s., *An. arabiensis* and *An. coluzzii*. Similarly, no *wsp* gene fragment amplification occurred from *wAnM*-infected *An. moucheti*. However, *wsp* sequences were obtained from both *Wolbachia*-infected individuals of *An.* species A from Katana. We also analysed the *wsp* sequences of 22 specimens of *An.* species A from Lwiro (near Katana) and found identical sequences to the two individuals from Katana. Phylogenetic analysis of the *wsp* sequences obtained for the *wAnS* strain, for both individuals from Katana (*wAnS* *wsp* DRC-KAT1, *wAnS* *wsp* DRC-KAT2) and three representative individuals from Lwiro (*wAnS* *wsp* DRC-LWI1, *wAnS* *wsp* DRC-LWI2, *wAnS* *wsp* DRC-LWI3) indicates *wAnS* is most closely related to *Wolbachia* strains of Supergroup B (such as *wPip*, *wAlbB*, *wMa* and *wNo*), which is consistent with 16S rRNA phylogeny. However, the improved phylogenetic resolution provided by *wsp* indicates they cluster separately (Figure 4b). Typing of the *wAnS* *wsp* nucleotide sequences highlighted that there were no exact matches to *wsp* alleles currently in the *Wolbachia* MLST database and, in addition, *wAnS* *wsp* sequences demonstrated novel amino acid motifs in three out of the four hypervariable regions (HVRs) when compared to those present in the MLST database (Table 2). All *Wolbachia* 16S and *wsp* sequences of sufficient quality to generate a consensus were deposited into GenBank (accession numbers MH605275–MH605285; listed in Supplementary Table 2).

MLST was undertaken to provide more accurate strain phylogenies. This was done for the novel *Wolbachia* strains *wAnM* and *wAnS* in addition to the resident *wAnga-Ghana* strain in *An. coluzzii* from Ghana. We were unable to amplify any of the five MLST genes from *Wolbachia*-infected *An. gambiae* s.s. and *An. arabiensis* from DRC (likely due to low infection densities). New alleles for all five MLST gene loci (sequences differed from those currently present in the MLST database) and novel allelic profiles confirm the diversity of these novel *Wolbachia* strains (Table 2). The phylogeny of these three novel strains based on concatenated sequences of all five MLST gene loci confirms they cluster within Supergroup B (Figure 5a). This also demonstrates the novelty as comparison with a wide range of strains (including

all isolates highlighted through partial matching during typing of each locus) shows these strains are distinct from currently available sequences (Figure 5a and Table 2). The concatenated phylogeny indicates that *wAnM* is most closely related to a Hemiptera strain: Isolate number 1616 found in *Bemisia tabaci* in Uganda, and a Coleoptera strain: Isolate number 20 found in *Tribolium confusum*. Concatenation of the MLST loci also indicates *wAnS* is closest to a group containing various Lepidoptera and Hymenoptera strains from multiple countries in Asia, Europe and America, as well as two mosquito strains: Isolate numbers 1830 and 1831, found in *Aedes cinereus* and *Coquillettidia richiardii* in Russia. This highlights the lack of concordance between *Wolbachia* strain phylogeny and their insect hosts across diverse geographical regions.

We also found evidence of potential strain variants in *wAnS* through variable MLST gene fragment amplification and resulting closest-match allele numbers. A second *wAnS*-infected sample from Katana, *An. sp.* A/1 (W+) DRC-KAT2, only successfully amplified *hcpA* and *coxA* gene fragments and although identical sequences were obtained for *wsp* (Figure 4b) and *hcpA*, genetic diversity was seen in the *coxA* sequences, with typing indicating a different, but still novel allele for the *coxA* sequence from this individual (*wAnS*(2) *coxA* DRC-KAT2) (Figure 5b). Further analysis of the *coxA* sequence as part of MLST allele submission from this variant suggested the possibility of a double infection, where two differing strains of *Wolbachia* are present. MLST gene fragment amplification was also variable for *wAnga-Ghana*-infected *An. coluzzii*, requiring two individuals to generate the five MLST gene sequences, and for the *hcpA* locus, more degenerate primers (*hcpA* F3/*hcpA* R3) were required to generate sequence of sufficient quality for analysis. This is likely due to the low density of this strain potentially influencing the ability to successfully amplify all MLST genes, in addition to the possibility of genetic variation in primer binding regions. Despite the sequences generated for this strain producing exact matches with alleles in the database for each of the five gene loci, the resultant allelic profile, and therefore strain type, did not produce a match, showing this *wAnga-Ghana* strain is also a novel strain type. The closest matches to the *wAnga-Ghana* allelic profile were with strains from two Lepidopteran species: Isolate number 609 found in *Fabriciana adippe* from Russia, and Isolate number 658 found in *Pammene fasciana* from Greece,

<table><caption>**Table 2.** Novel resident *Wolbachia* strain WSP typing and multilocus sequence typing (MLST) gene allelic profiles. Novel allele numbers (in bold) assigned by the *Wolbachia* MLST database for strains from *An.* species A (*wAnS*) and *An. moucheti* (*wAnM*) are shown, alongside the novel allelic profile from *An. coluzzii* (*wAnga-Ghana*), comprising exact matches to existing alleles present in the database for each gene locus. (HVR; Hypervariable regions within the *wsp* sequence.).</caption><thead><tr><th rowspan="2">Mosquito species</th><th rowspan="2">Wolbachia strain</th><th colspan="5">WSP typing allele numbers</th><th colspan="5">MLST gene allele numbers</th></tr><tr><th>wsp</th><th>HVR1</th><th>HVR2</th><th>HVR3</th><th>HVR4</th><th>gatB</th><th>coxA</th><th>hcpA</th><th>ftsZ</th><th>fbpA</th></tr></thead><tbody><tr><td>An. species A</td><td>wAnS</td><td>728</td><td>254</td><td>288</td><td>284</td><td>23</td><td>279</td><td>274</td><td>302</td><td>240</td><td>445</td></tr><tr><td>An. moucheti</td><td>wAnM</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>280</td><td>275</td><td>303</td><td>241</td><td>446</td></tr><tr><td>An. coluzzii</td><td>wAnga-Ghana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>9</td><td>64</td><td>3*</td><td>177</td><td>4</td></tr></tbody></table>

<footnote>*Alternative degenerate primers (set 3) were used to generate sequence from another *An. coluzzii* individual from the same location to complete the full allelic profile.</footnote>
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

**Figure 5.** *Wolbachia* multilocus sequence typing (MLST) phylogenetic analysis of resident *Wolbachia* strains in *An. coluzzii*, *An. moucheti* and *An. species A*. (A) Maximum Likelihood molecular phylogenetic analysis from concatenation of all five MLST gene loci for resident *Wolbachia* strains from *An. coluzzii* (*wAnga-Ghana; blue*), *An. moucheti* (*wAnM; green*) and *An. species A* (*wAnsA; red*). The tree with the highest log likelihood (-10606.13) is shown and drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 94 nucleotide sequences. There were a total of 2067 positions in the final dataset. Concatenated sequence data from *Wolbachia* strains downloaded from MLST database for comparison are shown with isolate numbers in brackets (IsoN). *Wolbachia* strains isolated from mosquito species highlighted in navy blue, bold. Strains isolated from other Dipteran species are shown in navy blue, from Coleoptera in olive green, from Hemiptera in purple, from Hymenoptera in teal blue, from Lepidoptera in maroon and from other, or unknown orders in black. (B) Maximum Likelihood molecular phylogenetic analysis for *coxA* gene locus for resident *Wolbachia* strains from *An. coluzzii* (*wAnga-Ghana; blue*), *An. moucheti* (*wAnM; green*) and *An. species A* (*wAnsA* and *wAnsA*(2); red). The tree with the highest log likelihood (-1921.11) is shown and drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 84 nucleotide sequences. There were a total of 402 positions in the final dataset. Sequence data for the *coxA* locus from *Wolbachia* strains downloaded from MLST database for comparison are shown in black and navy blue with isolate numbers (IsoN) from the MLST database shown in brackets. *Wolbachia* strains isolated from mosquito species highlighted in navy blue. GenBank sequence for *wAnga-Mali* *coxA* shown in maroon with accession number.
but each of these only produced a match for three out of the five loci. The concatenated phylogeny for this strain (Figure 5a) indicates that across the 5 MLST loci, *wAnga*-Ghana is actually most closely related to a Lepidopteran strain found in *Thersamo-nia thersamon* in Russia (Isolate number 132). The phylogeny of *Wolbachia* strains based on the *coxA* gene (Figure 5b) highlights the genetic diversity of both the *wAnSA* strain variants and also *wAnga*-Ghana, compared to the *wAnga*-Mali strain⁴⁰; *coxA* gene sequences are not available for *wAnga* strains from Burkina Faso³⁹. All *Wolbachia* MLST sequences were deposited into GenBank (accession numbers MH605286–MH605305; listed in Supplementary Table 3).

## Resident strain densities and relative abundance

The relative densities of *Wolbachia* strains were estimated using qPCR targeting the *ftsZ*⁵⁶ and *16S rRNA*⁴⁰ genes. qPCR analysis of *ftsZ* and *16S rRNA* indicated the amount of *Wolbachia* detected in *wAnSA*-infected and *wAnM*-infected females was three orders of magnitude higher (Ct values 20–22) than *Wolbachia*-infected *An. gambiae* s.s., *An. arabiensis* and *wAnga*-Ghana-infected *An. coluzzii* (Ct values 30–33). To account for variation in mosquito body size and DNA extraction efficiency, we compared the total amount of DNA for *Wolbachia*-infected mosquito extracts and conversely, we found less total DNA in the *wAnSA*-infected extract (1.36 ng/µl) and *wAnM*-infected extracts (5.85 ng/µl) compared to the mean of 6.64 ± 2.33 ng/µl for *wAnga*-Ghana-infected *An. coluzzii*. To estimate the relative abundance of resident *Wolbachia* strains in comparison to other bacterial species, we sequenced the bacterial microbiome using *16S rRNA* amplicon sequencing on *Wolbachia*-infected individuals. We found *wAnSA*, *wAnSA* (2) and *wAnM* *Wolbachia* strains were the dominant OTUs of these mosquito species (Figure 6). In contrast, the lower-density infection *wAnga*-Ghana strain represented only ~10% of the OTUs within the microbiome.

## *P. falciparum*, *Wolbachia* and *Asaia* prevalence

The prevalence of *P. falciparum* in female mosquitoes was extremely variable across countries and collection locations (Figure 1 and Table 1) with very high prevalence recorded in *An. gambiae* s.s. from villages close to Boke (52%) and Faranah (44%) in Guinea. Despite the collection of other *Anopheles* species in Guinea, *An. gambiae* s.s. was the only species to have detectable malaria parasite infections. In contrast, *P. falciparum* was detected in multiple major vector species from DRC, including *An. gambiae* s.s., *An. arabiensis* and *An. funestus* s.s. A high prevalence of *P. falciparum* was also detected in *An. gambiae* s.s. from Uganda for both collection years; 19% for 2013 and 36% for 2014. In contrast, no *P. falciparum* infections were detected in any of the *An. coluzzii* or *An. melas* collected in Ghana. In Madagascar, *P. falciparum* was detected in only two species; *An. gambiae* s.s. and *An. rufipes*. We compared the overall *P. falciparum* infection rates in *An. gambiae* s.s. mosquitoes collected across all locations from DRC to determine if there was any correlation with the presence of the low density *wAnga*-DRC *Wolbachia* resident strain. Overall, of the 128 mosquitoes collected, only 1.56% (n=2) had detectable *Wolbachia-Plasmodium* co-infections, compared to 10.16% (n=13) where we only detected *Wolbachia*. A further 11.72% (n=15) were only PCR-positive for *P. falciparum*. As expected, for the vast majority of mosquitoes (76.56%, n=98) we found no evidence of *Wolbachia* or *P. falciparum* present, resulting in no correlation across all samples (Fisher's exact *post hoc* test on unnormalized data, two-tailed, *P*=0.999). Interestingly, one *An. species* A female from Katana, DRC (infected with *wAnSA*) was co-infected with *P. falciparum*.

For all *Wolbachia*-infected females collected in our study (including *An. coluzzii* from Ghana and novel resident strains in *An. moucheti* and *An. species A*), we did not detect the presence of *Asaia*. No resident *Wolbachia* strain infections were detected

<figure><img src="image_13.png" /><figcaption>**Figure 6.** The relative abundance of resident *Wolbachia* strains in *Anopheles*. Bacterial genus level taxonomy was assigned to operational taxonomic units clustered with a 97% cut-off using the SILVA SSU v128 97% database, and individual genera comprising less than 1% of total abundance was merged into "Others".</figcaption></figure>
in *Anopheles* mosquitoes from Guinea, Uganda or Madagascar.
However, high *Asaia* and malaria parasite prevalence rates were
present in *Anopheles* mosquitoes from Uganda and Guinea (includ-
ing in multiple species in all four sites in Guinea). We compared
the overall *P. falciparum* infection rates in *An. gambiae* s.s.
collected across all locations from Guinea, with and without *Asaia*
bacteria, and found no overall correlation (Fisher's exact *post
hoc* test on unnormalized data, two-tailed, *P*=0.4902). There was
also no overall correlation between *Asaia* and *P. falciparum* infec-
tions in *An. gambiae* s.s. from Uganda for both 2013 (Fisher's
exact *post hoc* test on unnormalized data, two-tailed, *P*=0.601)
and 2014 (Fisher's exact *post hoc* test on unnormalized data,
two-tailed, *P*=0.282).

Asaia can be environmentally acquired at all life stages but can
also have the potential to be vertically and horizontally transmit-
ted between individual mosquitoes. Therefore, we performed
16S microbiome analysis on a sub-sample of Asaia-infected
An. gambiae s.s. from Kissidougou (Guinea), a location in
which high levels of Asaia were detected by qPCR (mean Asaia
Ct = 17.84 ± 2.27)⁶⁴. Asaia in these individuals is the dominant
bacterial species present (Figure 7a) but in Uganda we detected
much lower levels of Asaia (qPCR mean Ct = 33.33 ± 0.19)
and this was reflected in Asaia not being a dominant species in
microbiome analysis (Figure 7b). The alpha and beta diversity
of An. gambiae s.s. from Kissidougou, Guinea and Butemba,
Uganda shows much more overall diversity in the microbiome for

Uganda individuals (Supplementary Figure 1). Interestingly, 2/5 of
these individuals from Kissidougou (Guinea) were *P. falciparum*-infected compared to 3/5 individuals from Uganda. To determine
if the presence of *Asaia* had a quantifiable effect on the level of
*P. falciparum* detected, we normalized *P. falciparum* Ct values from qPCR (n = 61) (Supplementary Figure 2a) and compared gene ratios for *An. gambiae* s.s. mosquitoes from Guinea, with or without *Asaia* (Supplementary Figure 2b). Statistical analysis using student's t-tests revealed no significant difference
between normalized *P. falciparum* gene ratios between the *Asaia* positive (n = 33) and negative (n = 28) groups (p = 0.51, df = 59).
Larger variation of Ct values was seen for *Asaia* (n = 90) (Supplementary Figure 2c) suggesting the bacterial densities in
individual mosquitoes were more variable than *P. falciparum* parasite infection levels.

Discussion

Malaria transmission in Sub-Saharan Africa is highly depend-
ent on the local *Anopheles* vector species, but the primary vector
complexes recognised are *An. gambiae* s.l., *An. funestus* s.l.
*An. nili* s.l. and *An. moucheti* s.l.⁴¹,⁶⁵. *An. gambiae* s.s. and
*An. coluzzii* sibling species are considered the most important
malaria vectors in Sub-Saharan Africa and recent studies indicate
that *An. coluzzii* extends further north, and closer to the coast
than *An. gambiae* s.s. within West Africa⁶⁶. In our study, high
*Plasmodium* prevalence rates in *An. gambiae* s.s. across Guinea
would be consistent with high malaria parasite prevalence in

<figure><img src="image_14.png" /><figcaption>A</figcaption></figure>

Kissidougou (Guinea)

<figure><img src="image_15.png" /><figcaption>B</figcaption></figure>

Butemba (Uganda)

**Figure 7.** The relative abundance of bacteria in *An. gambiae* s.s. comparing two locations with contrasting *Asaia* infection densities. Bacterial genus level taxonomy was assigned to operational taxonomic units clustered with a 97% cut-off using the SILVA SSU v128 97% database, and individual genera comprising less than 1% of total abundance was merged into "Others".
humans (measured by rapid diagnostic tests) in Guéckédou pre-
fecture, and the overall national malaria prevalence, estimated to
be 44% in 2013⁶⁷. However, malaria prevalence has decreased in
the past few years with an overall prevalence across Guinea esti-
mated at 15% for 2016. Although our *P. falciparum* infection
prevalence rates were also high in DRC, recent studies have shown
comparable levels of infection with 35% of *An. gambiae* s.l. mos-
quitoes infected from Kinshasa⁶⁸. We detected *P. falciparum* in
*An. gambiae* s.s., *An. arabiensis*, *An. funestus* s.s. and *An. spe-
cies A* from DRC. Morphological differences have been widely
used for identification of malaria vectors but species complexes
(such as *An. gambiae* s.l. and *An. funestus* s.l.) require species-
diagnostic PCR assays. Historically, malaria parasite entomol-
ogy studies in Africa have focused predominantly on species
from these complexes, likely due to the fact that mosquitoes from
these complexes dominate the collections⁴³. In our study, we used
ITS2 sequencing to confirm secondary vector species that were
*P. falciparum*-infected given the difficulties of morphologi-
cal identification and recent studies demonstrating the inaccu-
racy of diagnostic species PCR-based molecular identification⁶⁹.
Our study is the first to report the detection of *P. falciparum*
in *An. rufipes* from Madagascar; previously this species was
considered a vector of *Plasmodium* species of non-human ori-
gin and has only very recently been implicated in human malaria
transmission⁷⁰. However, detection of *P. falciparum* parasites in
whole body mosquitoes does not confirm that the species plays
a significant role in transmission. Detection could represent
infected bloodmeal stages or oocysts present in the midgut wall
so further studies are warranted to determine the ability of this
species to transmit human malaria parasites.

The mosquito microbiota can modulate the mosquito immune
response and bacteria present in wild *Anopheles* populations
can influence malaria vector competence⁴,⁵. Endosymbiotic
*Wolbachia* bacteria are particularly widespread through insect
populations, but they were commonly thought to be absent
from *Anopheles* mosquitoes. However, the recent discovery of
*Wolbachia* strains in *An. gambiae* s.l. in Burkina Faso and Mali³⁹,⁴⁰,
in addition to our study showing infection in *Anopheles* from
Ghana and DRC, suggest resident strains could be widespread
across Sub-Saharan Africa. The discovery of resident strains in
Burkina Faso resulted from sequencing of the 16S rRNA
gene identifying *Wolbachia* sequences rather than screening
using *Wolbachia*-specific genes³⁹. Intriguingly, *Wolbachia* infec-
tions in these mosquitoes could not be detected using conven-
tional PCR targeting the *wsp* gene. As the *wsp* gene has often
been used in previous studies to detect strains in *Anopheles*
species²⁵,²⁷, this could explain why resident strains in the
*An. gambiae* complex have gone undetected until very recently.
Recent similar methods using 16S rRNA amplicon sequenc-
ing to determine the overall microbiota in wild mosquito pop-
ulations has provided evidence for *Wolbachia* infections in
*An. gambiae* s.l. in additional villages in Burkina Faso⁷¹ and
*Anopheles* species collected in Illinois, USA⁷². Our study describ-
ing resident *Wolbachia* strains in numerous species of *Anopheles*
malaria vectors also highlights the potential for *Wolbachia* to
be influencing malaria transmission, as postulated by previous
studies³⁹,⁴⁰,⁷³. No significant correlation was present in our

study for *Plasmodium* and *Wolbachia* prevalence in the 128 *An. gambiae* s.s. individuals from DRC. As the majority (77%) of samples had neither detectable *Wolbachia* resident strains or *P. falciparum*, a larger sample size would provide a more comprehensive assessment factoring in the *Plasmodium* parasite life stages. Although there is evidence from previous studies that *Wolbachia* is negatively correlated with *Plasmodium* in both Burkina Faso⁷³ and Mali⁴⁰, our infection prevalence rates for resident *Wolbachia* strains in *An. coluzzii* from Ghana (4%) and *An. gambiae* s.s. from the DRC were variable but low (8–24%). These results are more aligned to infection prevalence rates in *An. gambiae* s.l. from Burkina Faso (11%)³⁹ but much lower than those reported in Mali (60–80%)⁴⁰ where infection was associated with reduced prevalence and intensity of sporozoite infection in field-collected females.

The discovery of a resident *Wolbachia* strain in *An. moucheti*, a highly anthropophilic and efficient malaria vector found in the forested areas of Western and Central Africa⁴¹, suggests further studies are warranted that utilize large sample sizes to examine the influence of the *wAnM Wolbachia* strain on *Plasmodium* infection dynamics in this malaria vector. *An. moucheti* is often the most abundant vector, breeding in slow moving streams and rivers, contributing to year round malaria transmission in these regions⁷⁴,⁷⁵. This species has also been implicated as a main bridge vector species in the transmission of ape *Plasmodium* malaria in Gabon⁷⁶. There is thought to be high genetic diversity in *An. moucheti* populations⁷⁷,⁷⁸, which may either influence the prevalence of *Wolbachia* resident strains, or *Wolbachia* could be contributing to genetic diversity through its effect on host reproduction. A novel *Wolbachia* strain in *An. spe*cies A, present at high infection frequencies in Lwiro (close to Katana in DRC), also suggests more *Anopheles* species, includ-ing unidentified and potentially new species, could be infected with this widespread endosymbiotic bacterium. *An. species* A should be further investigated to determine if this species is a potential malaria vector, given our study demonstrated *P. falciparum* infection in one of two individuals screened and ELISA-positive samples of this species were reported from the Western Highlands of Kenya¹².

The variability of *Wolbachia* prevalence rates in *An. gambiae*
complex from locations within DRC and Ghana and previous
studies in Burkina Faso³⁹ and Mali⁴⁰ suggest the environment
is one factor that influences the presence or absence of resident
strains. In our study we found no evidence of *Wolbachia-Asaia*
co-infections across all countries, supporting laboratory stud-
ies that have shown these two bacterial species demonstrate
competitive exclusion in *Anopheles* species³⁶,³⁸. We also found
that *Asaia* infection densities (whole body mosquitoes) were
variable and location dependent which would correlate with
this bacterium being environmentally acquired at all life stages,
but also having the potential for both vertical and horizontal
transmission³⁷. Significant variations in overall *Asaia* prevalence
and density across different *Anopheles* species and locations
in our study would also correlate with our data indicating no evi-
dence of an association with *P. falciparum* prevalence in both
Guinea and Uganda populations. Further studies are needed to
determine the complex interaction between these two bacterial species and malaria in diverse *Anopheles* malaria vector species. Horizontal transfer of *Wolbachia* strains between species (even over large phylogenetic differences) has shaped the evolutionary history of this endosymbiont in insects, and there is evidence for loss of infection in host lineages over evolutionary time⁷⁹. Our results showing a novel strain present in *An. coluzzii* from Ghana (phylogenetically different to strains present in *An. gambiae* s.l. mosquitoes from both Burkina Faso and Mali), strain variants observed in *An. species A*, and the concatenated grouping of the novel *Anopheles* strains with strains found in different Orders of insects, support the lack of congruence between insect host and *Wolbachia* strain phylogenies⁸⁰.

Our qPCR and 16S microbiome analysis indicates the densities of wAnM and wAnsA strains are significantly higher than resident *Wolbachia* strains in *An. gambiae* s.l. However, caution must be taken as we were only able to analyse selected individuals, and larger collections of wild populations would be required to confirm these results. Native *Wolbachia* strains dominating the microbiome of *An. species A* and *An. moucheti* is consistent with other studies of resident strains in mosquitoes showing *Wolbachia* 16S rRNA gene amplicons vastly outnumber sequences from other bacteria in *Ae. albopictus* and *Cx. quinquefasciatus*⁸¹,⁸². The discovery of novel *Wolbachia* strains provides the rationale to undertake vector competence experiments to determine what effect these strains are having on malaria transmission. The tissue tropism of novel *Wolbachia* strains in malaria vectors will be particularly important to characterise given this will determine if these endosymbiotic bacteria are proximal to malaria parasites within the mosquito. It would also be important to determine the additional phenotypic effects novel resident *Wolbachia* strains have on their mosquito hosts. Some *Wolbachia* strains induce a reproductive phenotype termed cytoplasmic incompatibility (CI) that results in inviable offspring when an uninfected female mates with a *Wolbachia*-infected male. In contrast, *Wolbachia*-infected females produce viable progeny when they mate with both infected and uninfected male mosquitoes. This reproductive advantage over uninfected females allows *Wolbachia* to spread within mosquito populations.

## Conclusions

*Wolbachia* has been the focus of recent biocontrol strategies in which *Wolbachia* strains transferred into naïve mosquito species provide strong inhibitory effects on arboviruses¹⁶,¹⁸⁻²⁰,⁸³,⁸⁴ and malaria parasites³¹,³⁵. The discovery of two novel *Wolbachia* strains in *Anopheles* mosquitoes that are potentially present at much higher density than resident strains in the *An. gambiae* complex, also suggests the potential for these strains to be transinfected into other *Anopheles* species to produce inhibitory effects on *Plasmodium* parasites. *Wolbachia* transinfection success is partly attributed to the relatedness of donor and recipient host so the transfer of high density *Wolbachia* strains between *Anopheles* species may result in stable infections (or co-infections) that have strong inhibitory effects on *Plasmodium* development. Finally, if the resident strain present

in *An. moucheti* is at low infection frequencies in wild populations, an alternative strategy known as the incompatible insect technique (IIT) could be implemented where *Wolbachia*-infected males are released to suppress the wild populations through CI (reviewed by ²²). In summary, the important discovery of diverse novel *Wolbachia* strains in *Anopheles* species will help our understanding of how *Wolbachia* strains can potentially impact malaria transmission, through natural associations or being used as candidate strains for transinfection to create stable infections in other species.

## Data availability

ITS2 GenBank accession numbers are listed in Supplementary Table 1; *Wolbachia* 16S and wsp gene GenBank accession numbers are listed in Supplementary Table 2; *Wolbachia* MLST gene GenBank accession numbers are listed in Supplementary Table 3.

Raw PCR screening data is available at Open Science Framework: DOI: https://doi.org/10.17605/OSF.IO/MW6XZ⁶⁴.

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

## Grant information

CLJ and TW were supported by a Wellcome Trust /Royal Society grant awarded to TW (101285): http://www.wellcome.ac.uk; https://royalsociety.org. GLH is supported by NIH grants (R21AI124452 and R21AI129507), a University of Texas Rising Star award, the John S. Dunn Foundation Collaborative Research Award, the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation, and the Centers for Disease Control and Prevention (CDC) (Cooperative Agreement Number U01CK000512). The papers contents are solely the responsibility of the authors and do not necessarily represent the official views of the CDC or the Department of Health and Human Services. This work was also supported by a James W. McLaughlin postdoctoral fellowship at the University of Texas Medical Branch to SH. Field work in Uganda was funded by UK aid (through the Programme Partnership Arrangement grant to Malaria Consortium). YAA and ARM were supported by a NIH grant R01AI123074. SRI was funded by the U.S. President's Malaria Initiative.

*The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.*

## Acknowledgements

We would like to thank all the mosquito collectors and residents of the villages where collections took place. We would also like to thank John Gimnig, Bill Hawley and Barb Marston for reviewing our manuscript. This publication made use of the PubMLST website (https://pubmlst.org/wolbachia/) sited at the University of Oxford (Jolley & Maiden 2010, BMC Bioinformatics, 11:595). The development and maintenance of this site has been funded by the Wellcome Trust.
## Supplementary material

Supplementary Table 1. Additional sample details and ITS2 GenBank accession numbers.

Click here to access the data.

Supplementary Table 2. *Wolbachia* 16S and wsp GenBank accession numbers.

Click here to access the data.

Supplementary Table 3. *Wolbachia* MLST gene GenBank accession numbers.

Click here to access the data.

Supplementary Figure 1. Alpha and beta diversity of *An. gambiae* s.s. from Kissidougou, Guinea and Butemba, Uganda.

Click here to access the data.

Supplementary Figure 2. Prevalence of the bacterial species *Asaia* and malaria parasites in *An. gambiae* s.s. mosquitoes from Guinea.

Click here to access the data.

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# Open Peer Review

Current Referee Status:

<figure><img src="image_16.png" /></figure>

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

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

## Version 2

Referee Report 11 December 2018

https://doi.org/10.21956/wellcomeopenres.16284.r34345

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

Ottavia Romoli ID<fnref n="1" />, Mathilde Gendrin ID<fnref n="2" />,<fnref n="3" />

Our main comments have been addressed in the revised manuscript.

We still don't understand what An. species A is (and even An species O). It is not referenced in Vectorbase, so is it just a species that the authors were not able to identify, or a recently identified species that we are not aware of? A short explanation on this would be helpful, especially that Wolbachia was specifically found at high prevalence in this mosquito species.

Except for this small point, we think that the manuscript is sound and clear, and that conclusions are drawn adequately.

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

**We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.**

## Version 1

Referee Report 08 November 2018

https://doi.org/10.21956/wellcomeopenres.16091.r33880

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

Christophe Antonio-Nkondjio ID

Malaria Research Laboratory, Organisation for Coordination in the Fight Against Endemic Diseases in Central Africa, Yaoundé, Cameroon

Good study by Jefferies et al. presenting the distribution of Wolbachia strains in anopheles species from different sub-Saharan Africa countries.

**General comment**

In the method section the authors say DNA was extracted from whole mosquitoes or abdomen for their analysis. What are the chances that wolbachia infections cases reported in the paper could be due to parasites contain in the blood meal rather than true infection of mosquitoes?

<footnote n="1">¹ Microbiota of Insect Vectors Group, Institut Pasteur de la Guyane, Cayenne, French Guiana</footnote>

<footnote n="2">² Microbiota of Insect Vectors Group, Institut Pasteur de la Guyane, Cayenne, French Guiana</footnote>

<footnote n="3">³ Parasites and Vectors Department, Institut Pasteur, Paris, France</footnote>
# Methods

## Study sites & collection methods

* "Democratic Republic of the Congo" change to "Democratic Republic of Congo"

* Collection sites it will be interesting to indicate from the coordinates if it is Latitude North/South or longitude East/West the paper is also for non specialists in the domain.

**Figure 1:**

* B, C, D in the legend it is mentioned "P. falciparum prevalence" is it for human or mosquitoes please provide precision. (% Positive ??, % Negative ??)

* P. falciparum should be in italics.

* Figure 1A: It should be interesting to indicate the names of study sites. The authors could label the sites by using number for sites for each country 1, 2, 3 ... then providing in the legend what 1 is placed for.

**Figure 6:** legend not clear.

**Figure 7 B:** The legend is not clear (can't read anything).

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

Yes

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

Yes

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

Yes

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

Yes

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

Yes

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

Yes

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

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

Referee Report 05 November 2018

https://doi.org/10.21956/wellcomeopenres.16091.r34140

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Anne Duplouy

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

Lund University, Lund, Sweden
Jeffries and colleagues provide here a study of the prevalence and penetrance of *Wolbachia* infection in several species of *Anopheles* mosquitoes across several Sub-Saharan countries. To my knowledge this is the largest such study on the topics. This study adds important data on growing evidence that species of the genus *Anopheles* can host the infection, thus contrasting with previous reports suggesting that the bacterium was absent from these mosquitoes.

Furthermore, the authors investigate the tripartite occurrence between *Wolbachia*, the parasite *Plasmodium* and another symbiotic bacterium *Asaia*. The article supports previous studies suggesting niche competition between *Wolbachia* and *Asaia*, as none of the samples carry both bacteria. The study does not, however, provide field-based evidence that the presence of *Wolbachia* and/or *Asaia* in the mosquitoes would affect parasitism by *Plasmodium*.

This research is timely. With the development of new pest control strategies using *Wolbachia* as a natural biological agent against the transmission of several vector-borne diseases in the field, it is important to have a comprehensive understanding of the diversity of the natural infections already present in the field, but also of the different factors that could affect the efficiency of such control programs. Including the presence of competing natural infection by *Asaia* bacteria for example.

The study is well written and clear, with the sufficient information included to support future potential replication. I think this is a fine contribution to the current literature, I have only minor comments to the authors.

It might be worth modifying the text in the abstract, and the introduction, to specify that the previous reports of *Wolbachia* in *Anopheles* were only from 2 *West-African* countries, while the current study is providing data from 5 countries across the Sub-Saharan region.

**Method:**

Please provide information on how the maps of figure 1 were generated. Did you need any approval/licenses for using these maps?

Please provide information on collection permits, if any was needed from the different African countries.

What is CDC standing for in the method section? ‘CDC-light trap’

In the *Wolbachia* detection method section:

Edit typo: 'was used AS a positive control'

Table 1: What is the rational for the authors to provide the information by countries rather than by species? Isn't the most interesting point of the paper about the infection being reported in additional species of *Anopheles*?

Figure 2: Explain the significance of the difference square/circle/triangle shapes and filled vs empty shapes? Also state in legend that the codes given are the Genbank Accession numbers.

Figure 2: Where did you get the sequences from the *An. bwambae* and *An. quadriannulatus*? I think this info is missing from the method section.

*P. falciparum*, *Wolbachia* and *Asaia* prevalence section, paragraph 2:

Does your analysis include the *P. falciparum* and *Wolbachia* infected specimens? Would it make any difference to remove the *Wolbachia*-infected specimens from the analysis?
Discussion section, end of 4th paragraph:

'New' strain in *An. Coluzzii* from Ghana. 'New' sounds like the infection is more recent than any other infection found in this mosquito species, which the results are not supporting. Would 'unique' or 'different' be good enough?

Figure 7: Where is *Asaia* from Figure 7b? from the current picture it looks like *Asaia* is absent from those samples. Although the text states that the infection is not a dominant species of those samples. If *Asaia* is included in the 'Others' maybe it is worth specifying it in the legend, otherwise it could be added as a particular section of the graph like in Figure 7a to ease comparison of the two panels.

Figure S1: Why are some of the circles slightly larger than others? Is it that different samples are overlapping?

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

Yes

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

Yes

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

Yes

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

Yes

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

Partly

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

Yes

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

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

Referee Report 21 September 2018

https://doi.org/10.21956/wellcomeopenres.16091.r33884

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

**Mathilde Gendrin** [id 1,2](id 3)

Jeffries and co-authors performed a large scale analysis of the presence of Wolbachia in Anopheles mosquitoes from five countries in Africa. They found that in two of these countries, some mosquitoes were

<footnote n="1">¹ Microbiota of Insect Vectors Group, Institut Pasteur de la Guyane, Cayenne, French Guiana</footnote>

<footnote n="2">² Parasites and Vectors Department, Institut Pasteur, Paris, France</footnote>

<footnote n="3">³ Microbiota of Insect Vectors Group, Institut Pasteur de la Guyane, Cayenne, French Guiana</footnote>
infected with Wolbachia, confirming and widening the recent discovery of the presence of Wolbachia in Anopheles mosquitoes. This is the strongest point of the paper, as an independent confirmation is always welcome and as some populations of Anopheles are even found here to have a high prevalence of Wolbachia.

The authors also checked for the presence of Asaia sp. in the analysed mosquitoes, as this bacterium is thought to compete with Wolbachia in Anopheles. They did not find any mosquito co-infected by Asaia and Wolbachia. This is also an important finding as it corroborates studies performed in the laboratory, but this time with field-collected mosquitoes. They found that in mosquitoes coming from one population, Asaia was actually a dominant species, >99% of the microbiota. Figure 7a is not very clear as one expects the scale to go from 0 to 100%, therefore we suggest to use a discontinued axis to present these interesting results.

Finally the authors investigated the presence of Plasmodium in the studied mosquitoes, as Wolbachia is thought to interfere with some transmitted pathogens. This part is less convincing as the tests have been performed on DNA extraction from whole bodies or abdomens, while the presence of Plasmodium in head and thorax (or more specifically, in salivary glands) is a more suitable method to assess transmission potential. Moreover, the conclusions drawn on the interactions between Plasmodium and Wolbachia are not exactly clear. Considering that 10.16 + 1.56 = 11.72% mosquitoes are infected with Wolbachia and 11.72 + 1.56 = 13.28% are infected with Plasmodium, if there is no effect between Wolbachia and Plasmodium, you expect that 11.72% x 13.28% = 1.56% is infected by both. Surprisingly, this is exactly the result here. Biology is rarely so close to math, for so small numbers... The authors should thus state more clearly that their results favor no interactions, as confirmed by the p value which is very close to 1. On the contrary, the discussion currently suggests that the non significant correlation is due to small numbers. However, one cannot jump to conclusion on the inability of Wolbachia to interfere with Plasmodium, as these results have been performed on abdomens and whole bodies, therefore we do not know whether the co-infected mosquitoes had just blood fed (and/or carried early stages of Plasmodium).

To improve the clarity of the article, it would be interesting to have an additional figure or table summarizing the experimental set up, explaining which mosquitoes are included in which analysis and which Wolbachia strain is found in which mosquitoes.

We also have minor comments on the manuscript:

The expression « resident strain » is not clear to us.

16S « rRNA » and « rDNA »: a consistent word may be used, rRNA seems more consensual.

The total number of mosquitoes, of Wolbachia infected mosquitoes, of Asaia infected ones, etc would be interesting.

Page 3:

§2: Asaia is not an endosymbiont

§3: « have » needs probably to be removed in « than would have be identified using morphological identification alone »

§4 needs a first sentence identifying the gap of knowledge that the authors want to fill

§5: Can the authors clearly state whether some mosquitoes had blood in their midgut?

Page 4:

Figure 1: scale should be in km, miles is not an SI unit
§2: « DNA extraction and MOSQUITO species identification ». More generally, it is not always clear whether the authors speak about mosquitoes or Wolbachia strains.

§3: « as preliminary trials revealed this was the optimal method for both sensitivity and specificity »: please add « data not shown » or remove it

Page 5:

Instead of µL of DNA, the actual quantity in ng would be preferable.

All PCRs: primer sequences are needed

§3: « Both chromatograms (forward and reverse traces) from each sample WERE manually »

Pages 6-7

Table 1: probably some mistakes, e.g. An. gambiae in Mikalayi: 11.8% corresponds neither to 1/16 nor to 2/16, so all the numbers should be checked. It would be appropriate to enter the actual numbers in brackets, and to indicate the co-prevalence of Wolbachia and Plasmodium. The legend should be grouped below or above the table and the explanation about mosquitoes in bold is unclear.

In the text, numbers would be interesting rather than only proportions.

« previously named M molecular form OF AN. GAMBIAE » (or remove it, as this precision may now be superfluous). On the contrary, « An. species A » is barely introduced, it would be interesting to mention something about this species and its identification (besides the quick explanation in the introduction).

Page 13

« Approximately 1000-fold higher », it is very much of an approximation (variable Ct values and potential variations in 16S copy number): it may be good to rephrase, mentioning that 1000 is an order of magnitude rather than approximately.

§2: « An. moucheti (wAnM-infected) » comes at the 2nd occurrence of wAnM.

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

Yes

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

Yes

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

Partly

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

Yes

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

Yes

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

Partly

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

We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.
Author Response 13 Nov 2018

**Thomas Walker**, London School of Hygiene & Tropical Medicine, UK

Dear Mathilde and Ottavia,

Firstly many thanks for your thoughtful and comprehensive review of our manuscript. We have tried to address all your comments below in **bold**:

Figure 7a is not very clear as one expects the scale to go from 0 to 100%, therefore we suggest to use a discontinued axis to present these interesting results.

**We agree and have modified this figure for clarity**

Finally the authors investigated the presence of Plasmodium in the studied mosquitoes, as Wolbachia is thought to interfere with some transmitted pathogens. This part is less convincing as the tests have been performed on DNA extraction from whole bodies or abdomens, while the presence of Plasmodium in head and thorax (or more specifically, in salivary glands) is a more suitable method to assess transmission potential. Moreover, the conclusions drawn on the interactions between Plasmodium and Wolbachia are not exactly clear. Considering that 10.16 + 1.56 = 11.72% mosquitoes are infected with Wolbachia and 11.72 + 1.56 = 13.28% are infected with Plasmodium, if there is no effect between Wolbachia and Plasmodium, you expect that 11.72% x 13.28% = 1.56% is infected by both. Surprisingly, this is exactly the result here. Biology is rarely so close to math, for so small numbers... The authors should thus state more clearly that their results favor no interactions, as confirmed by the p value which is very close to 1. On the contrary, the discussion currently suggests that the non significant correlation is due to small numbers. However, one cannot jump to conclusion on the inability of Wolbachia to interfere with Plasmodium, as these results have been performed on abdomens and whole bodies, therefore we do not know whether the co-infected mosquitoes had just blood fed (and/or carried early stages of Plasmodium)

**We agree and have modified our discussion on these results to make more appropriate conclusions based on our data**

To improve the clarity of the article, it would be interesting to have an additional figure or table summarizing the experimental set up, explaining which mosquitoes are included in which analysis and which Wolbachia strain is found in which mosquitoes.

**Many thanks for this suggestion. After careful consideration, we feel that an additional figure or table is not needed given we have figure 1 showing which *Anopheles* species were Wolbachia-infected and from which locations within countries and have all the PCR screening data from all samples available from Open Science Framework: DOI 10.17605/OSF.IO/MW6XZ in addition to sample details for all accession numbers in the supplementary tables.**

However, we have also modified table 1 to provide the comparison between Plasmodium-infected, Wolbachia-infected, Asaia-infected, co-infected individuals and uninfected individuals across all collection sites.

We also have minor comments on the manuscript:
The expression « resident strain » is not clear to us.

‘Resident’ *Wolbachia* strains are considered to have resulted naturally and have an evolutionary association with the host (wAlbA and wAlbB in Ae. albopictus) rather than have been generated artificially through transinfection (eg. wMel in Ae. aegypti).

We have modified our introduction to make this clearer by the inclusion of 'those naturally present in wild insect populations'

16S « rRNA » and « rDNA »: a consistent word may be used, rRNA seems more consensual.

We agree with this. For Wolbachia screening and phylogeny including strains in Anopheles, 16S rRNA is most commonly used (de Oliveira et al. 2015: Werren & Windsor 2000; Gomes et al. 2017; Baldini et al. 2014). We have checked our manuscript and corrected these errors.

The total number of mosquitoes, of Wolbachia infected mosquitoes, of Asaia infected ones, etc would be interesting.

We agree and have modified table 1 to include the number of infected mosquitoes for all categories (including uninfected individuals).

Page 3:

§2: Asaia is not an endosymbiont

We agree and have modified throughout the manuscript to reflect this mistake

§3: « have » needs probably to be removed in « than would have been identified using morphological identification alone »

We agree have corrected this sentence

§4 needs a first sentence identifying the gap of knowledge that the authors want to fill

We agree and have added the following sentence: "Investigating the prevalence and diversity of *Wolbachia* strains naturally present in *Anopheles* populations across diverse malaria endemic countries would allow a greater understanding of how this bacterium could be influencing malaria transmission in field populations and provide candidate strains for transinfection"

§5: Can the authors clearly state whether some mosquitoes had blood in their midgut?

We did not fully determine the Sella score of the mosquitoes used in our study so our collection likely contained individuals that had undigested blood. However, we have the following sentences in our discussion which we feel acknowledges the limitations of our study:

"However, detection of *P. falciparum* parasites in whole body mosquitoes does not confirm that the species plays a significant role in transmission. Detection could represent infected bloodmeal stages or oocysts present in the midgut wall so further studies are warranted to determine this species ability to transmit human malaria parasites."

Page 4:

Figure 1: scale should be in km, miles is not an SI unit
We have changed this to km

§2: « DNA extraction and MOSQUITO species identification ». More generally, it is not always
clear whether the authors speak about mosquitoes or Wolbachia strains.

We have added the word 'mosquito' prior to species identification for clarity

§3: « as preliminary trials revealed this was the optimal method for both sensitivity and specificity »:
please add « data not shown » or remove it

We have removed this as it's been shown before in multiple previous publications and is a
well-established PCR assay for detection of *Plasmodium*.

Page 5:

Instead of µL of DNA, the actual quantity in ng would be preferable.

Although we did measure total DNA for selected samples and normalised *An. gambiae*
extracts to Ct values for a single copy *An. gambiae* rps17 housekeeping gene, we did not
do this for all species across all countries so for consistency we feel ul of DNA is more
representative of our work

All PCRs: primer sequences are needed

We have added all primer sequences were appropriate

§3: « Both chromatograms (forward and reverse traces) from each sample WERE manually »

We have changed this grammatical error

Pages 6-7

Table 1: probably some mistakes, e.g. *An. gambiae* in Mikalayi: 11.8% corresponds neither to 1/16 nor to 2/16, so all the numbers should be checked. It would be appropriate to enter the actual numbers in brackets, and to indicate the co-prevalence of Wolbachia and Plasmodium. The legend should be grouped below or above the table and the explanation about mosquitoes in bold is unclear.

In the text, numbers would be interesting rather than only proportions.

« previously named M molecular form OF AN. GAMBIAE » (or remove it, as this precision may now
be superfluous). On the contrary, « An. species A » is barely introduced, it would be interesting to
mention something about this species and its identification (besides the quick explanation in the
introduction).

We have modified table 1 for clarity including numbers and removed the reference to M
and S forms. The legend format is according to WOR guidelines and we have modified the
table legend for clarity.

As very little is known about *An. species A* and what we were able to find on this species
is presented in our discussion "*An. species A* should be further investigated to determine
if this species is a potential malaria vector, given our study demonstrated *P. falciparum*
infection in one of two individuals screened and ELISA-positive samples of this species
were reported from the Western Highlands of Kenya<sup>42</sup>."

Page 13

« Approximately 1000-fold higher », it is very much of an approximation (variable Ct values and potential variations in 16S copy number): it may be good to rephrase, mentioning that 1000 is an order of magnitude rather than approximately.
**We have modified this sentence**

§2: « An. moucheti (wAnM-infected) » comes at the 2nd occurrence of wAnM.

**We have corrected this by removing 'An. moucheti'**

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