# Ribosomal RNA (rRNA) sequences from 33 globally distributed mosquito species for improved metagenomics and species identification

Cassandra Koh, Lionel Frangeul, Hervé Blanc, Carine Ngoagouni, Sébastien

Boyer, Philippe Dussart, Nina Grau, Romain Girod, Jean-Bernard Duchemin,
Maria-Carla Saleh

► To cite this version:

Cassandra Koh, Lionel Frangeul, Hervé Blanc, Carine Ngoagouni, Sébastien Boyer, et al.. Ribosomal RNA (rRNA) sequences from 33 globally distributed mosquito species for improved metagenomics and species identification. eLife, inPress, 12, pp.e82762. 10.7554/eLife.82762. pasteur-03977506

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Submitted on 7 Feb 2023

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Distributed under a Creative Commons Attribution 4.0 International License
1 Ribosomal RNA (rRNA) sequences from 33 globally distributed mosquito species for improved metagenomics and species identification

3

4 Cassandra Koh¹*, Lionel Frangeul¹, Hervé Blanc¹, Carine Ngoagouni², Sébastien Boyer³, Philippe Dussart⁴, Nina Grau⁵, Romain Girod⁵, Jean-Bernard Duchemin⁶ and Maria-Carla Saleh¹*

6 ¹Viruses and RNA Interference Unit, Institut Pasteur, Université Paris Cité, CNRS UMR3569, Paris F-75015, France

8 ²Medical Entomology Laboratory, Institut Pasteur de Bangui, Bangui PO Box 923, Central African Republic

10 ³Medical and Veterinary Entomology Unit, Institut Pasteur du Cambodge, Phnom Penh 12201, Cambodia

12 ⁴Virology Unit, Institut Pasteur du Cambodge, Phnom Penh 12201, Cambodia

13 ⁵Medical Entomology Unit, Institut Pasteur de Madagascar, Antananarivo 101, Madagascar

14 ⁶Vectopôle Amazonien Emile Abonnenc, Institut Pasteur de la Guyane, Cayenne 97306, French Guiana

16 * To whom correspondence should be addressed.

17 Present address: Philippe Dussart, Institut Pasteur de Madagascar, Antananarivo 101, Madagascar;

18 Nina Grau, Sciences Economiques et Sociales de la Santé et Traitement de l'Information Médicale,

19 Faculté de Médecine, Marseille 13005, France

20 Corresponding authors: Cassandra Koh, cassandra.koh@pasteur.fr, +33 1 40 61 36 74; Maria-Carla Saleh, carla saleh@pasteur.fr, +33 1 45 68 85 47.

21
22 **ABSTRACT**

23 Total RNA sequencing (RNA-seq) is an important tool in the study of mosquitoes and the RNA viruses
24 they vector as it allows assessment of both host and viral RNA in specimens. However, there are two
25 main constraints. First, as with many other species, abundant mosquito ribosomal RNA (rRNA) serves
26 as the predominant template from which sequences are generated, meaning that the desired host and
27 viral templates are sequenced far less. Second, mosquito specimens captured in the field must be
28 correctly identified, in some cases to the sub-species level. Here, we generate mosquito ribosomal
29 RNA (rRNA) datasets which will substantially mitigate both of these problems. We describe a strategy
30 to assemble novel rRNA sequences from mosquito specimens and produce an unprecedented
31 dataset of 234 full-length 28S and 18S rRNA sequences of 33 medically important species from
32 countries with known histories of mosquito-borne virus circulation (Cambodia, the Central African
33 Republic, Madagascar, and French Guiana). These sequences will allow both physical and
34 computational removal of rRNA from specimens during RNAseq protocols. We also assess the utility
35 of rRNA sequences for molecular taxonomy and compare phylogenies constructed using rRNA
36 sequences versus those created using the gold standard for molecular species identification of
37 specimens—the mitochondrial *cytochrome c oxidase I* (COI) gene. We find that rRNA- and COI-
38 derived phylogenetic trees are incongruent and that 28S and concatenated 28S+18S rRNA
39 phylogenies reflect evolutionary relationships that are more aligned with contemporary mosquito
40 systematics. This significant expansion to the current rRNA reference library for mosquitoes will
41 improve mosquito RNA-seq metagenomics by permitting the optimization of species-specific rRNA
42 depletion protocols for a broader range of species and streamlining species identification by rRNA
43 sequence and phylogenetics.

44 **Keywords:** surveillance, RNA-seq, ribosomal RNA, molecular marker, metagenomics, mosquito
45 INTRODUCTION

46 Mosquitoes top the list of vectors for arthropod-borne diseases, being implicated in the transmission
47 of many human pathogens responsible for arboviral diseases, malaria, and lymphatic filariasis (WHO,
48 2017). Mosquito-borne viruses circulate in sylvatic (between wild animals) or urban (between
49 humans) transmission cycles driven by different mosquito species with their own distinct host
50 preferences. Although urban mosquito species are chiefly responsible for amplifying epidemics in
51 dense human populations, sylvatic mosquitoes maintain the transmission of these viruses among
52 forest-dwelling animal reservoir hosts and are implicated in spillover events when humans enter their
53 ecological niches (Valentine et al., 2019). Given that mosquito-borne virus emergence is preceded by
54 such spillover events, continuous surveillance and virus discovery in sylvatic mosquitoes is integral to
55 designing effective public health measures to pre-empt or respond to mosquito-borne viral epidemics.

56 Metagenomics on field specimens is the most powerful method in the toolkit for understanding
57 mosquito-borne disease ecology through the One Health lens (Webster et al., 2016). With next-
58 generation sequencing becoming more accessible, such studies have provided unprecedented
59 insights into the interfaces among mosquitoes, their environment, and their animal and human hosts.
60 As mosquito-associated viruses are mostly RNA viruses, RNA sequencing (RNA-seq) is especially
61 informative for surveillance and virus discovery. However, working with lesser studied mosquito
62 species poses several problems.

63 First, metagenomics studies based on RNA-seq are bedevilled by overabundant ribosomal RNAs
64 (rRNAs). These non-coding RNA molecules comprise at least 80% of the total cellular RNA
65 population (Gale & Crampton, 1989). Due to their length and their abundance, they are a sink for
66 precious next generation sequencing reads, decreasing the sensitivity of pathogen detection unless
67 depleted during library preparation. Yet the most common rRNA depletion protocols require prior
68 knowledge of rRNA sequences of the species of interest as they involve hybridizing antisense oligos
69 to the rRNA molecules prior to removal by ribonucleases (Fauver et al., 2019; Phelps et al., 2021) or
70 by bead capture (Kukutla et al., 2013). Presently, reference sequences for rRNAs are limited to only a
71 handful of species from three genera: *Aedes*, *Culex*, and *Anopheles* (Ruzzante et al., 2019). The lack
72 of reliable rRNA depletion methods could deter mosquito metagenomics studies from expanding their
73 sampling diversity, resulting in a gap in our knowledge of mosquito vector ecology. The inclusion of
74 lesser studied yet medically relevant sylvatic species is therefore imperative.
Second, species identification based on morphology is notoriously complicated for members of
species subgroups. This is especially the case among *Culex* subgroups. Sister species are often
sympatric and show at least some competence for a number of viruses, such as Japanese
encephalitis virus, St Louis encephalitic virus, and Usutu virus (Nchoutpouen et al., 2019). Although
they share many morphological traits, each of these species have distinct ecologies and host
preferences, thus the challenge of correctly identifying vector species can affect epidemiological risk
estimation for these diseases (Farajollahi et al., 2011). DNA molecular markers are often employed to
a limited degree of success to distinguish between sister species (Batovska et al., 2017; Zittra et al.,
2016).

To address the lack of full-length rRNA sequences in public databases, we sought to determine
the 28S and 18S rRNA sequences of a diverse set of Old and New World sylvatic mosquito species
from four countries representing three continents: Cambodia, the Central African Republic,
Madagascar, and French Guiana. These countries, due to their proximity to the equator, contain high
mosquito biodiversity (Foley et al., 2007) and have had long histories of mosquito-borne virus
circulation (Desdouits et al., 2015; Halstead, 2019; Héraud et al., 2022; Jacobi & Serie, 1972;
Ratsitorahina et al., 2008; Saluzzo et al., 2018; Zeller et al., 2016). Increased and continued
surveillance of local mosquito species could lead to valuable insights on mosquito virus biogeography.
Using a unique score-based read filtration strategy to remove interfering non-mosquito rRNA reads for
accurate *de novo* assembly, we produced a dataset of 234 novel full-length 28S and 18S rRNA
sequences from 33 mosquito species, 30 of which have never been recorded before.

We also explored the functionality of 28S and 18S rRNA sequences as molecular markers by
comparing their performance to that of the mitochondrial *cytochrome c oxidase subunit I* (COI) gene
for molecular taxonomic and phylogenetic investigations. The COI gene is the most widely used DNA
marker for molecular species identification and forms the basis of the Barcode of Life Data System
(BOLD) (Hebert et al., 2003; Ratnasingham & Hebert, 2007). Presently, full-length rRNA sequences
are much less represented compared to other molecular markers. However, given the availability of
relevant reference sequences, 28S and concatenated 28S+18S rRNA sequences can be the better
approach for molecular taxonomy and phylogenetic studies. We hope that our sequence dataset, with
its species diversity and eco-geographical breadth, and the assembly strategy we describe would
further facilitate the use of rRNA as markers. In addition, this dataset enables the design of species-
specific oligos for cost-effective rRNA depletion for a broader range of mosquito species and
streamlined molecular species identification during RNA-seq.

## 107 RESULTS

### 108 Poor rRNA depletion using a non-specific depletion method

During library preparations of mosquito samples for RNA-seq, routinely used methods for depleting
rRNA are commercial kits optimised for human or mice samples (Belda et al., 2019; Bishop-Lilly et al.,
2010; Chandler et al., 2015; N. Kumar et al., 2012; Weedall et al., 2015; Zakrzewski et al., 2018) or
through 80–100 base pair antisense probe hybridisation followed by ribonuclease digestion (Fauver et
al., 2019; Phelps et al., 2021). In cases where the complete reference rRNA sequence of the target
species is not known, oligos would be designed based on the rRNA sequence of the closest related
species (25, this study). These methods should deplete the conserved regions of rRNA sequences.
However, the variable regions remain at abundances high enough to compromise RNA-seq output. In
our hands, we have found that using probes designed for the *Ae. aegypti* rRNA sequence followed by
RNase H digestion according to the protocol published by Morlan *et al.* (2012) produced poor
depletion in *Ae. albopictus*, and in Culicine and Anopheline species (Figure 1), in which between 46–
94% of reads post-depletion were ribosomal. Additionally, the lack of reference rRNA sequences
compromises the *in silico* clean-up of remaining rRNA reads from sequencing data, as reads
belonging to variable regions would not be removed. To solve this and to enable RNA-seq
metagenomics on a broader range of mosquito species, we performed RNA-seq to generate
reference rRNA sequences for 33 mosquito species representing 10 genera from Cambodia, the
Central African Republic, Madagascar, and French Guiana. Most of these species are associated with
vector activity for various pathogens in their respective ecologies (Table 1). In parallel, we sequenced
the mitochondrial COI gene to perform molecular species identification of our samples and to
comparatively evaluate the use of rRNA as a molecular marker (Figure 2).
<figure><img src="image_2.png" /></figure>

129

Figure 1. Percentage of rRNA reads in mosquito total RNA-seq data after depletion using

pools of 5 individual mosquitoes were ribodepleted by
probe hybridisation followed by RNase H digestion according to the protocol by Morlan *et al.* (2012).

Percentages of remaining rRNA reads were calculated from the number of rRNA reads over total
reads per sample pool. Depletion efficiency decreases with taxonomic distance from *Ae. aegypti*
underlining the need for reference sequences for species of interest.

<table><caption>Table 1. Mosquito species represented in this study and their vector status.</caption><thead><tr><td>Mosquito taxonomy*</td><td>Origin**</td><td>Collection site (ecosystem type)</td><td>Vector for***</td><td>Reference</td></tr></thead><tbody><tr><td>Aedes (Fredardsius) vittatus</td><td>CF</td><td>rural (village)</td><td>ZIKV, CHIKV, YFV</td><td>(Diallo et al., 2020)</td></tr><tr><td>Aedes (Ochlerotatus) scapularis</td><td>GF</td><td>rural (village)</td><td>YFV</td><td>(Vasconcelos et al., 2001)</td></tr><tr><td>Aedes (Ochlerotatus) serratus</td><td>GF</td><td>rural (village)</td><td>YFV, OROV</td><td>(Cardoso et al., 2010; Romero-Alvarez & Escobar, 2018)</td></tr><tr><td>Aedes (Stegomyia) aegypti</td><td>CF</td><td>urban</td><td>DENV, ZIKV, CHIKV, YFV</td><td>(Kraemer et al., 2019)</td></tr><tr><td>Aedes (Stegomyia) albopictus</td><td>CF, KH</td><td>rural (village, nature reserve)</td><td>DENV, ZIKV, CHIKV, YFV, JEV</td><td>(Auerswald et al., 2021; Kraemer et al., 2019)</td></tr><tr><td>Aedes (Stegomyia) simpsoni</td><td>CF</td><td>rural (village)</td><td>YFV</td><td>(Mukwaya et al., 2000)</td></tr><tr><td>Anopheles (Anopheles) baezai</td><td>KH</td><td>rural (nature reserve)</td><td>unreported</td><td>–</td></tr><tr><td>Anopheles (Anopheles) coustani</td><td>MG, CF</td><td>rural (village)</td><td>RVFV, malaria</td><td>(Mwangangi et al., 2013; Nepomichene et al., 2018; Ratovonjato et al., 2011)</td></tr><tr><td>Anopheles (Cellia) funestus</td><td>MG, CF</td><td>rural (village)</td><td>ONNV, malaria</td><td>(Lutomiah et al., 2013; Tabue et al., 2017)</td></tr><tr><td>Anopheles (Cellia) gambiae</td><td>MG, CF</td><td>rural (village)</td><td>ONNV, malaria</td><td>(Brault et al., 2004)</td></tr><tr><td>Anopheles (Cellia) squamosus</td><td>MG</td><td>rural (village)</td><td>RVFV, malaria</td><td>(Ratovonjato et al., 2011; Stevenson et al., 2016)</td></tr></tbody></table>
<table><tr><td>Coquillettidia (Rhynchotaenia) venezuelensis</td><td>GF</td><td>rural (village)</td><td>OROV</td><td>(Travassos Da Rosa et al., 2017)</td></tr><tr><td>Culex (Culex) antennatus</td><td>MG</td><td>rural (village)</td><td>RVFV</td><td>(Nepomichene et al., 2018; Ratovonjato et al., 2011)</td></tr><tr><td>Culex (Culex) duttoni</td><td>CF</td><td>rural (village)</td><td>unreported</td><td>–</td></tr><tr><td>Culex (Culex) neavei</td><td>MG</td><td>rural (village)</td><td>USUV</td><td>(Nikolay et al., 2011)</td></tr><tr><td>Culex (Culex) orientalis</td><td>KH</td><td>rural (nature reserve)</td><td>JEV</td><td>(Kim et al., 2015)</td></tr><tr><td>Culex (Culex) perexiguus</td><td>MG</td><td>rural (village)</td><td>WNV, USUV</td><td>(Vázquez González et al., 2011)</td></tr><tr><td>Culex (Culex) pseudovishnui</td><td>KH</td><td>rural (nature reserve)</td><td>JEV</td><td>(Auerswald et al., 2021)</td></tr><tr><td>Culex (Culex) quinquefasciatus</td><td>MG, CF, KH</td><td>rural (village, nature reserve)</td><td>ZIKV, JEV, WNV, DENV, SLEV, RVFV, Wuchereria bancrofti</td><td>(Bhattacharya & Basu, 2016; Maquart et al., 2021; Ndiaye et al., 2016; Pereira Serra et al., 2016)</td></tr><tr><td>Culex (Culex) tritaeniorhynchus</td><td>MG, KH</td><td>rural (village, nature reserve)</td><td>JEV, WNV, RVFV</td><td>(Auerswald et al., 2021; Hayes et al., 1980; Jupp et al., 2002)</td></tr><tr><td>Culex (Melanoconion) spissipes</td><td>GF</td><td>rural (village)</td><td>VEEV</td><td>(Weaver et al., 2004)</td></tr><tr><td>Culex (Melanoconion) portesi</td><td>GF</td><td>rural (village)</td><td>VEEV, TONV</td><td>(Talaga et al., 2021; Weaver et al., 2004)</td></tr><tr><td>Culex (Melanoconion) pedroi</td><td>GF</td><td>rural (village)</td><td>EEEV, VEEV, MADV</td><td>(Talaga et al., 2021; M. J. Turell et al., 2008)</td></tr><tr><td>Culex (Oculeomyia) bitaeniorhynchus</td><td>MG, KH</td><td>rural (village, nature reserve)</td><td>JEV</td><td>(Auerswald et al., 2021)</td></tr><tr><td>Culex (Oculeomyia) poicilipes</td><td>MG</td><td>rural (village)</td><td>RVFV</td><td>(Ndiaye et al., 2016)</td></tr><tr><td>Eretmapodites intermedius</td><td>CF</td><td>rural (village)</td><td>unreported</td><td>–</td></tr><tr><td>Limatus durhamii</td><td>GF</td><td>rural (village)</td><td>ZIKV</td><td>(Barrio-Nuevo et al., 2020)</td></tr><tr><td>Mansonia (Mansonia) titillans</td><td>GF</td><td>rural (village)</td><td>VEEV, SLEV</td><td>(Hoyos-López et al., 2015; Michael J Turell, 1999)</td></tr><tr><td>Mansonia (Mansonioides) indiana</td><td>KH</td><td>rural (nature reserve)</td><td>JEV</td><td>(Arunachalam et al., 2004)</td></tr><tr><td>Mansonia (Mansonioides) uniformis</td><td>MG, CF, KH</td><td>rural (village, nature reserve)</td><td>RVFV, Wuchereria bancrofti</td><td>(Lutomiah et al., 2013; Ughasi et al., 2012)</td></tr><tr><td>Mimomyia (Etorleptomyia) mediolineata</td><td>MG</td><td>rural (village)</td><td>unreported</td><td>–</td></tr><tr><td>Psorophora (Janthinosoma) ferox</td><td>GF</td><td>rural (village)</td><td>ROCV</td><td>(Mitchell et al., 1986)</td></tr><tr><td>Uranotaenia (Uranotaenia) geometrica</td><td>GF</td><td>rural (village)</td><td>unreported</td><td>–</td></tr></table>

137 * () indicates subgenus

138 ** Origin countries are listed as their ISO alpha-2 codes: Central African Republic, CF; Cambodia, KH;

139 Madagascar, MG; French Guiana, GF.

140 ** dengue virus, DENV; Zika virus, ZIKV; chikungunya virus, CHIKV; Yellow Fever virus, YFV; Oropouche virus, OROV; Japanese encephalitis virus, JEV; Rift Valley Fever virus, RVFV; O'Nyong Nyong virus, ONNV; Usutu virus, USUV; West Nile virus, WNV; Saint Louis encephalitis virus, SLEV; Venezuelan equine encephalitis virus, VEEV; Tonate virus, TONV; Eastern equine encephalitis virus, EEEV; Madariaga virus, MADV; Rocio virus, ROCV.

145 rRNA reads filtering and sequence assembly

146 Assembling Illumina reads to reconstruct rRNA sequences from total mosquito RNA is not a

147 straightforward task. Apart from host rRNA, total RNA samples also contain rRNA from other
organisms associated with the host (microbiota, external parasites, or ingested diet). As rRNA
sequences share high homology in conserved regions, Illumina reads (150 bp) from non-host rRNA
can interfere with the contig assembly of host 28S and 18S rRNA.

Our score-based filtration strategy, described in detail in the Methods section, allowed us to
bioinformatically remove interfering rRNA reads and achieve successful *de novo* assembly of 28S and
18S rRNA sequences for all our specimens. Briefly, for each Illumina read, we computed a ratio of
BLAST scores against an Insecta library over scores against a Non-Insecta library (Figure 2A). Based
on their ratio of scores, reads could be segregated into four categories (Figure 2B): (i) reads mapping
only to the Insecta library, (ii) reads mapping better to the Insecta relative to Non-Insecta library, (iii)
reads mapping better to the Non-Insecta relative to the Insecta library, and (iv) reads mapping only to
the Non-Insecta library. By applying a conservative threshold at 0.8 to account for the non-
exhaustiveness of the SILVA database, we removed reads that likely do not originate from mosquito
rRNA. Notably, 15 of our specimens were engorged with vertebrate blood, a rich source of non-
mosquito rRNA (Appendix 1—table 1). The successful assembly of complete 28S and 18S rRNA
sequences for these specimens demonstrates that this strategy performs as expected even with high
amounts of non-host rRNA reads. This is particularly important in studies on field-captured
mosquitoes as females are often sampled already having imbibed a blood meal or captured using the
human landing catch technique.

We encountered challenges for three specimens morphologically identified as *Ma. africana*
(Specimen ID S33-S35) (Appendix 1—table 1). COI amplification by PCR did not produce any
product, hence COI sequencing could not be used to confirm species identity. In addition, the genome
assembler SPAdes (Bankevich et al., 2012) was only able to assemble partial length rRNA contigs,
despite the high number of reads with high scores against the Insecta library. Among other *Mansonia*
specimens, the partial length contigs shared the highest similarity with contigs obtained from sample
"Ma uniformis CF S51". We then performed a guided assembly using the 28S and 18S sequences of
this specimen as references, which successfully produced full-length contigs. In two of these
specimens (Specimen ID S34 and S35), our assembly initially produced two sets of 28S and 18S
rRNA sequences, one of which was similar to mosquito rRNA with low coverage and another with ten-
fold higher coverage and 95% nucleotide sequence similarity to a water mite of genus *Horreolanus*
known to parasitize mosquitoes. Our success in obtaining rRNA sequences for mosquito and water
mite shows that our strategy can be applied to metabarcoding studies where the input material comprises multiple insect species, provided that appropriate reference sequences of the target species or of a close relative are available.

Altogether, we were able to assemble 122 28S and 114 18S full-length rRNA sequences for 33 mosquito species representing 10 genera sampled from four countries across three continents. This dataset contains, to our knowledge, the first records for 30 mosquito species and for seven genera: *Coquillettidia*, *Mansonia*, *Limatus*, *Mimomyia*, *Uranotaenia*, *Psorophora*, and *Eretmapodites*.

Individual GenBank accession numbers for these sequences and specimen information are listed in Appendix 1—table 1.
<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Figure 2. Novel mosquito rRNA sequences were obtained using a unique filtering method. (A)</figcaption></figure>

Schematic of sequencing and bioinformatics analysis performed in this study to obtain full-length 18S and 28S rRNA sequences as well as COI DNA sequences. Nucleic acids were isolated from mosquito specimens for next generation (for rRNA) or Sanger (for COI) sequencing. Two in-house libraries were created from the SILVA rRNA gene database: Insecta and Non-Insecta, which comprises 8 585 sequences and 558 185 sequences, respectively. Following BLASTn analysis against these two libraries, each RNA-seq read is assigned a ratio of BLASTn scores to describe their relative nucleotide similarity to insect rRNA sequences. Based on these ratios of scores, RNA-seq reads can
196 then be filtered to remove non-mosquito reads prior to assembly with SPAdes to give full-length 18S
197 and 28S rRNA sequences. Image created with Biorender.com. (B) Based on their ratio of scores,
198 reads can be segregated into four categories, as shown on this ratio of scores vs. number of reads
199 plot for the representative specimen "Ae simpsoni CF S27": (i) reads with hits only in the Insecta
200 library (shaded in green), (ii) reads with a higher score against the Insecta library (shaded in blue), (iii)
201 reads with a higher score against the Non-Insecta library (shaded in yellow), and (iv) reads with no
202 hits in the Insecta library (shaded in red). We applied a conservative threshold at 0.8, indicated by the
203 black horizontal line, where only reads above this threshold are used in the assembly with SPAdes.
204 For this given specimen, 175 671 reads (96.3% of total reads) passed the ≥0.8 cut-off, 325 reads
205 (0.18% of total reads) had ratios of scores <0.8, while 6 423 reads (3.52%) did not have hits against
206 the Insecta library.

207 Comparative phylogeny of novel rRNA sequences relative to existing records

208 To verify the assembly accuracy of our rRNA sequences, we constructed a comprehensive
209 phylogenetic tree from the full-length 28S rRNA sequences generated from our study and included
210 relevant rRNA sequences publicly available from GenBank (Figure 3). We applied a search criterion
211 for GenBank sequences with at least 95% coverage of our sequence lengths (~4000 bp), aiming to
212 represent as many species or genera as possible. Although we rarely found records for the same
213 species included in our study, the resulting tree showed that our 28S sequences generally clustered
214 according to their respective species and subgenera, supported by moderate to good bootstrap
215 support at terminal nodes. Species taxa generally formed monophyletic clades, with the exception of
216 *An. gambiae* and *Cx. quinquefasciatus*. *An. gambiae* 28S rRNA sequences formed a clade with
217 closely related sequences from *An. arabiensis*, *An. merus*, and *An. coluzzii*, suggesting unusually
218 high interspecies homology for Anophelines or other members of subgenus *Cellia* (Figure 3, in purple,
219 subgenus *Cellia*). Meanwhile, *Cx. quinquefasciatus* 28S rRNA sequences formed a taxon
220 paraphyletic to sister species *Cx. pipiens* (Figure 3, in coral, subgenus *Culex*).
<figure><img src="image_5.png" /></figure>
**Figure 3.** 28S sequences generated from this study clustered with conspecifics or congenerics from existing **GenBank** records. A rooted phylogenetic tree based on full-length 28S sequences (3900 bp) from this study and from GenBank was inferred using the maximum-likelihood method and constructed to scale in MEGA X (S. Kumar et al., 2018) using an unknown *Horreolanus* species found among our samples as an outgroup. Values at each node indicate bootstrap support (%) from 500 replications. Sequences from GenBank are annotated with filled circles and their accession numbers are shown. For sequences from this study, each specimen label contains information on taxonomy, origin (in 2-letter country codes), and specimen ID number. Some specimens produced up to two consensus 28S sequences; this is indicated by the numbers 1 or 2 in the beginning of the specimen label. Specimen genera are indicated by colour: *Culex* in coral, *Anopheles* in purple, *Aedes* in dark blue, *Mansonia* in dark green, *Culiseta* in maroon, *Limatus* in light green, *Coquillettidia* in light blue, *Psorophora* in yellow, *Mimomyia* in teal, *Uranotaenia* in pink and *Eretmapodites* in brown. Scale bar at 0.05 is shown.

28S rRNA sequence-based phylogenetic reconstructions (Figure 3, with GenBank sequences; Figure 4—figure supplement 1, this study only) showed marked incongruence to that of 18S rRNA sequences (Figure 4—figure supplement 2). Although all rRNA trees show the bifurcation of family *Culicidae* into subfamilies *Anophelinae* (genus *Anopheles*, in purple) and *Culicinae* (all other genera), the recovered intergeneric phylogenetic relationships vary between the 28S and 18S rRNA trees and are weakly supported. The 18S rRNA tree also exhibited several taxonomic anomalies: (i) the lack of definitive clustering by species within the *Culex* subgenus (in coral) (ii) the lack of distinction between 18S rRNA sequences of *Cx. pseudovishnui* and *Cx. Tritaeniorhynchus* (in coral); (iii) the placement of *Ma* sp. 3 CF S35 (in dark green) within a *Culex* clade; and (iv) the lack of a monophyletic *Mimomyia* clade (in teal) (Figure 4—figure supplement 2). However, 28S and 18S rRNA sequences are encoded by linked loci in rDNA clusters and should not be analysed separately.

Indeed, when concatenated 28S+18S rRNA sequences were generated from the same specimens (Figure 4), the phylogenetic tree resulting from these sequences more closely resembles the 28S tree (Figure 3) with regard to the basal position of the *Mimomyia* clade (in teal) within the *Culicinae* subfamily with good bootstrap support in either tree (84% in 28S rRNA tree, 100% in concatenated 28S+18S rRNA tree). For internal nodes, bootstrap support values were higher in the concatenated tree compared to the 28S tree. Interestingly, the 28S+18S rRNA tree formed an *Aedini* tribe-clade
encompassing taxa from genera *Psorophora* (in yellow), *Aedes* (in dark blue), and *Eretmapodites* (in brown), possibly driven by the inclusion of 18S rRNA sequences. Concatenation also resolved the anomalies found in the 18S rRNA tree and added clarity to the close relationship between *Culex* (in coral) and *Mansonia* (in dark green) taxa. Of note, relative to the 28S tree (Figure 3) the *Culex* and *Mansonia* genera are no longer monophyletic in the concatenated 28S+18S rRNA tree (Figure 4).

Genus *Culex* is paraphyletic with respect to subgenus *Mansonoides* of genus *Mansonia* (Figure 3).

*Ma. titillans* and *Ma* sp. 4, which we suspect to be *Ma. pseudotitillans*, always formed a distinct branch in 28S or 18S rRNA phylogenies, thus possibly representing a clade of subgenus *Mansonia*.

The concatenated 28S+18S rRNA tree (Figure 4) recapitulates what is classically known about the systematics of our specimens, namely (i) the early divergence of subfamily *Anophelinae* from subfamily *Culicinae*, (ii) the division of genus *Anopheles* (in purple) into two subgenera, *Anopheles* and *Cellia*, (iii) the division of genus *Aedes* (in dark blue) into subgenera *Stegomyia* and *Ochlerotatus*, (iv) the divergence of the monophyletic subgenus *Melanoconion* within the *Culex* genus (in coral) (Harbach, 2007; Harbach & Kitching, 2016).
<figure><img src="image_6.png" /><figcaption>Figure 4. Concatenating 28S and 18S rRNA sequences produces phylogenetic relationships that are concordant with classical *Culicidae* systematics with higher bootstrap support than 28S sequences alone. This phylogenetic tree based on concatenated 28S+18S rRNA sequences (3900+1900 bp) generated from this study was inferred using the maximum-likelihood method and constructed to scale using MEGA X (S. Kumar et al., 2018) using an unknown *Horreolanus* species found among our samples as an outgroup. Values at each node indicate bootstrap support (%) from</figcaption></figure>
273 500 replications. Each specimen label contains information on taxonomy, origin (as indicated in 2-letter country codes), and specimen ID number. Some specimens produced up to two consensus 274 28S+18S rRNA sequences; this is indicated by the numbers 1 or 2 in the beginning of the specimen 275 label. Specimen genera are indicated by colour: *Culex* in coral, *Anopheles* in purple, *Aedes* in dark 276 blue, *Mansonia* in dark green, *Limatus* in light green, *Coquillettidia* in light blue, *Psorophora* in yellow, 277 *Mimomyia* in teal, *Uranotaenia* in pink and *Eretmapodites* in brown. Scale bar at 0.05 is shown. 278

279 rRNA as a molecular marker for taxonomy and phylogeny

280 We sequenced a 621 bp region of the COI gene to confirm morphological species identification of our 281 specimens and to compare the functionality of rRNA and COI sequences as molecular markers for 282 taxonomic and phylogenetic investigations. COI sequences were able to unequivocally determine the 283 species identity in most specimens except for the following cases. *An. coustani* COI sequences from 284 our study regardless of specimen origin shared remarkably high nucleotide similarity (>98%) with 285 several other *Anopheles* species such as *An. rhodesiensis*, *An. rufipes*, *An. ziemanni*, *An. tenebrosus*, 286 although *An. coustani* remained the most frequent and closest match. In the case of *Ae. simpsoni*, 287 three specimens had been morphologically identified as *Ae. opok* although their COI sequences 288 showed 97-100% similarity to that of *Ae. simpsoni*. As GenBank held no records of *Ae. opok* COI at 289 the time of this study, we instead aligned the putative *Ae. simpsoni* COI sequences against two sister 290 species of *Ae. opok*: *Ae. luteocephalus* and *Ae. africanus*. We found they shared only 90% and 89% 291 similarity, respectively. Given this significant divergence, we concluded these specimens to be *Ae.* 292 *simpsoni*. Ambiguous results were especially frequent among *Culex* specimens belonging to the *Cx.* 293 *pipiens* or *Cx. vishnui* subgroups, where the query sequence differed with either of the top two hits by 294 a single nucleotide. For example, between *Cx. quinquefasciatus* and *Cx. pipiens* of the *Cx. pipiens* 295 subgroup, and between *Cx. vishnui* and *Cx. tritaeniorhynchus* of the *Cx. vishnui* subgroup. 296

296 Among our three specimens of *Ma. titillans*, two appeared to belong to a single species that is 297 different from but closely related to *Ma. titillans*. We surmised that these specimens could instead be 298 *Ma. pseudotitillans* based on morphological similarity but were not able to verify this by molecular 299 means as no COI reference sequence is available for this species. These specimens are hence 300 putatively labelled as "Ma sp.4".

301 Phylogenetic reconstruction based on the COI sequences showed clustering of all species taxa 302 into distinct clades, underlining the utility of the COI gene in molecular taxonomy (Figure 5)(Hebert et
al., 2003; Ratnasingham & Hebert, 2007). However, species delineation among members of *Culex* subgroups were not as clear cut, although sister species were correctly placed as sister taxa (Figure 5, in coral). This is comparable to the 28S+18S rRNA tree (Figure 4, in coral) and is indicative of lower intraspecies distances relative to interspecies distances.

To evaluate the utility of 28S and 18S rRNA sequences for molecular taxonomy, we used the 28S+18S rRNA tree to discern the identity of six specimens for which COI sequencing could not be performed. These specimens include three unknown *Mansonia* species (Specimen ID S33–S35), a *Ma. uniformis* (Specimen ID S51), an *An. gambiae* (Specimen ID S47), and a *Ur. geometrica* (Specimen ID S113) (Appendix 1—table 1). Their positions in the 28S+18S rRNA tree relative to adjacent taxa confirms the morphological identification of all six specimens to the genus level and, for three of them, to the species level (Figure 4; *Mansonia* in dark green, *Anopheles* in purple, *Uranotaenia* in pink).

The phylogenetic relationships indicated by the COI tree compared to the 28S+18S rRNA tree present only few points of similarity, with key differences summarised in Table 2. COI-based phylogenetic inference indeed showed clustering of generic taxa into monophyletic clades albeit with very weak bootstrap support, except for genera *Culex* and *Mansonia* (Figure 5; *Culex* in coral, *Mansonia* in dark green). Contrary to the 28S+18S rRNA tree (Figure 4), *Culex* subgenus *Melanoconion* was depicted as a polyphyletic taxon with *Cx. spissipes* being a part of the greater *Culicini* clade with members from subgenera *Oculeomyia* and *Culex* while *Cx. pedroi* and *Cx. portesi* formed a distantly related clade. Among the *Mansonia* specimens, the two unknown *Ma* sp.4 specimens were not positioned as the nearest neighbours of *Ma. titillans* and instead appeared to have diverged earlier from most of the other taxa from the *Culicidae* family. Notably, the COI sequences of genus *Anopheles* (Figure 5, in purple) is not basal to the other members of *Culicidae* and is instead shown to be sister to *Culex* COI sequences (8% bootstrap support). This is a direct contrast to what is suggested by the rRNA phylogenies (Figures 3 and 4, Figure 4—figure supplements 1 and 2; *Anopheles* in purple), which suggests *Culex* (in coral) rRNA sequences to be among the most recently diverged. Bootstrap support for the more internal nodes of the COI trees were remarkably low compared to those of rRNA-based trees.

In all rRNA trees, it is clear that the interspecific and intersubgeneric evolutionary distances within the genus *Anopheles* are high relative to any other genera, indicating a greater degree of divergence
(Figure 3, Figure 3—figure supplement 1, Figure 4, Figure 4—figure supplements 1 and 2; *Anopheles* in purple). This is evidenced by the longer branch lengths connecting Anopheline species-clades to the node of the most recent common ancestor for subgenera *Anopheles* and *Cellia*. This feature is not evident in the COI tree, where the Anopheline interspecies distances are comparable to those within the *Culex*, *Aedes*, and *Mansonia* taxa (Figure 5; *Anopheles* in purple, *Culex* in coral, *Aedes* in dark blue, *Mansonia* in dark green).

<figure><img src="image_7.png" /><figcaption>Figure 5. COI sequences cluster by species but show phylogenetic relationships that contrast those derived from rRNA trees. A phylogenetic tree based on COI sequences (621–699 bp) was</figcaption></figure>
inferred using the maximum-likelihood method and constructed to scale using MEGA X (S. Kumar et al., 2018) with three water mite species to serve as outgroups. Outgroup sequences obtained from GenBank are annotated with filled circles and their accession numbers are shown. Values at each node indicate bootstrap support (%) from 500 replications. Each specimen label contains information on taxonomy, origin (as indicated in 2-letter country codes), and specimen ID. Specimen genera are indicted by colour: *Culex* in coral, *Anopheles* in purple, *Aedes* in dark blue, *Mansonia* in dark green, *Limatus* in light green, *Coquillettidia* in light blue, *Psorophora* in yellow, *Mimomyia* in teal, *Uranotaenia* in pink and *Eretmapodites* in brown. Scale bar at 0.05 is shown.

<table><caption>**Table 2.** Summary of differences between rRNA and COI phylogenies.</caption><thead><tr><td>Taxa</td><td>28S+18S rRNA phylogeny (Figure 4)</td><td>COI phylogeny (Figure 5)</td></tr></thead><tbody><tr><td>The <i>Anopheles</i> genus</td><td>forms a clade that is basal to the all other members of family <i>Culicidae</i>; interspecies branch lengths are notably long</td><td>forms a sister clade to the <i>Culex</i> genus, and is depicted to have diverged more recently; interspecies branch lengths are comparable to that of other genera</td></tr><tr><td>The <i>Ur. geometrica</i> species</td><td>forms a clade within the <i>Culicinae</i> subfamily lineage</td><td>forms a clade that is basal to the all other members of family <i>Culicidae</i></td></tr><tr><td>The <i>Aedini</i> tribe</td><td>forms a monophyletic clade comprising the genera <i>Aedes</i>, <i>Eretmapodites</i>, and <i>Psorophora</i>, with the latter being an early divergent lineage</td><td>does not form a monophyletic clade; the <i>Psorophora</i> clade is placed among <i>Aedes</i> taxa and the <i>Eretmapodites</i> clade is sister to a <i>Culex</i> subgenus <i>Melanoconion</i> clade</td></tr><tr><td>The <i>Culex</i> genus</td><td>splits into two monophyletic clades with the three French Guyanese species forming a closely-related minor clade</td><td>splits into two clades with two out of three French Guyanese species (<i>Cx. pedroi</i> and <i>Cx. portesi</i>) forming a distantly-related minor clade, while the third (<i>Cx. spissipes</i>) is a part of the greater clade</td></tr><tr><td>The <i>Mansonia</i> genus</td><td>is a polyphyletic group comprising two clades with the two French Guyanese taxa forming a distantly-related minor clade; the major clade is placed among <i>Culex</i> taxa</td><td>forms a subgenus <i>Mansonoides</i> clade as per the 28S+18S rRNA tree but the French Guyanese taxa do not cluster together; is depicted to have diverged earlier relative to other taxa in the assemblage</td></tr><tr><td>The Ma sp.4 species</td><td>forms a sister clade to <i>Ma. titillans</i> as part of a minor French Guyanese <i>Mansonia</i> clade</td><td>does not form a sister clade to <i>Ma. titillans</i>; instead is shown to have diverged earlier than all other members of family <i>Culicidae</i> after <i>Ur. geometrica</i></td></tr></tbody></table>

351

352 **On Culex subgroups**
353 *Culex* (subgenus *Culex*) specimens of this study comprise several closely related sister species
354 belonging to the *Cx. vishnui* and *Cx. univittatus* subgroups, which are notoriously difficult to
355 differentiate based on morphology. Accordingly, in the 28S+18S rRNA (Figure 4, in coral) and COI
356 (Figure 5, in coral) trees these species and their known sister species were clustered together within
357 the *Culex* (subgenus *Culex*) clade: *Cx. tritaeniorhynchus* with *Cx. pseudovishnui* (*Cx. vishnui*
358 subgroup); *Cx. perexiguus* with *Cx. neavei* (*Cx. univittatus* subgroup).

359 The use of the COI sequence to distinguish between members of the *Culex* subgroups was
360 limited. For example, for the two *Cx. quinquefasciatus* samples in our taxonomic assemblage
361 (Specimen ID S74 and S75) (Appendix 1—table 1), BLAST analyses of their COI sequences revealed
362 they are a single nucleotide away from *Cx. pipiens* or *Cx. quinquefasciatus* COI sequences (Appendix
363 2—table 1). In the 28S rRNA tree with GenBank sequences (Figure 3), two *Cx. pipiens* GenBank
364 sequences formed a clade sister to another containing three *Cx. quinquefasciatus* GenBank
365 sequences and the "Cx quinquefasciatus MG S74" sequence with 78% bootstrap support. This is in
366 accordance with other studies examining mitochondrial sequences (Sun et al., 2019) and
367 morphological attributes (Harbach et al., 2017). This shows that the 28S rRNA sequence can
368 distinguish the two species and confirms that "Cx quinquefasciatus MG S74" is indeed a *Cx.
369 quinquefasciatus* specimen. However, "Cx quinquefasciatus MG S75" is shown to be basal from other
370 sequences within this *Cx. pipiens* subgroup-clade with 100% bootstrap support. Given that *Cx.
371 quinquefasciatus* and *Cx. pipiens* are known to interbreed, it is plausible that this individual is a hybrid
372 of the two species (Farajollahi et al., 2011).

373 DISCUSSION

374 RNA-seq metagenomics on field-captured sylvatic mosquitoes is a valuable tool for tracking
375 mosquito viruses through surveillance and virus discovery. However, the lack of reference rRNA
376 sequences hinders good oligo-based depletion and efficient clean-up of RNA-seq data. Additionally,
377 *de novo* assembly of rRNA sequences is complicated due to regions that are highly conserved across
378 all distantly related organisms that could be present in a single specimen, i.e., microbiota, parasites,
379 or vertebrate blood meal. Hence, we sought to establish a method to bioinformatically filter out non-
380 host rRNA reads for the accurate assembly of novel 28S and 18S rRNA reference sequences.
We found that phylogenetic reconstructions based on 28S sequences or concatenated 28S+18S rRNA sequences were able to correctly cluster mosquito taxa according to species and corroborate current mosquito classification. This demonstrates that our bioinformatics methodology reliably generates bona fide 28S and 18S rRNA sequences, even in specimens parasitized by water mites or engorged with vertebrate blood. Further, we were able to use 28S+18S rRNA taxonomy for molecular species identification when COI sequences were unavailable or ambiguous, thus supporting the use of rRNA sequences as a marker. They have the advantage of circumventing the need to additionally isolate and sequence DNA from specimens, as RNA-seq reads can be directly mapped against reference sequences. Post-depletion, in our hands there are sufficient numbers of remaining reads (5–10% of reads per sample) for assembly of complete rRNA contigs (unpublished data).

Phylogenetic inferences based on 28S or 18S rRNA sequences alone do not recover the same interspecific relationships (Figure 4—figure supplements 1 and 2). Relative to 28S sequences, we observed more instances where multiple specimens have near-identical 18S rRNA sequences. This can occur for specimens belonging to the same species, but also for conspecifics sampled from different geographic locations, such as *An. coustani*, *An. gambiae*, or *Ae. albopictus*. More rarely, specimens from the same species subgroup, such as *Cx. pseudovishnui* and *Cx. tritaeniorhynchus*, also shared 18S rRNA sequences. This was surprising given that the 18S rRNA sequences in our dataset is 1900 bp long. Concatenation of 28S and 18S rRNA sequences resolved this issue, enabling species delineation even among sister species of *Culex* subgroups, where morphological identification meets its limits.

In Cambodia and other parts of Asia, the *Cx. vishnui* subgroup includes *Cx. tritaeniorhynchus*, *Cx. vishnui*, and *Cx. pseudovishnui*, which are important vectors of JEV (Maquart & Boyer, 2022). The former two were morphologically identified in our study but later revealed by COI sequencing to be a sister species. Discerning sister species of the *Cx. pipiens* subgroup is further complicated by interspecific breeding, with some populations showing genetic introgression to varying extents (Cornel et al., 2003). The seven sister species of this subgroup are practically indistinguishable based on morphology and require molecular methods to discern (Farajollahi et al., 2011; Zittra et al., 2016).

Indeed, the 621 bp COI sequence amplified in our study did not contain enough nucleotide divergence to allow clear identification, given that the COI sequence of *Cx. quinquefasciatus* specimens differed from that of *Cx. pipiens* by a single nucleotide. Batovska et al. (2017) found that
even the Internal Transcribed Spacer 2 (ITS2) rDNA region, another common molecular marker,
could not differentiate the two species. Other DNA molecular markers such as nuclear Ace-2 or CQ11
genes (Aspen & Savage, 2003; Zittra et al., 2016) or *Wolbachia pipientis* infection status (Cornel et
al., 2003) are typically employed in tandem. In our study, 28S rRNA sequence-based phylogeny
validated the identity of specimen "Cx quinquefasciatus MG S74" (Figure 3, in coral) and suggested
that specimen "Cx quinquefasciatus MG S75" might have been a *pipiens-quinquefasciatus* hybrid.
These examples demonstrate how 28S rRNA sequences, concatenated with 18S rRNA sequences or
alone, contain enough resolution to differentiate between *Cx. pipiens* and *Cx. quinquefasciatus*.
rRNA-based phylogeny thus allows for more accurate species identification and ecological
observations in the context of disease transmission. Additionally, tracing the genetic flow across
hybrid populations within the *Cx. pipiens* subgroup can inform estimates of vectorial capacity for each
species. As only one or two members from the *Cx. pipiens* and *Cx. vishnui* subgroups were
represented in our taxonomic assemblage, an explicit investigation including all member species of
these subgroups in greater sample numbers is warranted to further test the degree of accuracy with
which 28S and 18S rRNA sequences can delineate sister species.

Our study included French Guianese *Culex* species *Cx. spissipes* (group Spissipes), *Cx. pedroi*
(group Pedroi), and *Cx. portesi* (group Vomerifer). These species belong to the New World subgenus
*Melanoconion*, section Spissipes, with well-documented distribution in North and South Americas
(Sirivanakarn, 1982) and are vectors of encephalitic alphaviruses EEEV and VEEV among others
(Talaga et al., 2021; M. J. Turell et al., 2008; Weaver et al., 2004). Indeed, our rooted rRNA and COI
trees showed the divergence of the three *Melanoconion* species from the major *Culex* clade
comprising species broadly found across Africa and Asia (Auerswald et al., 2021; Farajollahi et al.,
2011; Nchoutpouen et al., 2019; Takhampunya et al., 2011). The topology of the concatenated
28S+18S rRNA tree places the *Cx. portesi* and *Cx. pedroi* species-clades as sister groups (92%
bootstrap support), with *Cx. spissipes* as a basal group within the *Melanoconion* clade (100%
bootstrap support) (Figure 4, in coral). This corroborates the systematics elucidated by Navarro and
Weaver (2004) using the ITS2 marker, and those by Sirivanakarn (1982) and Sallum and Forattini
(1996) based on morphology. Curiously, in the COI tree, *Cx. Spissipes* sequences were clustered
with unknown species Cx. Sp1, forming a clade sister to another containing other *Culex* (*Culex*) and
*Culex* (*Oculeomyia*) species, albeit with very low bootstrap support (Figure 5, in coral). Previous
441 phylogenetic studies based on the COI gene have consistently placed *Cx. spissipes* or the Spissipes group basal to other groups within the *Melanoconion* subgenus (Torres-Gutierrez et al., 2016, 2018).
442
443 However, these studies contain only *Culex* (*Melanoconion*) species in their assemblage, apart from
444 *Cx. quinquefasciatus* to act as an outgroup. This clustering of *Cx. spissipes* with non-*Melanoconion*
445 species in our COI phylogeny could be an artefact of a much more diversified assemblage rather than
446 a true phylogenetic link.

447 Taking advantage of our multi-country sampling, we examined whether rRNA or COI phylogeny
448 can be used to distinguish conspecifics originating from different geographies. Our assemblage
449 contains five of such species: *An. coustani*, *An. funestus*, *An. gambiae*, *Ae. albopictus*, and *Ma.*
450 *uniformis*. Among the rRNA trees, the concatenated 28S+18S and 28S rRNA trees were able to
451 discriminate between *Ma. uniformis* specimens from Madagascar, Cambodia, and the Central African
452 Republic (in dark green), and between *An. coustani* specimens from Madagascar and the Central
453 African Republic (in purple) (100% bootstrap support). In the COI tree, only *Ma. uniformis* was
454 resolved into geographical clades comprising specimens from Madagascar and specimens from
455 Cambodia (in dark green) (72% bootstrap support). No COI sequence was obtained from one *Ma.*
456 *uniformis* specimen from the Central African Republic. The 28S+18S rRNA sequences ostensibly
457 provided more population-level genetic information than COI sequences alone with better support.
458 The use of rRNA sequences in investigating the biodiversity of mosquitoes should therefore be
459 explored with a more comprehensive taxonomic assemblage.

460 The phylogenetic reconstructions based on rRNA or COI sequences in our study are hardly
461 congruent (Table 2), but two principal differences stand out. First, the COI phylogeny does not
462 recapitulate the early divergence of *Anophelinae* from *Culicinae* (Figure 5). This is at odds with other
463 studies estimating mosquito divergence times based on mitochondrial genes (Logue et al., 2013;
464 Lorenz et al., 2021) or nuclear genes (Reidenbach et al., 2009). The second notable feature in the
465 rRNA trees is the remarkably large interspecies and intersubgeneric evolutionary distances within
466 genus *Anopheles* relative to genera in the *Culicinae* subfamily (Figure 3, Figure 3—figure supplement
467 1, Figure 4, Figure 4—figure supplements 1 and 2; *Anopheles* in purple) but this is not apparent in the
468 COI tree. The hyperdiversity among *Anopheles* taxa may be attributed to the earlier diversification of
469 the *Anophelinae* subfamily in the early Cretaceous period compared to that of the *Culicinae* subfamily,
470 a difference of at least 40 million years (Lorenz et al., 2021). The differences in rRNA and COI tree
471 topologies indicate a limitation in using COI alone to determine evolutionary relationships. Importantly,
472 drawing phylogenetic conclusions from short DNA barcodes such as COI has been cautioned against
473 due to its weak phylogenetic signal (Hajibabaei et al., 2006). The relatively short length of our COI
474 sequences (621–699 bp) combined with the 100-fold higher nuclear substitution rate of mitochondrial
475 genomes relative to nuclear genomes (Arctander, 1995) could result in homoplasy (Danforth et al.,
476 2005), making it difficult to clearly discern ancestral sequences and correctly assign branches into
477 lineages, as evidenced by the poor nodal bootstrap support at genus-level branches. Indeed, in the
478 study by Lorenz et al. (2021), a phylogenetic tree constructed using a concatenation of all 13 protein-
479 coding genes of the mitochondrial genome was able to resolve ancient divergence events. This
480 affirms that while COI sequences can be used to reveal recent speciation events, longer or multi-gene
481 molecular markers are necessary for studies into deeper evolutionary relationships (Danforth et al.,
482 2005).

483 In contrast to Anophelines where 28S rRNA phylogenies illustrated higher interspecies divergence
484 compared to COI phylogeny, two specimens of an unknown *Mansonia* species, "Ma sp.4 GF S103"
485 and "Ma sp.4 GF S104", provided an example where interspecies relatedness based on their COI
486 sequences is greater than that based on their rRNA sequences in relation to "Ma titillans GF S105".
487 While all rRNA trees placed "Ma titillans GF S105" as a sister taxon with 100% bootstrap support, the
488 COI tree placed Ma sp.4 basal to all other species except *Ur. Geometrica* (Figure 5; *Mansonia* in dark
489 green, *Uranotaenia* in pink). This may hint at a historical selective sweep in the mitochondrial
490 genome, whether arising from geographical separation, mutations, or linkage disequilibrium with
491 inherited symbionts (Hurst & Jiggins, 2005), resulting in the disparate mitochondrial haplogroups
492 found in French Guyanese Ma sp.4 and *Ma. titillans*. In addition, both haplogroups are distant from
493 those associated with members of subgenus *Mansonoides*. To note, the COI sequences of "Ma sp.4
494 GF S103" and "Ma sp.4 GF S104" share 87.12 and 87.39% nucleotide similarity, respectively, to that
495 of "Ma titillans GF S105". Interestingly, the endosymbiont *Wolbachia pipientis* has been detected in
496 *Ma. titillans* sampled from Brazil (De Oliveira et al., 2015), which may contribute to the divergence of
497 "Ma titillans GF S105" COI sequence away from those of Ma sp.4. This highlights other caveats of
498 using a mitochondrial DNA marker in determining evolutionary relationships (Hurst & Jiggins, 2005),
499 which nuclear markers such as 28S and 18S rRNA sequences may be immune to.
Total RNA-seq is a valuable tool for surveillance and virus discovery in sylvatic mosquitoes but it is impeded by the lack of full-length rRNA reference sequences. Here we presented a rRNA sequence assembly strategy and a dataset of 234 newly generated mosquito 28S and 18S rRNA sequences. Our work has expanded the current mosquito rRNA reference library by providing, to our knowledge, the first full-length rRNA records for 30 species in public databases and paves the way for the assembly of many more. These novel rRNA sequences can improve mosquito metagenomics based on RNA-seq by enabling physical and computational removal of rRNA from specimens and streamlined species identification using rRNA markers.

Given that a reference sequence is available, rRNA markers could serve as a better approach for mosquito taxonomy and phylogeny than COI markers. In analysing the same set of specimens by their COI and rRNA sequences, we showed that rRNA sequences can discriminate between members of a species subgroup as well as conspecifics from different geographies. Phylogenetic inferences from a tree based on 28S rRNA sequences alone or on concatenated 28S+18S rRNA sequences are more aligned with contemporary mosquito systematics, showing evolutionary relationships that agree with other phylogenetic studies. While COI-based phylogeny can reveal recent speciation events, rRNA sequences may be better suited for investigations of deeper evolutionary relationships as they are less prone to selective sweeps and homoplasy. The advantages and disadvantages of rRNA and COI sequences as molecular markers are summarised in Table 3. Further studies are necessary to reveal how rRNA sequences compare against other nuclear or mitochondrial DNA marker systems (Batovska et al., 2017; Beebe, 2018; Behura, 2006; Ratnasingham & Hebert, 2007; Reidenbach et al., 2009; Vezenegho et al., 2022).

**Table 3.** Comparison of 28S or concatenated 28S+18S rRNA and COI sequences as molecular markers.

28S+18S rRNA

<table><thead><tr><td>Advantages</td><td>Disadvantages</td></tr></thead><tbody><tr><td>In RNA-seq metagenomics studies, molecular taxonomy of specimens based on rRNA sequences can be done from RNA-seq data without additional sample preparation or sequencing.</td><td>RNA-seq costs more than Sanger sequencing.</td></tr><tr><td>28S rRNA and concatenated 28S+18S rRNA sequences can resolve the identity of specimens where COI sequences were ambiguous, particularly between members of species subgroups.</td><td>Reference rRNA sequences are currently much more limited in breadth compared to other established molecular markers.</td></tr></tbody></table>
<table><tr><td>28S rRNA and concatenated 28S+18S rRNA sequences can distinguish conspecifics from different geographies for certain species.<br/><br/>Phylogenetic inferences based on 28S rRNA and concatenated 28S+18S rRNA sequences show relationships that are more concordant to contemporary mosquito systematics elucidated by other studies and may be a more suitable marker to study deep evolutionary relationships.<br/><br/>Being longer and nuclear-encoded, 28S or concatenated 28S+18S rRNA sequences are immune to homoplasy or to selective sweeps that may affect genomes of inherited symbionts such as mitochondria.</td><td></td></tr></table>

<table><thead><tr><th colspan="2">COI</th></tr><tr><th>Advantages</th><th>Disadvantages</th></tr></thead><tbody><tr><td>With a larger reference database, the COI is a versatile marker for molecular taxonomy.<br/><br/>Being a shorter DNA marker, the COI gene is cost-and time-effective to amplify, sequence, and characterise.<br/><br/>Universal primer sets to amplify the COI marker have been developed and tested for many diverse species.</td><td>All species taxa clustered into distinct clades but with weaker bootstrap support at internal nodes relative to those of the 28S+18S rRNA tree.<br/><br/>For <i>An. coustani</i>, and members of <i>Culex</i> species subgroups such as <i>Cx. quinquefasciatus</i> and <i>Cx. tritaeniorhynchus</i>, COI is unable to unequivocally confirm species identity as species can differ by just one nucleotide. Other molecular markers are often used in tandem.</td></tr></tbody></table>

524

## 525 MATERIALS AND METHODS

### 526 Sample collection

527 Mosquito specimens were sampled from 2019 to 2020 by medical entomology teams from the Institut Pasteur de Bangui (Central African Republic, Africa; CF), Institut Pasteur de Madagascar (Madagascar, Africa; MG), Institut Pasteur du Cambodge (Cambodia, Asia; KH), and Institut Pasteur de la Guyane (French Guiana, South America; GF). Adult mosquitoes were sampled using several techniques including CDC light traps, BG sentinels, and human-landing catches. Sampling sites are sylvatic locations including rural settlements in the Central African Republic, Madagascar, and French Guiana and national parks in Cambodia. Mosquitoes were morphologically identified using taxonomic identification keys (Edwards, 1941; Grjebine, 1966; Huang & Ward, 1981; Oo et al., 2006; Rattanarithikul et al., 2007, 2010; Rattanarithikul, Harbach, et al., 2005; Rattanarithikul, Harrison, et al., 2005; Rattanarithikul, Harrison, Harbach, et al., 2006; Rattanarithikul, Harrison, Panthusiri, et al., 2006; Rueda, 2004) on cold tables before preservation by flash freezing in liquid nitrogen and

528 Pasteur de Bangui (Central African Republic, Africa; CF), Institut Pasteur de Madagascar (Madagascar, Africa; MG), Institut Pasteur du Cambodge (Cambodia, Asia; KH), and Institut Pasteur de la Guyane (French Guiana, South America; GF). Adult mosquitoes were sampled using several techniques including CDC light traps, BG sentinels, and human-landing catches. Sampling sites are sylvatic locations including rural settlements in the Central African Republic, Madagascar, and French Guiana and national parks in Cambodia. Mosquitoes were morphologically identified using taxonomic identification keys (Edwards, 1941; Grjebine, 1966; Huang & Ward, 1981; Oo et al., 2006; Rattanarithikul et al., 2007, 2010; Rattanarithikul, Harbach, et al., 2005; Rattanarithikul, Harrison, et al., 2005; Rattanarithikul, Harrison, Harbach, et al., 2006; Rattanarithikul, Harrison, Panthusiri, et al., 2006; Rueda, 2004) on cold tables before preservation by flash freezing in liquid nitrogen and
transportation in dry ice to Institut Pasteur Paris for analysis. A list of the 112 mosquito specimens included in our taxonomic assemblage and their related information are provided in Appendix 1—table 1. To note, specimen ID S53, S80, and S81 were removed from our assemblage as their species identity could not be determined by COI or rRNA sequencing.

### RNA and DNA isolation

Nucleic acids were isolated from mosquito specimens using TRIzol reagent according to manufacturer's protocol (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Single mosquitoes were homogenised into 200 µL of TRIzol reagent and other of the reagents within the protocol were volume-adjusted accordingly. Following phase separation, RNA were isolated from the aqueous phase while DNA were isolated from the remaining interphase and phenol-chloroform phase. From here, RNA is used to prepare cDNA libraries for next generation sequencing while DNA is used in PCR amplification and Sanger sequencing of the mitochondrial *cytochrome c oxidase subunit I* (COI) gene as further described below.

### Probe depletion of rRNA

We tested a selective rRNA depletion protocol by Morlan et al. (2012) on several mosquito species from the *Aedes*, *Culex*, and *Anopheles* genera. We designed 77 tiled 80 bp DNA probes antisense to the *Ae. aegypti* 28S, 18S, and 5.8S rRNA sequences. A pool of probes at a concentration of 0.04 µM were prepared. To bind probes to rRNA, 1 µL of probes and 2 µL of Hybridisation Buffer (100 mM Tris-HCl and 200 mM NaCl) was added to rRNA samples to a final volume of 20 µL and subjected to a slow-cool incubation starting at 95 °C for 2 minutes, then cooling to 22 °C at a rate of 0.1 °C per second, ending with an additional 5 minutes at 22 °C. The resulting RNA:DNA hybrids were treated with 2.5 µL Hybridase™ Thermostable RNase H (Epicentre, Illumina, Madison, Wisconsin, USA) and incubated at 37 °C for 30 minutes. To remove DNA probes, the mix was treated with 1 µL DNase I (Invitrogen) and purified with Agencourt RNAClean XP Beads (Beckman Coulter, Brea, California, USA). The resulting RNA is used for total RNA sequencing to check depletion efficiency.

### Total RNA sequencing

To obtain rRNA sequences, RNA samples were quantified on a Qubit Fluorometer (Invitrogen) using the Qubit RNA BR Assay kit (Invitrogen) for concentration adjustment. Non-depleted total RNA was used for library preparation for next generation sequencing using the NEBNext Ultra II RNA Library
Preparation Kit for Illumina (New England Biolabs, Ipswich, Massachusetts, USA) and the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (New England Biolabs). Sequencing was performed on a NextSeq500 sequencing system (Illumina, San Diego, California, USA). Quality control of fastq data and trimming of adapters were performed with FastQC and cutadapt, respectively.

## 28S and 18S rRNA assembly

To obtain 28S and 18S rRNA contigs, we had to first clean our fastq library by separating the reads representing mosquito rRNA from all other reads. To achieve this, we used the SILVA RNA sequence database to create 2 libraries: one containing all rRNA sequences recorded under the "Insecta" node of the taxonomic tree, the other containing the rRNA sequences of many others nodes distributed throughout the taxonomic tree, hence named "Non-Insecta" (Quast et al., 2013). Each read was aligned using the nucleotide Basic Local Alignment Search Tool (BLASTn, https://blast.ncbi.nlm.nih.gov/) of the National Center for Biotechnology Information (NCBI) against each of the two libraries and the scores of the best high-scoring segment pairs from the two BLASTns are subsequently used to calculate a ratio of Insecta over Non-Insecta scores (Altschul et al., 1990). Only reads with a ratio greater than 0.8 were used in the assembly. The two libraries being non-exhaustive, we chose this threshold of 0.8 to eliminate only reads that were clearly of a non-insect origin. Selected reads were assembled with the SPAdes genome assembler using the "-rna" option, allowing more heterogeneous coverage of contigs and kmer lengths of 31, 51 and 71 bases (Bankevich et al., 2012). This method successfully assembled rRNA sequences for all specimens, including a parasitic *Horreolanus* water mite (122 sequences for 28S and 114 sequences for 18S).

Initially, our filtration technique had two weaknesses. First, there is a relatively small number of complete rRNA sequences in the Insecta library from SILVA. To compensate for this, we carried out several filtration cycles, each time adding in the complete sequences produced in previous cycles to the Insecta library. Second, when our mosquito specimens were parasitized by other insects, it was not possible to bioinformatically filter out rRNA reads belonging to the parasite. For these rare cases, we used the "--trusted-contigs" option of the SPAdes assembler (Bankevich et al., 2012), giving it access to the 28S and 18S rRNA sequences of the mosquito closest in terms of taxonomic distance. By doing this, the assembler was able to reconstruct the rRNA of the mosquito as well as the rRNA of the parasitizing insect. All assembled rRNA sequences from this study have been deposited in
597 GenBank with accession numbers OM350214–OM350327 for 18S rRNA sequences and OM542339–
598 OM542460 for 28S rRNA sequences.

599 **COI amplicon sequencing**

600 The mitochondrial COI gene was amplified from DNA samples using the universal “Folmer” primer set
601 LCO1490 (5’- GGTCAACAAATCATAAAGATATTGG -3’) and HCO2198 (5’-
602 TAAACTTCAGGGTGACCAAAAAATCA-3’), as per standard COI marker sequencing practices,
603 producing a 658 bp product (Folmer et al., 1994). PCRs were performed using Phusion High-Fidelity
604 DNA Polymerase (Thermo Fisher Scientific). Every 50 µL reaction contained 10 µL of 5X High Fidelity
605 buffer, 1 µL of 10 mM dNTPs, 2.5 µL each of 10 mM forward (LCO1490) and reverse (HCO2198)
606 primer, 28.5 µL of water, 5 µL of DNA sample, and 0.5 µL of 2 U/µL Phusion DNA polymerase. A 3-
607 step cycling incubation protocol was used: 98 °C for 30 seconds; 35 cycles of 98 °C for 10 seconds,
608 60 °C for 30 seconds, and 72 °C for 15 seconds; 72 °C for 5 minutes ending with a 4 °C hold. PCR
609 products were size-verified using gel electrophoresis and then gel-purified using the QIAquick Gel
610 Extraction Kit (Qiagen, Hilden, Germany). Sanger sequencing of the COI amplicons were performed
611 by Eurofins Genomics, Ebersberg, Germany.

612 **COI sequence analysis**

613 Forward and reverse COI DNA sequences were end-trimmed to remove bases of poor quality (Q
614 score < 30). At the 5' ends, sequences were trimmed at the same positions such that all forward
615 sequences start with 5'- TTTTGG and all reverse sequences start with 5'- GGNTCT. Forward and
616 reverse sequences were aligned using BLAST to produce a 621 bp consensus sequence. In cases
617 where good quality sequences extends beyond 621 bp, forward and reverse sequences were
618 assembled using Pearl (https://www.gear-genomics.com/pearl/) and manually checked for errors
619 against trace files (Rausch et al., 2019, 2020). We successfully assembled a total of 106 COI
620 sequences. All assembled COI sequences from this study have been deposited in GenBank with
621 accession numbers OM630610–OM630715.

622 **COI validation of morphology-based species identification**

623 We analysed assembled COI sequences with BLASTn against the nucleotide collection (nr/nt)
624 database to confirm morphology-based species identification. BLAST analyses revealed 32 cases
625 where top hits indicated a different species identity, taking <95% nucleotide sequence similarity as the
threshold to delineate distinct species (Appendix 2—table 1). In these cases, the COI sequence of the specimen was then BLAST-aligned against a GenBank record representing the morphological species to verify that the revised identity is a closer match by a significant margin, i.e., more than 2% nucleotide sequence similarity. All species names reported hereafter reflect identities determined by COI sequence except for cases where COI-based identities were ambiguous, in which case morphology-based identities were retained. In cases where matches were found within a single genus but of multiple species, specimens were indicated as an unknown member of their genus (e.g., *Culex* sp.). Information of the highest-scoring references for all specimens, including details of ambiguous BLASTn results, are recorded in Appendix 2—table 1.

Within our COI sequences, we found six unidentified *Culex* species (including two that matched to GenBank entries identified only to the genus level), four unidentified *Mansonia* species, and one unidentified *Mimomyia* species. For *An. baezai*, no existing GenBank records were found at the time this analysis was performed.

## Phylogenetic analysis

Multiple sequence alignment (MSA) were performed on assembled COI and rRNA sequences using the MUSCLE software (Edgar, 2004; Madeira et al., 2019). As shown in Figure 3—figure supplement 2, the 28S rRNA sequences contain many blocks of highly conserved nucleotides, which makes the result of multiple alignment particularly obvious. We therefore did not test other alignment programs. The multiple alignment of the COI amplicons is even more evident since no gaps are necessary for this alignment.

Phylogenetic tree reconstructions were performed with the MEGA X software using the maximum-likelihood method (S. Kumar et al., 2018). Default parameters were used with bootstrapping with 500 replications to quantify confidence level in branches. For rRNA trees, sequences belonging to an unknown species of parasitic water mite (genus *Horreolanus*) found in our specimens served as an outgroup taxon. In addition, we created and analysed a separate dataset combining our 28S rRNA sequences and full-length 28S rRNA sequences from GenBank totalling 169 sequences from 58 species (12 subgenera). To serve as outgroups for the COI tree, we included sequences obtained from GenBank of three water mite species, *Horreolanus orphanus* (KM101004), *Sperchon fuxiensis* (MH916807), and *Arrenurus* sp. (MN362807).
655 **DECLARATIONS**

656 **Availability of data and materials**

657 The multiple sequence alignments that gave rise to the presented phylogenetic reconstructions are
658 included as source data files linked to the relevant figures. All sequences generated in this study have
659 been deposited in GenBank under the accession numbers OM350214–OM350327 for 18S rRNA
660 sequences, OM542339–OM542460 for 28S rRNA sequences, and OM630610–OM630715 for COI
661 sequences (Appendix 1—table 1).

662 **Competing interests**

663 The authors declare that they have no financial or non-financial competing interests.

664 **Impact statement**

665 A score-based read filtration strategy enables assembly of full-length ribosomal RNA sequences for
666 lesser-studied mosquito species, opening the doors to the use of these sequences as a novel
667 molecular marker for taxonomic and phylogenetic studies.

668 **Funding**

669 This work was supported by the Defence Advanced Research Projects Agency PREEMPT program
670 managed by Dr. Rohit Chitale and Dr. Kerri Dugan [Cooperative Agreement HR001118S0017] (the
671 content of the information does not necessarily reflect the position or the policy of the U.S.
672 government, and no official endorsement should be inferred).

673 **Acknowledgements**

674 We thank members of the Saleh lab for valuable discussions and to Dr Louis Lambrechts for critical
675 reading of the manuscript. We especially thank all medical entomology staff of IP Bangui, IP
676 Cambodge (Sony Yean, Kimly Heng, Kalyan Chhuoy, Sreynik Nhek, Moeun Chhum, Kimhuor Sour
677 and Pierre-Olivier Maquart), IP Madagascar, and IP Guyane for assistance in field missions,
678 laboratory work, and logistics, and Inès Partouche from IP Paris for laboratory assistance. We are
679 also grateful to Dr Catherine Dauga for advice on phylogenetic analyses, and to Amandine Guidez for
680 providing a French Guiana-specific COI reference library. Finally, we thank our reviewers, including
681 Leslie Vosshall and Katherine Young, for their constructive reviews of this manuscript.
682 FIGURE SUPPLEMENTS

683 Figure 3—figure supplement 1

684

685 Interspecific and intersubgeneric distances within the genus *Anopheles* indicate a greater

686 degree of divergence than those within any other genera of family *Culicidae*. The phylogenetic

687 tree presented in Figure 3 based on 28S sequences from this study and from GenBank (annotated

688 with filled circles) is depicted here in radial format to illustrate how the branch lengths separating

689 Anopheline taxa are longer relative to other members of family *Culicidae*. An unknown Horreolanus
species found among our samples serves as an outgroup. For sequences from this study, each specimen label contains information on taxonomy, origin (in 2-letter country codes), and specimen ID number. Specimen genera are indicated by colour: *Culex* in coral, *Anopheles* in purple, *Aedes* in dark blue, *Mansonia* in dark green, *Limatus* in light green, *Coquillettidia* in light blue, *Psorophora* in yellow, *Mimomyia* in teal, *Uranotaenia* in pink and *Eretmapodites* in brown. Scale bar at 0.05 is shown.

<figure><img src="image_8.png" /><figcaption>**Figure 3—figure supplement 2**</figcaption></figure>

Sequence conservation among 169 28S rRNA sequences obtained from this study and from GenBank combined. Multiple sequence alignment was performed on 28S rRNA sequences, 3900 bp in length. Each bar represents a 25-bp sliding window of the 28S rRNA sequence alignment where the y-axis values are the lowest percentage nucleotide identity found.

<caption>**Figure 4—figure supplement 1**</caption>
<figure><img src="image_9.png" /></figure>
703 **Phylogenetic tree based on 28S rRNA sequences generated from this study (3900 bp).** This tree 704 was inferred using maximum-likelihood method and constructed to scale in MEGA X (S. Kumar et al., 705 2018) using an unknown *Horreolanus* species found among our samples as an outgroup. Values at 706 each node indicate bootstrap support (%) from 500 replications. For sequences from this study, each 707 specimen label contains information on taxonomy, origin (in 2-letter country codes), and specimen ID 708 number. Some specimens produced up to two consensus 28S rRNA sequences; this is indicated by 709 the numbers 1 or 2 in the beginning of the specimen label. Specimen genera are indicated by colour: 710 *Culex* in coral, *Anopheles* in purple, *Aedes* in dark blue, *Mansonia* in dark green, *Limatus* in light 711 green, *Coquillettidia* in light blue, *Psorophora* in yellow, *Mimomyia* in teal, *Uranotaenia* in pink and 712 *Eretmapodites* in brown. Scale bar at 0.05 is shown.

713 **Figure 4—figure supplement 2**
<figure><img src="image_10.png" /><figcaption>Phylogenetic tree based on 18S rRNA sequences (1900 bp). This tree was inferred using maximum-likelihood method and constructed to scale in MEGA X (S. Kumar et al., 2018) using an unknown *Horreolanus* species found among our samples as an outgroup. Values at each node indicate bootstrap support (%) from 500 replications. For sequences from this study, each specimen label contains information on taxonomy, origin (in 2-letter country codes), and specimen ID number. One 18S rRNA sequence was obtained for each specimen. Specimen genera are indicated by colour: Culex in coral, Anopheles in purple, Aedes in dark blue, Mansonia in dark green, Limatus in light green, Coquillettidia in light blue, Psorophora in yellow, Mimomyia in teal, Uranotaenia in pink and Eretmapodites in brown. Scale bar at 0.05 is shown.</figcaption></figure>
<table><thead><tr><th>Sequence ID</th><th>Taxonomy [Genus (subgenus) species]</th><th>Origin</th><th>Collection site</th><th>Collection period</th><th>Blood engorged (Y/N)</th><th>Sample ID</th><th>COI accession number</th><th>18S rRNA accession number</th><th>28S rRNA accession number</th></tr></thead><tbody><tr><td>Ae_albopictus_KH_S1</td><td>Aedes (Stegomyia) albopictus</td><td>Cambodia</td><td>Rattanakiri</td><td>Dec 2019</td><td>N</td><td>1</td><td>OM630613</td><td>OM350214</td><td>OM542460</td></tr><tr><td>Ae_albopictus_KH_S2</td><td>Aedes (Stegomyia) albopictus</td><td>Cambodia</td><td>Rattanakiri</td><td>Dec 2019</td><td>N</td><td>2</td><td>OM630614</td><td>OM350220</td><td>OM542373</td></tr><tr><td>Ae_albopictus_KH_S3</td><td>Aedes (Stegomyia) albopictus</td><td>Cambodia</td><td>Rattanakiri</td><td>Dec 2019</td><td>N</td><td>3</td><td>OM630615</td><td>OM350316</td><td>OM542374</td></tr><tr><td>An_baezai_KH_S4</td><td>baezai</td><td>Cambodia</td><td>Koh Kong</td><td>Mar 2019</td><td>N</td><td>4</td><td>OM630631</td><td>OM350327</td><td>OM542357</td></tr><tr><td>An_baezai_KH_S5</td><td>baezai</td><td>Cambodia</td><td>Koh Kong</td><td>Mar 2019</td><td>N</td><td>5</td><td>OM630632</td><td>OM350233</td><td>OM542440</td></tr><tr><td>An_baezai_KH_S6</td><td>baezai</td><td>Cambodia</td><td>Koh Kong</td><td>Mar 2019</td><td>N</td><td>6</td><td>OM630633</td><td>OM350234</td><td>OM542358</td></tr><tr><td>Cx_pseudovishnui_KH_S7</td><td>pseudovishnui</td><td>Cambodia</td><td>Rattanakiri</td><td>Dec 2019</td><td>N</td><td>7</td><td>OM630689</td><td>OM350285</td><td>OM542413</td></tr><tr><td>Cx_pseudovishnui_KH_S8</td><td>pseudovishnui</td><td>Cambodia</td><td>Rattanakiri</td><td>Dec 2019</td><td>N</td><td>8</td><td>OM630690</td><td>OM350286</td><td>OM542414</td></tr><tr><td>Cx_pseudovishnui_KH_S9</td><td>pseudovishnui</td><td>Cambodia</td><td>Rattanakiri</td><td>Dec 2019</td><td>N</td><td>9</td><td>OM630691</td><td>OM350287</td><td>OM542415</td></tr><tr><td>Ma_indiana_KH_S10</td><td>indiana</td><td>Cambodia</td><td>Battambong</td><td>Nov 2019</td><td>N</td><td>10</td><td>OM630698</td><td>OM350295</td><td>OM542422</td></tr><tr><td>MaUniformis_KH_S11</td><td>uniformis</td><td>Cambodia</td><td>Battambong</td><td>Nov 2019</td><td>N</td><td>11</td><td>OM630699</td><td>OM350296</td><td>OM542423</td></tr><tr><td>Ma_indiana_KH_S12</td><td>Mansonia</td><td>Cambodia</td><td>Battambong</td><td>Nov 2019</td><td>N</td><td>12</td><td>OM630700</td><td>OM350297</td><td>OM542424</td></tr></tbody></table>
<table>
  <tr>
    <td>Cx_sp.1_KH_S13</td>
    <td>Culex sp.1<br/>Culex (Culex)<br/>orientalis<br/>Mansonia<br/>(Mansonioides)</td>
    <td>Cambodia</td>
    <td>Prek Toal</td>
    <td>Feb 2019</td>
    <td>N</td>
    <td>13</td>
    <td>OM630672</td>
    <td>OM350267</td>
    <td>OM542395</td>
  </tr>
  <tr>
    <td>Cx_orientalis_KH_S14</td>
    <td>Culex sp.1<br/>Culex (Culex)<br/>orientalis<br/>Mansonia<br/>(Mansonioides)</td>
    <td>Cambodia</td>
    <td>Prek Toal</td>
    <td>Feb 2019</td>
    <td>N</td>
    <td>14</td>
    <td>OM630673</td>
    <td>OM350268</td>
    <td>OM542396</td>
  </tr>
  <tr>
    <td>MaUniformis_KH_S15</td>
    <td>uniformis<br/>Mansonia<br/>(Mansonioides)</td>
    <td>Cambodia</td>
    <td>Battambong</td>
    <td>Nov 2019</td>
    <td>N</td>
    <td>15</td>
    <td>OM630705</td>
    <td>OM350303</td>
    <td>OM542430</td>
  </tr>
  <tr>
    <td>MaUniformis_KH_S16</td>
    <td>uniformis<br/>Mansonia<br/>(Mansonioides)</td>
    <td>Cambodia</td>
    <td>Battambong</td>
    <td>Nov 2019</td>
    <td>N</td>
    <td>16</td>
    <td>OM630706</td>
    <td>OM350305</td>
    <td>OM542432</td>
  </tr>
  <tr>
    <td>MaUniformis_KH_S17</td>
    <td>uniformis<br/>Culex<br/>(Oculeomyia)</td>
    <td>Cambodia</td>
    <td>Battambong</td>
    <td>Nov 2019</td>
    <td>N</td>
    <td>17</td>
    <td>OM630707</td>
    <td>OM350304</td>
    <td>OM542431</td>
  </tr>
  <tr>
    <td>Cx_bitaeniorhynchus_KH_S18</td>
    <td>bitaeniorhynchus<br/>Culex<br/>(Oculeomyia)</td>
    <td>Cambodia</td>
    <td>Battambong</td>
    <td>Nov 2019</td>
    <td>N</td>
    <td>18</td>
    <td>OM630656</td>
    <td>OM350255</td>
    <td>OM542381</td>
  </tr>
  <tr>
    <td>Cx_bitaeniorhynchus_KH_S19</td>
    <td>bitaeniorhynchus<br/>Culex<br/>(Oculeomyia)</td>
    <td>Cambodia</td>
    <td>Battambong</td>
    <td>Nov 2019</td>
    <td>N</td>
    <td>19</td>
    <td>OM630657</td>
    <td>OM350256</td>
    <td>OM542382</td>
  </tr>
  <tr>
    <td>Cx_bitaeniorhynchus_KH_S20</td>
    <td>bitaeniorhynchus<br/>Culex (Culex)</td>
    <td>Cambodia</td>
    <td>Battambong</td>
    <td>Nov 2019</td>
    <td>N</td>
    <td>20</td>
    <td>OM630658</td>
    <td>OM350257</td>
    <td>OM542383,<br/>OM542384</td>
  </tr>
  <tr>
    <td>Cx_tritaeniorhynchus_KH_S21</td>
    <td>tritaeniorhynchus<br/>Culex (Culex)</td>
    <td>Cambodia</td>
    <td>Battambong</td>
    <td>Nov 2019</td>
    <td>N</td>
    <td>21</td>
    <td>OM630680</td>
    <td>OM350277</td>
    <td>OM542404</td>
  </tr>
  <tr>
    <td>Cx_tritaeniorhynchus_KH_S22</td>
    <td>tritaeniorhynchus<br/>Culex (Culex)</td>
    <td>Cambodia</td>
    <td>Battambong</td>
    <td>Nov 2019</td>
    <td>N</td>
    <td>22</td>
    <td>OM630681</td>
    <td>OM350278</td>
    <td>OM542405</td>
  </tr>
  <tr>
    <td>Cx_tritaeniorhynchus_KH_S23</td>
    <td>tritaeniorhynchus<br/>Aedes<br/>(Stegomyia)</td>
    <td>Cambodia</td>
    <td>Battambong</td>
    <td>Nov 2019</td>
    <td>N</td>
    <td>23</td>
    <td>OM630682</td>
    <td>OM350279</td>
    <td>OM542406</td>
  </tr>
  <tr>
    <td>Ae_aegypti_CF_S24</td>
    <td>aegypti<br/>Aedes<br/>(Stegomyia)</td>
    <td>Republic<br/>Central<br/>African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>24</td>
    <td>OM630610</td>
    <td>OM350314</td>
    <td>OM542339</td>
  </tr>
  <tr>
    <td>Ae_aegypti_CF_S25</td>
    <td>aegypti<br/>Aedes<br/>(Stegomyia)</td>
    <td>Republic<br/>Central<br/>African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>25</td>
    <td>OM630611</td>
    <td>OM350215</td>
    <td>OM542340</td>
  </tr>
  <tr>
    <td>Ae_aegypti_CF_S26</td>
    <td>aegypti<br/>Aedes<br/>(Stegomyia)</td>
    <td>Republic<br/>Central<br/>African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>26</td>
    <td>OM630612</td>
    <td>OM350216</td>
    <td>OM542341</td>
  </tr>
  <tr>
    <td>Ae_simpsoni_CF_S27</td>
    <td>simpsoni<br/>Aedes<br/>(Stegomyia)</td>
    <td>Republic<br/>Central<br/>African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>27</td>
    <td>OM630619</td>
    <td>OM350221</td>
    <td>OM542345</td>
  </tr>
  <tr>
    <td>Ae_simpsoni_CF_S28</td>
    <td>simpsoni<br/>Aedes<br/>(Stegomyia)</td>
    <td>Republic<br/>Central<br/>African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>28</td>
    <td>OM630620</td>
    <td>OM350222</td>
    <td>OM542346</td>
  </tr>
</table>
<table>
  <tr>
    <td>Ae_simpsoni_CF_S29</td>
    <td>simpsoni<br>Aedes<br>(Stegomyia)</td>
    <td>Republic<br>Central<br>African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>29</td>
    <td>OM630621</td>
    <td>OM350223</td>
    <td>OM542347</td>
  </tr>
  <tr>
    <td>Ae_vittatus_CF_S30</td>
    <td>vittatus<br>Aedes<br>(Fredwardsius)</td>
    <td>Republic<br>Central<br>African</td>
    <td>Gbozo</td>
    <td>Aug 2019</td>
    <td>Y</td>
    <td>30</td>
    <td>OM630628</td>
    <td>OM350230</td>
    <td>OM542439</td>
  </tr>
  <tr>
    <td>Ae_vittatus_CF_S31</td>
    <td>vittatus<br>Aedes<br>(Fredwardsius)</td>
    <td>Republic<br>Central<br>African</td>
    <td>Gbozo</td>
    <td>Aug 2019</td>
    <td>N</td>
    <td>31</td>
    <td>OM630629</td>
    <td>OM350231</td>
    <td>OM542355</td>
  </tr>
  <tr>
    <td>Ae_vittatus_CF_S32</td>
    <td>vittatus</td>
    <td>Republic<br>Central<br>African</td>
    <td>Gbozo</td>
    <td>Aug 2019</td>
    <td>N</td>
    <td>32</td>
    <td>OM630630</td>
    <td>OM350232</td>
    <td>OM542356</td>
  </tr>
  <tr>
    <td>Ma_sp.1_CF_S33</td>
    <td>Mansonia sp.1</td>
    <td>Republic<br>Central<br>African</td>
    <td>Bayanga</td>
    <td>Nov 2019</td>
    <td>Y</td>
    <td>33</td>
    <td>N/A</td>
    <td>OM350294</td>
    <td>OM542449</td>
  </tr>
  <tr>
    <td>Ma_sp.2_CF_S34</td>
    <td>Mansonia sp.2</td>
    <td>Republic<br>Central<br>African</td>
    <td>Bayanga</td>
    <td>Nov 2019</td>
    <td>Y</td>
    <td>34</td>
    <td>N/A</td>
    <td>OM350322</td>
    <td>OM542450,<br>OM542456</td>
  </tr>
  <tr>
    <td>Ho_sp.1_CF_S34</td>
    <td>Horreolanus sp.1</td>
    <td>Republic<br>Central<br>African</td>
    <td>Bayanga</td>
    <td>Nov 2019</td>
    <td>-</td>
    <td>34</td>
    <td>N/A</td>
    <td>OM350325</td>
    <td>OM542457</td>
  </tr>
  <tr>
    <td>Ho_sp.2_CF_S34</td>
    <td>Horreolanus sp.2</td>
    <td>Republic<br>Central<br>African</td>
    <td>Bayanga</td>
    <td>Nov 2019</td>
    <td>-</td>
    <td>34</td>
    <td>N/A</td>
    <td>OM350326</td>
    <td>OM542458</td>
  </tr>
  <tr>
    <td>Ma_sp.3_CF_S35</td>
    <td>Mansonia sp.3<br>Aedes<br>(Stegomyia)</td>
    <td>Republic<br>Central<br>African</td>
    <td>Bayanga</td>
    <td>Nov 2019</td>
    <td>Y</td>
    <td>35</td>
    <td>N/A</td>
    <td>OM350323</td>
    <td>OM542451</td>
  </tr>
  <tr>
    <td>Ae_albopictus_CF_S36</td>
    <td>albopictus<br>Aedes<br>(Stegomyia)</td>
    <td>Republic<br>Central<br>African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>36</td>
    <td>OM630616</td>
    <td>OM350217</td>
    <td>OM542342</td>
  </tr>
  <tr>
    <td>Ae_albopictus_CF_S37</td>
    <td>albopictus<br>Aedes<br>(Stegomyia)</td>
    <td>Republic<br>Central<br>African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>37</td>
    <td>OM630617</td>
    <td>OM350218</td>
    <td>OM542343</td>
  </tr>
  <tr>
    <td>Ae_albopictus_CF_S38</td>
    <td>albopictus<br>Anopheles<br>(Anopheles)</td>
    <td>Republic<br>Central<br>African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>38</td>
    <td>OM630618</td>
    <td>OM350219</td>
    <td>OM542344</td>
  </tr>
  <tr>
    <td>An_coustani_CF_S39</td>
    <td>coustani<br>Anopheles<br>(Anopheles)</td>
    <td>Republic<br>Central<br>African</td>
    <td>Pissa</td>
    <td>Jan 2020</td>
    <td>N</td>
    <td>39</td>
    <td>OM630634</td>
    <td>OM350235</td>
    <td>OM542359</td>
  </tr>
  <tr>
    <td>An_coustani_CF_S40</td>
    <td>coustani</td>
    <td>Republic</td>
    <td>Pissa</td>
    <td>Jan 2020</td>
    <td>N</td>
    <td>40</td>
    <td>OM630635</td>
    <td>OM350236</td>
    <td>OM542360</td>
  </tr>
</table>
<table>
  <tr>
    <td>An_coustani_CF_S41</td>
    <td>Anopheles (Anopheles) coustani</td>
    <td>Central African Republic</td>
    <td>Pissa</td>
    <td>Jan 2020</td>
    <td>N</td>
    <td>41</td>
    <td>OM630636</td>
    <td>OM350237</td>
    <td>OM542361</td>
  </tr>
  <tr>
    <td>An funestus_CF_S42</td>
    <td>Anopheles (Cellia) funestus</td>
    <td>Republic Central African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>Y</td>
    <td>42</td>
    <td>OM630640</td>
    <td>OM350241</td>
    <td>OM542365</td>
  </tr>
  <tr>
    <td>An funestus_CF_S43</td>
    <td>Anopheles (Cellia) funestus</td>
    <td>Republic Central African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>Y</td>
    <td>43</td>
    <td>OM630641</td>
    <td>OM350242</td>
    <td>OM542366</td>
  </tr>
  <tr>
    <td>An funestus_CF_S44</td>
    <td>Anopheles (Cellia) funestus</td>
    <td>Republic Central African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>Y</td>
    <td>44</td>
    <td>OM630642</td>
    <td>OM350243</td>
    <td>OM542367</td>
  </tr>
  <tr>
    <td>An gambiae_CF_S45</td>
    <td>Anopheles (Cellia) gambiae</td>
    <td>Republic Central African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>Y</td>
    <td>45</td>
    <td>OM630645</td>
    <td>OM350245</td>
    <td>OM542369, OM542370</td>
  </tr>
  <tr>
    <td>An gambiae_CF_S46</td>
    <td>Anopheles (Cellia) gambiae</td>
    <td>Republic Central African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>Y</td>
    <td>46</td>
    <td>OM630646</td>
    <td>OM350246</td>
    <td>OM542371</td>
  </tr>
  <tr>
    <td>An gambiae_CF_S47</td>
    <td>Anopheles (Cellia) gambiae</td>
    <td>Republic Central African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>Y</td>
    <td>47</td>
    <td>N/A</td>
    <td>OM350247</td>
    <td>OM542372</td>
  </tr>
  <tr>
    <td>Cx_sp.2_CF_S48</td>
    <td>Culex sp.2</td>
    <td>Republic Central African</td>
    <td>Bayanga</td>
    <td>Nov 2019</td>
    <td>Y</td>
    <td>48</td>
    <td>OM630669</td>
    <td>OM350269</td>
    <td>OM542446</td>
  </tr>
  <tr>
    <td>Cx_sp.2_CF_S49</td>
    <td>Culex sp.2</td>
    <td>Republic Central African</td>
    <td>Bayanga</td>
    <td>Nov 2019</td>
    <td>Y</td>
    <td>49</td>
    <td>OM630670</td>
    <td>OM350315</td>
    <td>OM542397</td>
  </tr>
  <tr>
    <td>Cx_sp.2_CF_S50</td>
    <td>Culex sp.2 Mansonia (Mansonioides) uniformis</td>
    <td>Republic Central African</td>
    <td>Bayanga</td>
    <td>Nov 2019</td>
    <td>Y</td>
    <td>50</td>
    <td>OM630671</td>
    <td>OM350270</td>
    <td>OM542398</td>
  </tr>
  <tr>
    <td>Ma uniformis_CF_S51</td>
    <td>Culex (Culex) duttoni</td>
    <td>Republic Central African</td>
    <td>Bouar</td>
    <td>May 2019</td>
    <td>Y</td>
    <td>51</td>
    <td>N/A</td>
    <td>OM350301</td>
    <td>OM542428</td>
  </tr>
  <tr>
    <td>Cx duttoni_CF_S52</td>
    <td>Eretmapodites intermedius</td>
    <td>Republic Central African</td>
    <td>Mbaiki</td>
    <td>Jan 2019</td>
    <td>Y</td>
    <td>52</td>
    <td>OM630704</td>
    <td>OM350302</td>
    <td>OM542429</td>
  </tr>
  <tr>
    <td>Er intermedius_CF_S54</td>
    <td>Eretmapodites intermedius</td>
    <td>Republic Central African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>54</td>
    <td>OM630692</td>
    <td>OM350288</td>
    <td>OM542416</td>
  </tr>
  <tr>
    <td>Er intermedius_CF_S55</td>
    <td>Eretmapodites intermedius</td>
    <td>Republic Central African</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>55</td>
    <td>OM630693</td>
    <td>OM350289</td>
    <td>OM542417</td>
  </tr>
  <tr>
    <td>Er intermedius_CF_S56</td>
    <td>Eretmapodites</td>
    <td>Central</td>
    <td>Pissa</td>
    <td>Jun 2019</td>
    <td>N</td>
    <td>56</td>
    <td>OM630694</td>
    <td>OM350290</td>
    <td>OM542418</td>
  </tr>
</table>
<table><tr><td>Cx_antennatus_MG_S57</td><td>Culex (Culex) antennatus</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>57</td><td>OM630653</td><td>OM350253</td><td>OM542379</td></tr><tr><td>Cx_antennatus_MG_S58</td><td>Culex (Culex) antennatus</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>58</td><td>OM630654</td><td>OM350319</td><td>OM542444</td></tr><tr><td>Cx_antennatus_MG_S59</td><td>Culex (Culex) antennatus</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>59</td><td>OM630655</td><td>OM350254</td><td>OM542380</td></tr><tr><td>Cx_perexiguus_MG_S60</td><td>Culex (Culex) perexiguus</td><td>Madagascar</td><td>Amparafaravola</td><td>Feb 2019</td><td>N</td><td>60</td><td>OM630660</td><td>OM350258</td><td>OM542386</td></tr><tr><td>Cx_sp.3_MG_S61</td><td>Culex sp.3</td><td>Madagascar</td><td>Ambato Boeny</td><td>Aug 2019</td><td>N</td><td>61</td><td>OM630661</td><td>OM350259</td><td>OM542387</td></tr><tr><td>Cx_sp.4_MG_S62</td><td>Culex sp.4</td><td>Madagascar</td><td>Ambato Boeny</td><td>Aug 2019</td><td>N</td><td>62</td><td>OM630662</td><td>OM350260</td><td>OM542388</td></tr><tr><td>Cx_sp.3_MG_S63</td><td>Culex sp.3</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>63</td><td>OM630686</td><td>OM350282</td><td>OM542410</td></tr><tr><td>Mi_sp.1_MG_S64</td><td>Mimomyia sp.1</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>64</td><td>OM630687</td><td>OM350283</td><td>OM542411</td></tr><tr><td>Cx_sp.3_MG_S65</td><td>Culex sp.3</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>65</td><td>OM630688</td><td>OM350284</td><td>OM542412</td></tr><tr><td>An_coustani_MG_S66</td><td>Anopheles (Anopheles) coustani</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>66</td><td>OM630637</td><td>OM350238</td><td>OM542362</td></tr><tr><td>An_coustani_MG_S67</td><td>Anopheles (Anopheles) coustani</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>67</td><td>OM630638</td><td>OM350239</td><td>OM542363</td></tr><tr><td>An_squamosus_MG_S68</td><td>squamosus</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>68</td><td>OM630639</td><td>OM350240</td><td>OM542364</td></tr><tr><td>An.funestus_MG_S69</td><td>Anopheles (Cellia) funestus</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>69</td><td>OM630643</td><td>OM350244</td><td>OM542368</td></tr><tr><td>An.funestus_MG_S70</td><td>Anopheles (Cellia) funestus</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2020</td><td>N</td><td>70</td><td>OM630644</td><td>OM350317</td><td>OM542441</td></tr><tr><td>An_gambiae_MG_S71</td><td>Anopheles (Cellia) gambiae</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>71</td><td>OM630647</td><td>OM350249</td><td>OM542442</td></tr><tr><td>An_gambiae_MG_S72</td><td>Anopheles (Cellia) gambiae</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>72</td><td>OM630648</td><td>OM350248</td><td>OM542443</td></tr><tr><td>An_gambiae_MG_S73</td><td>Anopheles (Cellia) gambiae</td><td>Madagascar</td><td>Ambato Boeny</td><td>Feb 2019</td><td>N</td><td>73</td><td>OM630649</td><td>OM350318</td><td>OM542459</td></tr><tr><td>Cx_quinquefasciatus_MG_S74</td><td>Culex (Culex) quinquefasciatus</td><td>Madagascar</td><td>Amparafaravola</td><td>Feb 2019</td><td>N</td><td>74</td><td>OM630674</td><td>OM350271</td><td>OM542399</td></tr><tr><td>Cx_quinquefasciatus_MG_S75</td><td>Culex (Culex) quinquefasciatus</td><td>Madagascar</td><td>Amparafaravola</td><td>Feb 2019</td><td>N</td><td>75</td><td>OM630675</td><td>OM350272</td><td>OM542447</td></tr><tr><td>Cx_perexiguus_MG_S76</td><td>Culex (Culex) perexiguus</td><td>Madagascar</td><td>Mampikony</td><td>Aug 2019</td><td>N</td><td>76</td><td>OM630676</td><td>OM350273</td><td>OM542400</td></tr></table>
<table>
  <tr>
    <td>Mauniformis_MG_S77</td>
    <td>Mansonia (Mansonioides) uniformis</td>
    <td>Madagascar</td>
    <td>Ambato Boeny</td>
    <td>Feb 2019</td>
    <td>N</td>
    <td>77</td>
    <td>OM630708</td>
    <td>OM350306</td>
    <td>OM542433</td>
  </tr>
  <tr>
    <td>Mauniformis_MG_S78</td>
    <td>Mansonia (Mansonioides) uniformis</td>
    <td>Madagascar</td>
    <td>Ambato Boeny</td>
    <td>Feb 2019</td>
    <td>N</td>
    <td>78</td>
    <td>OM630709</td>
    <td>OM350307</td>
    <td>OM542434</td>
  </tr>
  <tr>
    <td>Mauniformis_MG_S79</td>
    <td>Mansonia (Mansonioides) uniformis</td>
    <td>Madagascar</td>
    <td>Ambato Boeny</td>
    <td>Feb 2019</td>
    <td>N</td>
    <td>79</td>
    <td>OM630710</td>
    <td>OM350308</td>
    <td>OM542435<br>OM542385</td>
  </tr>
  <tr>
    <td>Cxpoicilipes_MG_S82</td>
    <td>Culex poicilipes</td>
    <td>Madagascar</td>
    <td>Mampikony</td>
    <td>Feb 2019</td>
    <td>N</td>
    <td>82</td>
    <td>OM630659</td>
    <td>OM350320</td>
    <td>OM542445</td>
  </tr>
  <tr>
    <td>Mi mediolineata_MG_S83</td>
    <td>Mimomyia mediolineata</td>
    <td>Madagascar</td>
    <td>Ambato Boeny</td>
    <td>Feb 2019</td>
    <td>N</td>
    <td>83</td>
    <td>OM630683</td>
    <td>OM350280</td>
    <td>OM542407<br>OM542408</td>
  </tr>
  <tr>
    <td>Cxneavei_MG_S84</td>
    <td>Culex (Culex) neavei</td>
    <td>Madagascar</td>
    <td>Ambato Boeny</td>
    <td>Feb 2019</td>
    <td>N</td>
    <td>84</td>
    <td>OM630684</td>
    <td>OM350281</td>
    <td>OM542409</td>
  </tr>
  <tr>
    <td>Aescapularis_GF_S85</td>
    <td>(Ochlerotatus) scapularis</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>85</td>
    <td>OM630624</td>
    <td>OM350224</td>
    <td>OM542348,<br>OM542349</td>
  </tr>
  <tr>
    <td>Aescapularis_GF_S86</td>
    <td>(Ochlerotatus) scapularis</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>86</td>
    <td>OM630622</td>
    <td>OM350225</td>
    <td>OM542350</td>
  </tr>
  <tr>
    <td>Aescapularis_GF_S87</td>
    <td>(Ochlerotatus) scapularis</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>87</td>
    <td>OM630623</td>
    <td>OM350226</td>
    <td>OM542351</td>
  </tr>
  <tr>
    <td>Aeserratus_GF_S88</td>
    <td>(Ochlerotatus) serratus</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>88</td>
    <td>OM630625</td>
    <td>OM350227</td>
    <td>OM542352</td>
  </tr>
  <tr>
    <td>Aeserratus_GF_S89</td>
    <td>(Ochlerotatus) serratus</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>89</td>
    <td>OM630626</td>
    <td>OM350228</td>
    <td>OM542353</td>
  </tr>
  <tr>
    <td>Aeserratus_GF_S90</td>
    <td>(Ochlerotatus) serratus</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>90</td>
    <td>OM630627</td>
    <td>OM350229</td>
    <td>OM542354</td>
  </tr>
  <tr>
    <td>Cqvenezuelensis_GF_S91</td>
    <td>venezuelensis</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>91</td>
    <td>OM630650</td>
    <td>OM350250</td>
    <td>OM542375</td>
  </tr>
  <tr>
    <td>Cqvenezuelensis_GF_S92</td>
    <td>venezuelensis</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>92</td>
    <td>OM630651</td>
    <td>OM350251</td>
    <td>OM542376<br>OM542377</td>
  </tr>
  <tr>
    <td>Cqvenezuelensis_GF_S93</td>
    <td>venezuelensis</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>93</td>
    <td>OM630652</td>
    <td>OM350252</td>
    <td>OM542378</td>
  </tr>
  <tr>
    <td>Cxportesi_GF_S94</td>
    <td>Culex sp. BTLHVDV-2014</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>94</td>
    <td>OM630666</td>
    <td>OM350264</td>
    <td>OM542392</td>
  </tr>
  <tr>
    <td>Cxportesi_GF_S95</td>
    <td>Culex sp. BTLHVDV-2014</td>
    <td>French</td>
    <td>Hameau</td>
    <td></td>
    <td>N</td>
    <td>95</td>
    <td>OM630667</td>
    <td>OM350265</td>
    <td>OM542393</td>
  </tr>
</table>
<table>
  <tr>
    <td>Cx_portesi_GF_S96</td>
    <td>Culex sp.<br>BTLHVDV-2014<br>Culex<br>(Melanoconion)</td>
    <td>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jul 2019</td>
    <td>N</td>
    <td>96</td>
    <td>OM630668</td>
    <td>OM350266</td>
    <td>OM542394</td>
  </tr>
  <tr>
    <td>Cx_spissipes_GF_S97</td>
    <td>sp. DJS-2020<br>Culex<br>(Melanoconion)</td>
    <td>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jul 2019</td>
    <td>N</td>
    <td>97</td>
    <td>OM630677</td>
    <td>OM350274</td>
    <td>OM542401</td>
  </tr>
  <tr>
    <td>Cx_spissipes_GF_S98</td>
    <td>sp. DJS-2020<br>Culex<br>(Melanoconion)</td>
    <td>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jul 2019</td>
    <td>N</td>
    <td>98</td>
    <td>OM630678</td>
    <td>OM350275</td>
    <td>OM542402</td>
  </tr>
  <tr>
    <td>Cx_spissipes_GF_S99</td>
    <td>sp. DJS-2020<br>Culex<br>(Melanoconion)</td>
    <td>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jul 2019</td>
    <td>N</td>
    <td>99</td>
    <td>OM630679</td>
    <td>OM350276</td>
    <td>OM542403</td>
  </tr>
  <tr>
    <td>Li_durhamii_GF_S100</td>
    <td>Limatus durhamii<br>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jul 2019</td>
    <td>N</td>
    <td>100</td>
    <td>OM630695</td>
    <td>OM350291</td>
    <td>OM542419</td>
  </tr>
  <tr>
    <td>Li_durhamii_GF_S101</td>
    <td>Limatus durhamii<br>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jul 2019</td>
    <td>N</td>
    <td>101</td>
    <td>OM630696</td>
    <td>OM350292</td>
    <td>OM542420</td>
  </tr>
  <tr>
    <td>Li_durhamii_GF_S102</td>
    <td>Limatus durhamii<br>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jul 2019</td>
    <td>N</td>
    <td>102</td>
    <td>OM630697</td>
    <td>OM350293</td>
    <td>OM542421</td>
  </tr>
  <tr>
    <td>Ma_sp.4_GF_S103</td>
    <td>Mansonia sp.4<br>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jan 2020</td>
    <td>N</td>
    <td>103</td>
    <td>OM630701</td>
    <td>OM350298</td>
    <td>OM542425</td>
  </tr>
  <tr>
    <td>Ma_sp.4_GF_S104</td>
    <td>Mansonia sp.4<br>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jan 2020</td>
    <td>N</td>
    <td>104</td>
    <td>OM630702</td>
    <td>OM350299</td>
    <td>OM542426</td>
  </tr>
  <tr>
    <td>Ma_titillans_GF_S105</td>
    <td>Mansonia<br>(Mansonia)<br>titillans<br>Culex<br>(Melanoconion)</td>
    <td>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Jan 2020</td>
    <td>N</td>
    <td>105</td>
    <td>OM630703</td>
    <td>OM350300</td>
    <td>OM542427</td>
  </tr>
  <tr>
    <td>Cx pedroi_GF_S106</td>
    <td>pedroi<br>Culex<br>(Melanoconion)</td>
    <td>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Nov 2020</td>
    <td>N</td>
    <td>106</td>
    <td>OM630663</td>
    <td>OM350261</td>
    <td>OM542389</td>
  </tr>
  <tr>
    <td>Cx pedroi_GF_S107</td>
    <td>pedroi<br>Culex<br>(Melanoconion)</td>
    <td>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Nov 2020</td>
    <td>N</td>
    <td>107</td>
    <td>OM630664</td>
    <td>OM350262</td>
    <td>OM542390</td>
  </tr>
  <tr>
    <td>Cx pedroi_GF_S108</td>
    <td>pedroi<br>Psorophora<br>ferox</td>
    <td>French<br>Guiana</td>
    <td>Hameau<br>Prefontaine</td>
    <td>Nov 2020</td>
    <td>N</td>
    <td>108</td>
    <td>OM630665</td>
    <td>OM350263</td>
    <td>OM542391</td>
  </tr>
  <tr>
    <td>Ps_ferox_GF_S109</td>
    <td>ferox<br>Psorophora<br>ferox</td>
    <td>French<br>Guiana</td>
    <td>Iracoubo</td>
    <td>2009</td>
    <td>N</td>
    <td>109</td>
    <td>OM630711</td>
    <td>OM350309</td>
    <td>OM542436</td>
  </tr>
  <tr>
    <td>Ps_ferox_GF_S110</td>
    <td>ferox<br>Psorophora<br>ferox</td>
    <td>French<br>Guiana</td>
    <td>Iracoubo</td>
    <td>2009</td>
    <td>N</td>
    <td>110</td>
    <td>OM630712</td>
    <td>OM350310</td>
    <td>OM542437</td>
  </tr>
  <tr>
    <td>Ps_ferox_GF_S111</td>
    <td>ferox<br>Uranotaenia<br>(Uranotaenia)</td>
    <td>French<br>Guiana</td>
    <td>Iracoubo</td>
    <td>2009</td>
    <td>N</td>
    <td>111</td>
    <td>OM630713</td>
    <td>OM350324</td>
    <td>OM542452</td>
  </tr>
  <tr>
    <td>Ur_geometry GF_S112</td>
    <td>geometry<br>French<br>Guiana</td>
    <td></td>
    <td></td>
    <td>2010</td>
    <td>N</td>
    <td>112</td>
    <td>OM630714</td>
    <td>OM350311</td>
    <td>OM542453</td>
  </tr>
  <tr>
    <td>Ur_geometry GF_S113</td>
    <td>Uranotaenia<br>French</td>
    <td></td>
    <td></td>
    <td>2010</td>
    <td>N</td>
    <td>113</td>
    <td>N/A</td>
    <td>OM350312</td>
    <td>OM542454</td>
  </tr>
</table>
<table><tr><td>Ur_geometrya_GF_S114</td><td>(Uranotaenia) geometrica<br/>Uranotaenia (Uranotaenia) geometrica<br/>Culex (Culex) tritaeniorhynchus</td><td>French<br/>Guiana</td><td>2010</td><td>N</td><td>114</td><td>OM630715</td><td>OM350313</td><td>OM542438,<br/>OM542455</td></tr><tr><td>Cx_tritaeniorhynchus_MG_S115</td><td>tritaeniorhynchus</td><td>Madagascar</td><td>Feb 2019</td><td>N</td><td>115</td><td>OM630685</td><td>OM350321</td><td>OM542448</td></tr></table>

727

728

<table><caption>Appendix 2—table 1. COI sequence BLAST analyses summary (XLSX).</caption><thead><tr><th>Sequence ID</th><th>Sequence length</th><th>Morphological identification</th><th>BLASTn top hit species</th><th>BLASTn top hit accession</th><th>Query coverage</th><th>E-value</th><th>% identity</th><th>Comments</th></tr></thead><tbody><tr><td>Ae_albopictus_KH_S1</td><td>699</td><td>Aedes albopictus</td><td>Aedes albopictus</td><td>MK714006.1</td><td>99%</td><td>0.0</td><td>99.71%</td><td></td></tr><tr><td>Ae_albopictus_KH_S2</td><td>695</td><td>Aedes albopictus</td><td>Aedes albopictus</td><td>MK714006.1</td><td>100%</td><td>0.0</td><td>99.71%</td><td></td></tr><tr><td>Ae_albopictus_KH_S3</td><td>695</td><td>Aedes albopictus</td><td>Aedes albopictus</td><td>MK714006.1</td><td>100%</td><td>0.0</td><td>99.71%</td><td></td></tr><tr><td>An_baezai_KH_S4</td><td>658</td><td>Anopheles baezai</td><td>Anopheles darlingi</td><td>MF381626.1</td><td>100%</td><td>0.0</td><td>92.71%</td><td>An baezai not found in GenBank databases</td></tr><tr><td>An_baezai_KH_S5</td><td>670</td><td>Anopheles baezai</td><td>Anopheles darlingi</td><td>MF381626.1</td><td>99%</td><td>0.0</td><td>92.81%</td><td>An baezai not found in GenBank databases</td></tr><tr><td>An_baezai_KH_S6</td><td>659</td><td>Anopheles baezai</td><td>Anopheles darlingi</td><td>MF381626.1</td><td>100%</td><td>0.0</td><td>92.72%</td><td>An baezai not found in GenBank databases<br/>95% similarity to Cx vishnui, 94% similarity with Cx tritaeniorhynchus</td></tr><tr><td>Cx_pseudovishnui_KH_S7</td><td>660</td><td>Culex vishnui</td><td>Culex pseudovishnui</td><td>MW321882.1</td><td>98%</td><td>0.0</td><td>98.92%</td><td>95% similarity to Cx vishnui, 94% similarity with Cx tritaeniorhynchus</td></tr><tr><td>Cx_pseudovishnui_KH_S8</td><td>659</td><td>Culex vishnui</td><td>Culex pseudovishnui</td><td>MW321882.1</td><td>98%</td><td>0.0</td><td>99.38%</td><td>95% similarity to Cx vishnui, 94% similarity with Cx tritaeniorhynchus</td></tr><tr><td>Cx_pseudovishnui_KH_S9</td><td>659</td><td>Culex vishnui</td><td>Culex pseudovishnui</td><td>MW321882.1</td><td>98%</td><td>0.0</td><td>98.92%</td><td>95% similarity to Cx vishnui, 94% similarity with Cx tritaeniorhynchus</td></tr><tr><td>Ma_indiana_KH_S10</td><td>660</td><td>Mansonia indiana</td><td>Mansonia indiana</td><td>MK637632.1</td><td>98.00%</td><td>0.0</td><td>99.54%</td><td>89.99% from Ma indiana</td></tr><tr><td>Mauniformis_KH_S11</td><td>686</td><td>Mansonia indiana</td><td>Mansonia uniformis</td><td>MK757484.1</td><td>99%</td><td>0.0</td><td>99.71%</td><td>MK637632.1</td></tr><tr><td>Ma_indiana_KH_S12</td><td>693</td><td>Mansonia indiana</td><td>Mansonia indiana</td><td>MK637632.1</td><td>97%</td><td>0.0</td><td>99.41%</td><td></td></tr><tr><td>Cx_sp.1_KH_S13</td><td>687</td><td>Culex quinquefasciatus</td><td>Culex (Lophoceraomyia) sp. 5</td><td>MW321904.1</td><td>98%</td><td>0.0</td><td>94.39%</td><td>90% from Cx quinquefasciatus</td></tr><tr><td>Cx_orientalis_KH_S14</td><td>662</td><td>Culex</td><td>Culex orientalis</td><td>MW228488.1</td><td>97%</td><td>0.0</td><td>98.29%</td><td>GU188856.2</td></tr></tbody></table>
<table><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td></td><td></td><td>quinquefasciatus</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MaUniformis_KH_S15</td><td>658</td><td>Mansonia uniformis</td><td>Mansonia uniformis</td><td>MK757484.1</td><td>100.00%</td><td>0.0</td><td>99.54%</td><td></td></tr><tr><td>MaUniformis_KH_S16</td><td>654</td><td>Mansonia uniformis</td><td>Mansonia uniformis</td><td>MK757484.1</td><td>100.00%</td><td>0.0</td><td>99.39%</td><td></td></tr><tr><td>MaUniformis_KH_S17</td><td>657</td><td>Mansonia uniformis</td><td>Mansonia uniformis</td><td>MK757484.1</td><td>99.00%</td><td>0.0</td><td>99.54%</td><td></td></tr><tr><td>CxBitaeniorhynchus_KH_S18</td><td>658</td><td>Culex bitaeniorhynchus</td><td>Culex bitaeniorhynchus</td><td>HQ398898.1</td><td>97.00%</td><td>0.0</td><td>99.69%</td><td></td></tr><tr><td>CxBitaeniorhynchus_KH_S19</td><td>650</td><td>Culex bitaeniorhynchus</td><td>Culex bitaeniorhynchus</td><td>HQ398898.1</td><td>98.00%</td><td>0.0</td><td>99.84%</td><td></td></tr><tr><td>CxBitaeniorhynchus_KH_S20</td><td>652</td><td>Culex bitaeniorhynchus</td><td>Culex bitaeniorhynchus</td><td>HQ398898.1</td><td>98.00%</td><td>0.0</td><td>99.38%</td><td></td></tr><tr><td>CxTritaeniorhynchus_KH_S21</td><td>695</td><td>Culex tritaeniorhynchus</td><td>Culex tritaeniorhynchus</td><td>MH374857.1</td><td>100%</td><td>0.0</td><td>99.57%</td><td>99.69% to Cx tritaeniorhynchus<br>MF179213.1</td></tr><tr><td>CxTritaeniorhynchus_KH_S22</td><td>690</td><td>Culex tritaeniorhynchus</td><td>Culex tritaeniorhynchus</td><td>MT876103.1</td><td>100%</td><td>0.0</td><td>99.57%</td><td></td></tr><tr><td>CxTritaeniorhynchus_KH_S23</td><td>663</td><td>Culex tritaeniorhynchus</td><td>Culex tritaeniorhynchus</td><td>MT876103.1</td><td>99%</td><td>0.0</td><td>98.79%</td><td></td></tr><tr><td>AeAegypti_CF_S24</td><td>689</td><td>Aedes aegypti</td><td>Aedes aegypti</td><td>MN299016.1</td><td>100%</td><td>0.0</td><td>99.56%</td><td></td></tr><tr><td>AeAegypti_CF_S25</td><td>660</td><td>Aedes aegypti</td><td>Aedes aegypti</td><td>MN299024.1</td><td>100.00%</td><td>0.0</td><td>99.70%</td><td></td></tr><tr><td>AeAegypti_CF_S26</td><td>660</td><td>Aedes aegypti</td><td>Aedes aegypti</td><td>MN299024.1</td><td>100.00%</td><td>0.0</td><td>99.70%</td><td></td></tr><tr><td>AeSimsoni_CF_S27</td><td>644</td><td>Aedes opok</td><td>Aedes simpsoni</td><td>LC473669.1</td><td>97.00%</td><td>0.0</td><td>97.77%</td><td>Ae opok not found in GenBank,<br>sequence is 90% and 89% away<br>from Ae luteocephalus and Ae<br>africanus, sister species of Ae<br>opok.</td></tr><tr><td>AeSimsoni_CF_S28</td><td>649</td><td>Aedes opok</td><td>Aedes simpsoni</td><td>MN552302.1</td><td>99.00%</td><td>0.0</td><td>100.00%</td><td>Ae opok not found in GenBank,<br>sequence is 90% and 89% away<br>from Ae luteocephalus and Ae<br>africanus, sister species of Ae<br>opok.</td></tr><tr><td>AeSimsoni_CF_S29</td><td>627</td><td>Aedes opok</td><td>Aedes simpsoni</td><td>MN552302.1</td><td>98.00%</td><td>0.0</td><td>98.87%</td><td>Ae opok not found in GenBank,<br>sequence is 90% and 89% away<br>from Ae luteocephalus and Ae<br>africanus, sister species of Ae<br>opok.</td></tr><tr><td>AeVittatus_CF_S30</td><td>623</td><td>Aedes vittatus</td><td>Aedes vittatus</td><td>MN552298.1</td><td>100.00%</td><td>0.0</td><td>99.84%</td><td></td></tr><tr><td>AeVittatus_CF_S31</td><td>622</td><td>Aedes vittatus</td><td>Aedes vittatus</td><td>MN552298.1</td><td>100.00%</td><td>0.0</td><td>99.68%</td><td></td></tr></tbody></table>
<table><tbody><tr><td>Ae_vittatus_CF_S32</td><td>621</td><td>Aedes vittatus<br>Mansonia</td><td>Aedes vittatus</td><td>MN552298.1</td><td>100.00%</td><td>0.0</td><td>99.68%</td><td></td></tr><tr><td>Ma_sp.1_CF_S33</td><td>-</td><td>africana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>No COI obtained</td></tr><tr><td>Ma_sp.2_CF_S34</td><td>-</td><td>Mansonia<br>africana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>No COI obtained</td></tr><tr><td>Ma_sp.3_CF_S35</td><td>-</td><td>africana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>No COI obtained</td></tr><tr><td>Ae_albopictus_CF_S36</td><td>627</td><td>Aedes albopictus</td><td>Aedes albopictus</td><td>MK995332.1</td><td>100.00%</td><td>0.0</td><td>99.84%</td><td></td></tr><tr><td>Ae_albopictus_CF_S37</td><td>621</td><td>Aedes albopictus</td><td>Aedes albopictus</td><td>MK995332.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td><td></td></tr><tr><td>Ae_albopictus_CF_S38</td><td>621</td><td>Anopheles<br>coustani</td><td>Anopheles<br>coustani</td><td>MK585968.1</td><td>100.00%</td><td>0.0</td><td>99.84%</td><td></td></tr><tr><td>An_coustani_CF_S39</td><td>621</td><td>Anopheles<br>coustani</td><td>Anopheles<br>coustani</td><td>MK585959.1</td><td>100.00%</td><td>0.0</td><td>99.03%</td><td></td></tr><tr><td>An_coustani_CF_S40</td><td>621</td><td>Anopheles<br>coustani</td><td>Anopheles<br>coustani</td><td>MK585968.1</td><td>94.00%</td><td>0.0</td><td>99.70%</td><td></td></tr><tr><td>An_coustani_CF_S41</td><td>699</td><td>Anopheles<br>coustani</td><td>Anopheles<br>coustani</td><td>MK300231.1</td><td>100.00%</td><td>0.0</td><td>99.71%</td><td></td></tr><tr><td>An.funestus_CF_S42</td><td>696</td><td>funestus<br>Anopheles</td><td>funestus<br>Anopheles</td><td>MT375215.1</td><td>100.00%</td><td>0.0</td><td>99.85%</td><td></td></tr><tr><td>An.funestus_CF_S43</td><td>660</td><td>funestus<br>Anopheles</td><td>funestus<br>Anopheles</td><td>MT375215.1</td><td>100.00%</td><td>0.0</td><td>99.70%</td><td></td></tr><tr><td>An.funestus_CF_S44</td><td>658</td><td>funestus<br>Anopheles</td><td>funestus<br>Anopheles</td><td>MT375215.1</td><td>100.00%</td><td>0.0</td><td>99.70%</td><td></td></tr><tr><td>An_gambiae_CF_S45</td><td>660</td><td>gambiae<br>Anopheles</td><td>gambiae<br>Anopheles</td><td>MG930895.1</td><td>86.00%</td><td>0.0</td><td>99.79%</td><td></td></tr><tr><td>An_gambiae_CF_S46</td><td>659</td><td>gambiae<br>Anopheles</td><td>gambiae<br>Anopheles</td><td>MT375223.1</td><td>89.00%</td><td>0.0</td><td>100.00%</td><td></td></tr><tr><td>An_gambiae_CF_S47</td><td>-</td><td>gambiae<br>Culex</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>No COI obtained</td></tr><tr><td>Cx_sp.2_CF_S48</td><td>653</td><td>quinquefasciatus<br>Culex</td><td>Culex corniger</td><td>KM593015.1</td><td>100.00%</td><td>0.0</td><td>94.95%</td><td>94% to all other Cx species</td></tr><tr><td>Cx_sp.2_CF_S49</td><td>660</td><td>quinquefasciatus<br>Culex</td><td>Culex nigripalpus</td><td>KM593058.1</td><td>99.00%</td><td>0.0</td><td>94.65%</td><td>94% to all other Cx species</td></tr><tr><td>Cx_sp.2_CF_S50</td><td>658</td><td>quinquefasciatus<br>Mansonia</td><td>Culex bidens</td><td>MH931446.1</td><td>100.00%</td><td>0.0</td><td>94.68%</td><td>94% to all other Cx species</td></tr><tr><td>MaUniformis_CF_S51</td><td>-</td><td>uniformis<br>Mansonia</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>No COI obtained</td></tr><tr><td>Cx_duttoni_CF_S52</td><td>621</td><td>uniformis<br>Eretmapodites</td><td>Culex duttoni</td><td>LC473629.1</td><td>100.00%</td><td>0.0</td><td>99.68%</td><td></td></tr><tr><td>Er_intermedius_CF_S54</td><td>620</td><td>sp.<br>Eretmapodites</td><td>Eretmapodites<br>intermedius</td><td>MN552305.1</td><td>100.00%</td><td>0.0</td><td>99.52%</td><td></td></tr><tr><td>Er_intermedius_CF_S55</td><td>621</td><td>sp.</td><td>Eretmapodites<br>intermedius</td><td>MN552305.1</td><td>100.00%</td><td>0.0</td><td>99.68%</td><td></td></tr></tbody></table>
<table><tr><td>Er_intermedius_CF_S56</td><td>621</td><td>Eretmapodites<br>sp.<br>Culex</td><td>Eretmapodites<br>intermedius</td><td>MN552305.1</td><td>100.00%</td><td>0.0</td><td>99.68%</td></tr><tr><td>Cx_antennatus_MG_S57</td><td>621</td><td>antennatus<br>Culex</td><td>Culex antennatus</td><td>LC473659.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td></tr><tr><td>Cx_antennatus_MG_S58</td><td>621</td><td>antennatus<br>Culex</td><td>Culex antennatus</td><td>LC473659.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td></tr><tr><td>Cx_antennatus_MG_S59</td><td>621</td><td>antennatus<br>Culex</td><td>Culex antennatus</td><td>LC473659.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td></tr><tr><td>Cx_perexiguus_MG_S60</td><td>621</td><td>Culex decens</td><td>Culex perexiguus<br>Unknown Culex<br>species</td><td>LC473634.1</td><td>100.00%</td><td>0.0</td><td>99.84%</td></tr><tr><td>Cx_sp.3_MG_S61</td><td>685</td><td>Culex decens</td><td>Unknown Culex<br>species</td><td>KU380436.1</td><td>96.00%</td><td>0.0</td><td>96.05%</td></tr><tr><td>Cx_sp.4_MG_S62</td><td>687</td><td>Culex decens</td><td>Unknown Culex<br>species</td><td>MT993494.1</td><td>99.00%</td><td>0.0</td><td>95.63%</td></tr><tr><td>Cx_sp.3_MG_S63</td><td>687</td><td>Culex univittatus</td><td>Unknown Culex<br>species<br>Mimomyia</td><td>KU380436.1<br>LC473719.1</td><td>95.00%</td><td>0.0</td><td>96.50%</td></tr><tr><td>Mi_sp.1_MG_S64</td><td>694</td><td>Culex univittatus</td><td>mimomyiaformis<br>Unknown Culex<br>species</td><td>KU380436.1</td><td>95.00%</td><td>0.0</td><td>96.50%</td></tr><tr><td>Cx_sp.3_MG_S65</td><td>691</td><td>Culex univittatus<br>Anopheles<br>coustani</td><td>Anopheles<br>coustani</td><td>NC_050693.1</td><td>99.00%</td><td>0.0</td><td>99.40%</td></tr><tr><td>An_coustani_MG_S66</td><td>669</td><td>Anopheles<br>coustani</td><td>Anopheles<br>coustani</td><td>NC_050693.1</td><td>99.00%</td><td>0.0</td><td>99.08%</td></tr><tr><td>An_coustani_MG_S67</td><td>659</td><td>Anopheles<br>coustani</td><td>Anopheles<br>coustani</td><td>NC_050693.1</td><td>99.00%</td><td>0.0</td><td>99.08%</td></tr><tr><td>An_squamosus_MG_S68</td><td>653</td><td>Anopheles<br>coustani</td><td>Anopheles<br>squamosus</td><td>MK776741.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td></tr><tr><td>An.funestus_MG_S69</td><td>654</td><td>Anopheles<br>funestus</td><td>Anopheles<br>funestus</td><td>MT375215.1</td><td>100.00%</td><td>0.0</td><td>99.85%</td></tr><tr><td>An.funestus_MG_S70</td><td>654</td><td>Anopheles<br>funestus</td><td>Anopheles<br>funestus</td><td>MG742199.1</td><td>100.00%</td><td>0.0</td><td>99.69%</td></tr><tr><td>An_gambiae_MG_S71</td><td>654</td><td>Anopheles<br>gambiae</td><td>Anopheles<br>gambiae</td><td>MT375222.1</td><td>100.00%</td><td>0.0</td><td>99.85%</td></tr><tr><td>An_gambiae_MG_S72</td><td>654</td><td>Anopheles<br>gambiae</td><td>Anopheles<br>gambiae</td><td>MT375222.1</td><td>100.00%</td><td>0.0</td><td>99.85%</td></tr><tr><td>An_gambiae_MG_S73</td><td>622</td><td>Anopheles<br>gambiae</td><td>Anopheles<br>gambiae</td><td>MT375222.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td></tr><tr><td>Cx_quinquefasciatus_MG_S74</td><td>654</td><td>Culex<br>quinquefasciatus<br>Culex</td><td>Culex pipiens<br>Culex</td><td>MT199095.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td></tr><tr><td>Cx_quinquefasciatus_MG_S75</td><td>647</td><td>Culex<br>quinquefasciatus<br>Culex</td><td>Culex<br>quinquefasciatus</td><td>MH423504.1</td><td>100.00%</td><td>0.0</td><td>98.15%</td></tr><tr><td>Cx_perexiguus_MG_S76</td><td>621</td><td>Culex<br>quinquefasciatus<br>Mansonia</td><td>Culex perexiguus<br>Mansonia</td><td>LC473634.1</td><td>100.00%</td><td>0.0</td><td>99.52%</td></tr><tr><td>Mauniformis_MG_S77</td><td>621</td><td>uniformis</td><td>uniformis</td><td>KU187165.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td></tr></table>
<table>
  <tr>
    <td>Mauniformis_MG_S78</td>
    <td>621</td>
    <td>Mansonia uniformis</td>
    <td>Mansonia uniformis</td>
    <td>KU187165.1</td>
    <td>100.00%</td>
    <td>0.0</td>
    <td>100.00%</td>
  </tr>
  <tr>
    <td>Mauniformis_MG_S79</td>
    <td>626</td>
    <td>Mansonia uniformis</td>
    <td>Mansonia uniformis</td>
    <td>KU187157.1</td>
    <td>100.00%</td>
    <td>0.0</td>
    <td>99.68%</td>
  </tr>
  <tr>
    <td>Cxpoicilipes_MG_S82</td>
    <td>689</td>
    <td>bitaeniorhynchus Culex</td>
    <td>Culex poicilipes</td>
    <td>LC473618.1</td>
    <td>95.00%</td>
    <td>0.0</td>
    <td>99.70%</td>
  </tr>
  <tr>
    <td>Mimediolineata_MG_S83</td>
    <td>694</td>
    <td>tritaeniorhynchus Culex</td>
    <td>Mimomyia mediolineata</td>
    <td>LC473723.1</td>
    <td>94.00%</td>
    <td>0.0</td>
    <td>99.39%</td>
  </tr>
  <tr>
    <td>Cxneavei_MG_S84</td>
    <td>671</td>
    <td>tritaeniorhynchus</td>
    <td>Culex neavei</td>
    <td>LC473635.1</td>
    <td>98.00%</td>
    <td>0.0</td>
    <td>99.85%</td>
  </tr>
  <tr>
    <td>Aescapularis_GF_S85</td>
    <td>659</td>
    <td>Aedes scapularis</td>
    <td>Aedes scapularis</td>
    <td>MN997484.1</td>
    <td>97.00%</td>
    <td>0.0</td>
    <td>98.76%</td>
  </tr>
  <tr>
    <td>Aescapularis_GF_S86</td>
    <td>658</td>
    <td>Aedes scapularis</td>
    <td>Aedes scapularis</td>
    <td>MF172265.1</td>
    <td>97.00%</td>
    <td>0.0</td>
    <td>99.38%</td>
  </tr>
  <tr>
    <td>Aescapularis_GF_S87</td>
    <td>654</td>
    <td>Aedes scapularis</td>
    <td>Aedes scapularis</td>
    <td>MF172265.1</td>
    <td>98.00%</td>
    <td>0.0</td>
    <td>99.22%</td>
  </tr>
  <tr>
    <td>Aeserratus_GF_S88</td>
    <td>660</td>
    <td>Aedes serratus</td>
    <td>Aedes serratus</td>
    <td>MF172269.1</td>
    <td>97.00%</td>
    <td>0.0</td>
    <td>98.91%</td>
  </tr>
  <tr>
    <td>Aeserratus_GF_S89</td>
    <td>660</td>
    <td>Aedes serratus</td>
    <td>Aedes serratus</td>
    <td>MF172268.1</td>
    <td>97.00%</td>
    <td>0.0</td>
    <td>99.22%</td>
  </tr>
  <tr>
    <td>Aeserratus_GF_S90</td>
    <td>654</td>
    <td>Aedes serratus</td>
    <td>Aedes serratus</td>
    <td>MF172268.1</td>
    <td>98.00%</td>
    <td>0.0</td>
    <td>99.07%</td>
  </tr>
  <tr>
    <td>Cqvenezuelensis_GF_S91</td>
    <td>658</td>
    <td>venezuelensis Coquillettidia</td>
    <td>venezuelensis Coquillettidia</td>
    <td>MN997703.1</td>
    <td>97.00%</td>
    <td>0.0</td>
    <td>97.98%</td>
  </tr>
  <tr>
    <td>Cqvenezuelensis_GF_S92</td>
    <td>621</td>
    <td>venezuelensis Coquillettidia</td>
    <td>venezuelensis Coquillettidia</td>
    <td>MN997703.1</td>
    <td>100.00%</td>
    <td>0.0</td>
    <td>98.07%</td>
  </tr>
  <tr>
    <td>Cqvenezuelensis_GF_S93</td>
    <td>621</td>
    <td>venezuelensis</td>
    <td>venezuelensis</td>
    <td>MN997703.1</td>
    <td>100.00%</td>
    <td>0.0</td>
    <td>97.75%</td>
  </tr>
  <tr>
    <td>Cxportesi_GF_S94</td>
    <td>653</td>
    <td>Culex portesi</td>
    <td>Culex portesi</td>
    <td>in-house reference library</td>
    <td>98.5-100%</td>
    <td></td>
    <td>reference sequence provided by Amandine Guidez, IP Guyane</td>
  </tr>
  <tr>
    <td>Cxportesi_GF_S95</td>
    <td>693</td>
    <td>Culex portesi</td>
    <td>Culex portesi</td>
    <td>in-house reference library</td>
    <td>98.5-100%</td>
    <td></td>
    <td>reference sequence provided by Amandine Guidez, IP Guyane</td>
  </tr>
  <tr>
    <td>Cxportesi_GF_S96</td>
    <td>687</td>
    <td>Culex portesi</td>
    <td>Culex portesi</td>
    <td>in-house reference library</td>
    <td>98.5-100%</td>
    <td></td>
    <td>reference sequence provided by Amandine Guidez, IP Guyane</td>
  </tr>
  <tr>
    <td>Cxspissipes_GF_S97</td>
    <td>672</td>
    <td>Culex spissipes</td>
    <td>Culex spissipes</td>
    <td>in-house reference library</td>
    <td>98.5-100%</td>
    <td></td>
    <td>reference sequence provided by Amandine Guidez, IP Guyane</td>
  </tr>
  <tr>
    <td>Cxspissipes_GF_S98</td>
    <td>663</td>
    <td>Culex spissipes</td>
    <td>Culex spissipes</td>
    <td>in-house reference library</td>
    <td>98.5-100%</td>
    <td></td>
    <td>reference sequence provided by Amandine Guidez, IP Guyane</td>
  </tr>
  <tr>
    <td>Cxspissipes_GF_S99</td>
    <td>660</td>
    <td>Culex spissipes</td>
    <td>Culex spissipes</td>
    <td>in-house reference library</td>
    <td>98.5-100%</td>
    <td></td>
    <td>reference sequence provided by Amandine Guidez, IP Guyane</td>
  </tr>
  <tr>
    <td>Li durhamii_GF_S100</td>
    <td>653</td>
    <td>Limatus durhamii</td>
    <td>Limatus durhamii</td>
    <td>MF172330.1</td>
    <td>98.00%</td>
    <td>0.0</td>
    <td>99.84%</td>
  </tr>
</table>
<table><tr><td>Li_durhamii_GF_S101</td><td>621</td><td>Limatus durhamii</td><td>Limatus durhamii MF172330.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td><td></td></tr><tr><td>Li_durhamii_GF_S102</td><td>699</td><td>Limatus durhamii<br>Mansonia</td><td>Limatus durhamii MF172330.1</td><td>94.00%</td><td>0.0</td><td>100.00%</td><td>87.12% to Ma titillans</td></tr><tr><td>Ma_sp.4_GF_S103</td><td>621</td><td>titillans<br>Mansonia</td><td>Mansonia sp. MT329066.1</td><td>100.00%</td><td>0.0</td><td>99.84%</td><td>MN968244.1</td></tr><tr><td>Ma_sp.4_GF_S104</td><td>695</td><td>titillans<br>Mansonia</td><td>Mansonia sp. MT329066.1</td><td>95.00%</td><td>0.0</td><td>99.85%</td><td>MN968244.1</td></tr><tr><td>Ma_titillans_GF_S105</td><td>669</td><td>titillans</td><td>Mansonia titillans MN968244.1</td><td>98.00%</td><td>0.0</td><td>99.70%</td><td></td></tr><tr><td>Cx pedroi_GF_S106</td><td>653</td><td>Culex pedroi</td><td>Culex pedroi KX779887.1</td><td>98.00%</td><td>0.0</td><td>98.60%</td><td></td></tr><tr><td>Cx pedroi_GF_S107</td><td>661</td><td>Culex pedroi</td><td>Culex pedroi KX779887.1</td><td>97.00%</td><td>0.0</td><td>98.76%</td><td></td></tr><tr><td>Cx pedroi_GF_S108</td><td>621</td><td>Culex pedroi<br>Psorophora<br>ferox</td><td>Culex pedroi KX779887.1</td><td>99.00%</td><td>0.0</td><td>98.87%</td><td></td></tr><tr><td>Ps_ferox_GF_S109</td><td>633</td><td>Psorophora<br>ferox</td><td>Psorophora ferox MF172349.1</td><td>100.00%</td><td>0.0</td><td>99.68%</td><td></td></tr><tr><td>Ps_ferox_GF_S110</td><td>621</td><td>Psorophora<br>ferox</td><td>Psorophora ferox MF172349.1</td><td>100.00%</td><td>0.0</td><td>99.68%</td><td></td></tr><tr><td>Ps_ferox_GF_S111</td><td>621</td><td>Psorophora<br>ferox</td><td>Psorophora ferox MF172347.1</td><td>99.00%</td><td>0.0</td><td>99.51%</td><td></td></tr><tr><td>Ur_geometrya_GF_S112</td><td>621</td><td>geometrica<br>Uranotaenia</td><td>geometrica<br>Uranotaenia NC_044662.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td><td></td></tr><tr><td>Ur_geometrya_GF_S113</td><td>-</td><td>geometrica<br>Uranotaenia</td><td>Uranotaenia<br>geometrica<br>Culex<br>tritaeniorhynchus</td><td>-</td><td>-</td><td>-</td><td>No COI obtained</td></tr><tr><td>Ur_geometrya_GF_S114</td><td>621</td><td>geometrica<br>Culex<br>tritaeniorhynchus</td><td>geometrica<br>Culex<br>tritaeniorhynchus NC_044662.1</td><td>100.00%</td><td>0.0</td><td>100.00%</td><td></td></tr><tr><td>Cx_tritaeniorhynchus_MG_S115</td><td>653</td><td>tritaeniorhynchus</td><td>MK861440.1</td><td>100.00%</td><td>0.0</td><td>98.77%</td><td></td></tr></table>
729 SOURCE DATA FILES

730 **Figure 3—source data 1.** Multiple sequence alignment of 169 28S rRNA sequences from this study
731 and from GenBank (FASTA).

732 **Figure 4—source data 1.** Multiple sequence alignment of 122 28S rRNA sequences, including two
733 sequences from *Horreolanus sp.* (FASTA).

734 **Figure 4—source data 2.** Multiple sequence alignment of 114 18S rRNA sequences, including two
735 sequences from *Horreolanus sp.* (FASTA).

736 **Figure 5—source data 1.** Multiple sequence alignment of 106 COI sequences (FASTA).

737 REFERENCES

738 Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment
739 search tool. *Journal of Molecular Biology*, *215*(3), 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2

741 Arctander, P. (1995). Comparison of a mitochondrial gene and a corresponding nuclear pseudogene.
742 *Proceedings of the Royal Society B: Biological Sciences*, **262**(1363), 13–19.

743 https://doi.org/10.1098/rspb.1995.0170

744 Arunachalam, N., Philip Samuel, P., Hiriyan, J., Thenmozhi, V., & Gajanana, A. (2004). Japanese
745 encephalitis in Kerala, South India: Can Mansonia (Diptera: Culicidae) play a supplemental role
746 in transmission? *Journal of Medical Entomology*, **41**(3), 456–461. https://doi.org/10.1603/0022-2585-41.3.456

748 Aspen, S., & Savage, H. M. (2003). Polymerase chain reaction assay identifies North American
749 members of the Culex pipiens complex based on nucleotide sequence differences in the
750 acetylcholinesterase gene Ace.2. *Journal of the American Mosquito Control Association*, **19**(4),
751 323–328.

752 Auerswald, H., Maquart, P. O., Chevalier, V., & Boyer, S. (2021). Mosquito vector competence for
753 japanese encephalitis virus. *Viruses*, **13**(6), 1154. https://doi.org/10.3390/v13061154

754 Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., Lesin, V. M.,
755 Nikolenko, S. I., Pham, S., Prjibelski, A. D., Pyshkin, A. V., Sirotkin, A. V., Vyahhi, N., Tesler, G.,
756 Alekseyev, M. A., & Pevzner, P. A. (2012). SPAdes: A new genome assembly algorithm and its
757 applications to single-cell sequencing. *Journal of Computational Biology*, **19**(5), 455–477.
758 https://doi.org/10.1089/cmb.2012.0021

759 Barrio-Nuevo, K. M., Cunha, M. S., Luchs, A., Fernandes, A., Rocco, I. M., Mucci, L. F., DE Souza, R.

760 P., Medeiros-Sousa, A. R., Ceretti-Junior, W., & Marrelli, M. T. (2020). Detection of Zika and

761 dengue viruses in wildcaught mosquitoes collected during field surveillance in an environmental
762 protection area in São Paulo, Brazil. *PLoS ONE*, 15(10), e0227239.

763 https://doi.org/10.1371/journal.pone.0227239

764 Batovska, J., Cogan, N. O. I., Lynch, S. E., & Blacket, M. J. (2017). Using next-generation sequencing

765 for DNA barcoding: Capturing allelic variation in ITS2. *G3: Genes, Genomes, Genetics*, 7(1),

766 19-29. https://doi.org/10.1534/G3.116.036145/-/DC1

767 Beebe, N. W. (2018). DNA barcoding mosquitoes: Advice for potential prospectors. *Parasitology*,

768 145(5), 622-633. https://doi.org/10.1017/S0031182018000343

769 Behura, S. K. (2006). Molecular marker systems in insects: current trends and future avenues.

770 *Molecular Ecology*, 15(11), 3087-3113. https://doi.org/10.1111/j.1365-294X.2006.03014.x

771 Belda, E., Nanfack-Minkeu, F., Eigtmeier, K., Carissimo, G., Holm, I., Diallo, M., Diallo, D., Vantaux,

772 A., Kim, S., Sharakhov, I. V., & Vernick, K. D. (2019). De novo profiling of RNA viruses in

773 Anopheles malaria vector mosquitoes from forest ecological zones in Senegal and Cambodia.

774 BMC Genomics, 20(1), 664. https://doi.org/10.1186/s12864-019-6034-1

775 Bhattacharya, S., & Basu, P. (2016). The Southern House Mosquito, Culex quinquefasciatus: profile

776 of a smart vector. *Journal of Entomology and Zoology Studies JEZS*, 4(2), 73-81.

777 Bishop-Lilly, K. A., Turell, M. J., Willner, K. M., Butani, A., Nolan, N. M. E., Lentz, S. M., Akmal, A.,

778 Mateczun, A., Brahmbhatt, T. N., Sozhamannan, S., Whitehouse, C. A., & Read, T. D. (2010).

779 Arbovirus detection in insect vectors by Rapid, high- throughput pyrosequencing. *PLoS*

780 *Neglected Tropical Diseases*, 4(11), e878. https://doi.org/10.1371/journal.pntd.0000878

781 Brault, A. C., Foy, B. D., Myles, K. M., Kelly, C. L. H., Higgs, S., Weaver, S. C., Olson, K. E., Miller, B.

782 R., & Powers, A. M. (2004). Infection patterns of o'nyong nyong virus in the malaria-transmitting

783 mosquito, Anopheles gambiae. *Insect Molecular Biology*, 13(6), 625-635.

784 https://doi.org/10.1111/j.0962-1075.2004.00521.x

785 Cardoso, J. da C., de Almeida, M. A. B., dos Santos, E., da Fonseca, D. F., Sallum, M. A. M., Noll, C.

786 A., Monteiro, H. A. d. O., Cruz, A. C. R., Carvalho, V. L., Pinto, E. V., Castro, F. C., Neto, J. P.

787 N., Segura, M. N. O., & Vasconcelos, P. F. C. (2010). Yellow fever virus in Haemagogus
788 leucocelaenus and Aedes serratus mosquitoes, Southern Brazil, 2008. Emerging Infectious Diseases, 16(12), 1918–1924. https://doi.org/10.3201/eid1612.100608

790 Chandler, J. A., Liu, R. M., & Bennett, S. N. (2015). RNA Shotgun Metagenomic Sequencing of Northern California (USA) Mosquitoes Uncovers Viruses, Bacteria, and Fungi. Frontiers in Microbiology, 6, 185. https://doi.org/10.3389/fmicb.2015.00185

793 Cornel, A. J., Mcabee, R. D., Rasgon, J., Stanich, M. A., Scott, T. W., & Coetzee, M. (2003). Differences in Extent of Genetic Introgression between Sympatric Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in California and South Africa. Journal of Medical Entomology, 40(1), 36–51. https://doi.org/10.1603/0022-2585-40.1.36

797 Danforth, B. N., Lin, C. P., & Fang, J. (2005). How do insect nuclear ribosomal genes compare to protein-coding genes in phylogenetic utility and nucleotide substitution patterns? Systematic Entomology, 30(4), 549–562. https://doi.org/10.1111/J.1a365-3113.2005.00305.X

800 De Oliveira, C. D., Gonçalves, D. S., Baton, L. A., Shimabukuro, P. H. F., Carvalho, F. D., & Moreira, L. A. (2015). Broader prevalence of Wolbachia in insects including potential human disease vectors. Bulletin of Entomological Research, 105(3), 305–315. https://doi.org/10.1017/S0007485315000085

804 Desdouits, M., Kamgang, B., Berthet, N., Tricou, V., Ngoagouni, C., Gessain, A., Manuguerra, J. C., Nakouné, E., & Kazanji, M. (2015). Genetic characterization of Chikungunya virus in the Central African Republic. Infection, Genetics and Evolution, 33, 25–31. https://doi.org/10.1016/J.MEEGID.2015.04.006

808 Diallo, D., Fall, G., Diagne, C. T., Gaye, A., Ba, Y., Dia, I., Faye, O., & Diallo, M. (2020). Concurrent amplification of Zika, chikungunya, and yellow fever virus in a sylvatic focus of arboviruses in Southeastern Senegal, 2015. BMC Microbiology, 20, 181. https://doi.org/10.1186/s12866-020-01866-9

812 Edgar, R. C. (2004). MUSCLE: A multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics, 5, 113. https://doi.org/10.1186/1471-2105-5-113

814 Edwards, F. W. (1941). Mosquitoes of the Ethiopian Region: III. Culicine Adults and Pupae. Order of the Trustees.

816 Farajollahi, A., Fonseca, D. M., Kramer, L. D., & Marm Kilpatrick, A. (2011). “Bird biting” mosquitoes and human disease: A review of the role of Culex pipiens complex mosquitoes in epidemiology.
818 *Infection, Genetics and Evolution*, 11(7), 1577-1585. doi: 10.1016/j.meegid.2011.08.013

819 Fauver, J. R., Akter, S., Morales, A. I. O., Black, W. C., Rodriguez, A. D., Stenglein, M. D., Ebel, G.

820 D., & Weger-Lucarelli, J. (2019). A reverse-transcription/RNase H based protocol for depletion of
821 mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and
822 pathogen discovery. *Virology*, 528, 181-197. https://doi.org/10.1016/j.virol.2018.12.020

823 Foley, D. H., Rueda, L. M., & Wilkerson, R. C. (2007). Insight into Global Mosquito Biogeography from
824 Country Species Records. *Journal of Medical Entomology*, 44(4), 554-567.
825 https://doi.org/10.1093/JMEDENT/44.4.554

826 Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. (1994). DNA primers for amplification of
827 mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. *Molecular
828 Marine Biology and Biotechnology*, 3(5), 294-299.

829 Gale, K., & Crampton, J. (1989). The ribosomal genes of the mosquito, Aedes aegypti. *European
830 Journal of Biochemistry*, 185(2), 311-317. https://doi.org/10.1111/j.1432-1033.1989.tb15117.x

831 Grjebine, A. (1966). *Insectes Diptères Culicidae Anophelinae*. ORSTOM / CNRS.

832 Hajibabaei, M., Singer, G. A. C., & Hickey, D. A. (2006). Benchmarking DNA barcodes: An
833 assessment using available primate sequences. *Genome*, 49(7), 851-854.
834 https://doi.org/10.1139/G06-025/SUPPL_FILE/G06-025B.PDF

835 Halstead, S. B. (2019). Travelling arboviruses: A historical perspective. *Travel Medicine and
836 Infectious Disease*, 31, 101471. https://doi.org/10.1016/J.TMAID.2019.101471

837 Harbach, R. E. (2007). The Culicidae (Diptera): A review of taxonomy, classification and phylogeny.
838 *Zootaxa*, 1668(1), 591-638. https://doi.org/10.11646/zootaxa.1668.1.28

839 Harbach, R. E., Culverwell, C. L., & Kitching, I. J. (2017). Phylogeny of the nominotypical subgenus of
840 Culex (Diptera: Culicidae): insights from analyses of anatomical data into interspecific
841 relationships and species groups in an unresolved tree. *Systematics and Biodiversity*, 15(4),
842 296-306. https://doi.org/10.1080/14772000.2016.1252439

843 Harbach, R. E., & Kitching, I. J. (2016). The phylogeny of Anophelinae revisited: Inferences about the
844 origin and classification of Anopheles (Diptera: Culicidae). *Zoologica Scripta*, 45(1), 34-47.
845 https://doi.org/10.1111/zsc.12137

846 Hayes, C. G., Basit, A., Bagar, S., & Akhter, R. (1980). Vector competence of Culex tritaeniorhynchus
847 (Diptera: Culicidae) for West Nile virus. *Journal of Medical Entomology*.
848 https://doi.org/10.1093/jmedent/17.2.172

849 Hebert, P. D. N., Cywinska, A., Ball, S. L., & DeWaard, J. R. (2003). Biological identifications through DNA barcodes. *Proceedings of the Royal Society B: Biological Sciences*, 270(1512), 313–321.

851 https://doi.org/10.1098/rspb.2002.2218

852 Héraud, J.-M., Andriamandimby, S. F., Olive, M.-M., Guis, H., Miatrana Rasamoelina, V., & Tantely, L. (2022). Arthropod-Borne Viruses of Madagascar. In S. M. Goodman (Ed.), *The New Natural History of Madagascar* (pp. 285–291). Princeton University Press.

855 Hoyos-López, R., Soto, S. U., Rúa-Uribe, G., & Gallego-Gómez, J. C. (2015). Molecular identification of saint louis encephalitis virus genotype IV in Colombia. *Memorias Do Instituto Oswaldo Cruz*, 110(6), 719–725. https://doi.org/10.1590/0074-02760280040

858 Huang, Y., & Ward, R. A. (1981). A Pictorial Key for the Identification of the Mosquitoes Associated with Yellow Fever in Africa. *Mosquito Systematics*.

860 Hurst, G. D. D., & Jiggins, F. M. (2005). Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: The effects of inherited symbionts. *Proceedings of the Royal Society B: Biological Sciences*, 272, 1525–1534.

863 https://doi.org/10.1098/rspb.2005.3056

864 Jacobi, J. C., & Serie, C. (1972). Prevalence of group B arbovirus infections in French Guiana in 1967-69. *Medecine d'Afrique Noire*, 19(3), 225–226.

866 Jupp, P. G., Kemp, A., Grobbelaar, A., Leman, P., Burt, F. J., Alahmed, A. M., Al Mujalli, D., Al Khamees, M., & Swanepoel, R. (2002). The 2000 epidemic of Rift Valley fever in Saudi Arabia: Mosquito vector studies. *Medical and Veterinary Entomology*. https://doi.org/10.1046/j.1365-2915.2002.00371.x

869

870 Kim, H., Cha, G. W., Jeong, Y. E., Lee, W. G., Chang, K. S., Roh, J. Y., Yang, S. C., Park, M. Y., Park, C., & Shin, E. H. (2015). Detection of Japanese encephalitis virus genotype V in Culex orientalis and Culex pipiens (Diptera: Culicidae) in Korea. *PLoS ONE*, 10(2), e0116547.

873 https://doi.org/10.1371/journal.pone.0116547

874 Kraemer, M. U. G., Reiner, R. C., Brady, O. J., Messina, J. P., Gilbert, M., Pigott, D. M., Yi, D., Johnson, K., Earl, L., Marczak, L. B., Shirude, S., Davis Weaver, N., Bisanzio, D., Perkins, T. A., Lai, S., Lu, X., Jones, P., Coelho, G. E., Carvalho, R. G., ... Golding, N. (2019). Past and future spread of the arbovirus vectors Aedes aegypti and Aedes albopictus. *Nature Microbiology*, 4(5),
878 854-863. https://doi.org/10.1038/s41564-019-0376-y

879 Kukutla, P., Steritz, M., & Xu, J. (2013). Depletion of ribosomal RNA for mosquito gut metagenomic
RNA-seq. *Journal of Visualized Experiments*, 74, 50093. https://doi.org/10.3791/50093

881 Kumar, N., Creasy, T., Sun, Y., Flowers, M., Tallon, L. J., & Dunning Hotopp, J. C. (2012). Efficient
subtraction of insect rRNA prior to transcriptome analysis of Wolbachia-Drosophila lateral gene
transfer. *BMC Research Notes*, 5, 230. https://doi.org/10.1186/1756-0500-5-230

884 Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular evolutionary
genetics analysis across computing platforms. *Molecular Biology and Evolution*, 35(6), 1547-
1549. https://doi.org/10.1093/molbev/msy096

887 Logue, K., Chan, E. R., Phipps, T., Small, S. T., Reimer, L., Henry-Halldin, C., Sattabongkot, J., Siba,
P. M., Zimmerman, P. A., & Serre, D. (2013). Mitochondrial genome sequences reveal deep
divergences among Anopheles punctulatus sibling species in Papua New Guinea. *Malaria
Journal*, 12(1), 1-11. https://doi.org/10.1186/1475-2875-12-64/FIGURES/3

891 Lorenz, C., Alves, J. M. P., Foster, P. G., Suesdek, L., & Sallum, M. A. M. (2021). Phylogeny and
temporal diversification of mosquitoes (Diptera: Culicidae) with an emphasis on the Neotropical
fauna. *Systematic Entomology*, 46(4), 798-811. https://doi.org/10.1111/SYEN.12489

894 Lutomiah, J., Bast, J., Clark, J., Richardson, J., Yalwala, S., Oullo, D., Mutisya, J., Mulwa, F., Musila,
L., Khamadi, S., Schnabel, D., Wurapa, E., & Sang, R. (2013). Abundance, diversity, and
distribution of mosquito vectors in selected ecological regions of Kenya: Public health
implications. *Journal of Vector Ecology*, 38(1), 134-142. https://doi.org/10.1111/j.1948-
7134.2013.12019.x

899 Madeira, F., Park, Y. M., Lee, J., Buso, N., Gur, T., Madhusoodanan, N., Basutkar, P., Tivey, A. R. N.,
900 Potter, S. C., Finn, R. D., & Lopez, R. (2019). The EMBL-EBI search and sequence analysis
tools APIs in 2019. *Nucleic Acids Research*, 47(W1), W636-W641.
901
902 https://doi.org/10.1093/nar/gkz268

903 Maquart, P. O., & Boyer, S. (2022). Culex vishnui. *Trends in Parasitology, Vector of the Month*, 491-
492. https://doi.org/10.1016/J.PT.2022.01.003

905 Maquart, P. O., Sokha, C., & Boyer, S. (2021). Mosquito diversity (Diptera: Culicidae) and medical
906 importance, in a bird sanctuary inside the flooded forest of Prek Toal, Cambodia. *Journal of
Asia-Pacific Entomology*, 24(4), 1221-1227. https://doi.org/10.1016/j.aspen.2021.08.001
908 Mitchell, C. J., Forattini, O. P., & Miller, B. R. (1986). Vector competence experiments with Rocio virus
909 and three mosquito species from the epidemic zone in Brazil. *Revista de Saúde Pública, 20*(3),
910 171-177. https://doi.org/10.1590/s0034-89101986000300001

911 Morlan, J. D., Qu, K., & Sinicropi, D. V. (2012). Selective depletion of rRNA enables whole
912 transcriptome profiling of archival fixed tissue. *PLoS ONE*, 7(8), e42882.
913 https://doi.org/10.1371/journal.pone.0042882

914 Mukwaya, L. G., Kayondo, J. K., Crabtree, M. B., Savage, H. M., Biggerstaff, B. J., & Miller, B. R.
915 (2000). Genetic differentiation in the yellow fever virus vector, Aedes simpsoni complex, in
916 Africa: Sequence variation in the ribosomal DNA internal transcribed spacers of anthropophilic
917 and non-anthropophilic populations. *Insect Molecular Biology*, 9(1), 85-91. doi: 10.1046/j.1365-
918 2583.2000.00161.x

919 Mwangangi, J. M., Muturi, E. J., Muriu, S. M., Nzovu, J., Midega, J. T., & Mbogo, C. (2013). The role
920 of Anopheles arabiensis and Anopheles coustani in indoor and outdoor malaria transmission in
921 Taveta District, Kenya. *Parasites and Vectors*, 6, 114. https://doi.org/10.1186/1756-3305-6-114

922 Navarro, J. C., & Weaver, S. C. (2004). Molecular phylogeny of the Vomerifer and Pedroi Groups in
923 the spissipes section of the subgenus Culex (Melanoconion). *Journal of Medical Entomology*,
924 41(4), 575-581. https://doi.org/10.1603/0022-2585-41.4.575

925 Nchoutpouen, E., Talipouo, A., Djiappi-Tchamen, B., Djamouko-Djonkam, L., Kopya, E., Ngadjeu, C.
926 S., Doumbe-Belisse, P., Awono-Ambene, P., Kekeunou, S., Wondji, C. S., & Antonio-Nkondjio,
927 C. (2019). Culex species diversity, susceptibility to insecticides and role as potential vector of
928 Lymphatic filariasis in the city of Yaoundé, Cameroon. *PLoS Neglected Tropical Diseases*,
929 13(4), e0007229. https://doi.org/10.1371/journal.pntd.0007229

930 Ndiaye, E. H., Fall, G., Gaye, A., Bob, N. S., Talla, C., Diagne, C. T., Diallo, D., Ba, Y., Dia, I., Kohl,
931 A., Sall, A. A., & Diallo, M. (2016). Vector competence of Aedes vexans (Meigen), Culex
932 poicilipes (Theobald) and Cx. quinquefasciatus Say from Senegal for West and East African
933 lineages of Rift Valley fever virus. *Parasites and Vectors*, 9, 94. https://doi.org/10.1186/s13071-
934 016-1383-y

935 Nepomichene, T. N. J. J., Raharimalala, F. N., Andriamandimby, S. F., Ravalohery, J. P., Failloux, A.
936 B., Heraud, J. M., & Boyer, S. (2018). Vector competence of Culex antennatus and Anopheles
coustani mosquitoes for Rift Valley fever virus in Madagascar. *Medical and Veterinary
938 *Entomology*, 32(2), 259-262. https://doi.org/10.1111/mve.12291

939 Nikolay, B., Diallo, M., Boye, C. S. B., & Sall, A. A. (2011). Usutu virus in Africa. *Vector-Borne and*

940 *Zoonotic Diseases*, 11(11), 1417-1423. https://doi.org/10.1089/vbz.2011.0631

941 Oo, T. T., Kaiser, A., & Becker, N. (2006). Illustrated keys to the anopheline mosquitoes of Myanmar.

942 *Journal of Vector Ecology*, 31(1), 9-16. https://doi.org/10.3376/1081-

943 1710(2006)31[9:ikttam]2.0.co;2

944 Pereira Serra, O., Fernandes Cardoso, B., Maria Ribeiro, A. L., dos Santos, F. A. L., & Dezengrini

945 Shessarenko, R. (2016). Mayaro virus and dengue virus 1 and 4 natural infection in culicids

946 from Cuiabá, state of Mato Grosso, Brazil. *Memórias Do Instituto Oswaldo Cruz*, 111(1), 20-29.

947 https://doi.org/10.1590/0074-02760150270

948 Phelps, W. A., Carlson, A. E., & Lee, M. T. (2021). Optimized design of antisense oligomers for

949 targeted rRNA depletion. *Nucleic Acids Research*, 49(1), e5.

950 https://doi.org/10.1093/nar/gkaa1072

951 Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., & Glöckner, F. O.

952 (2013). The SILVA ribosomal RNA gene database project: Improved data processing and web-

953 based tools. *Nucleic Acids Research*, 41 (Database issue), D590-D596.

954 https://doi.org/10.1093/nar/gks1219

955 Ratnasingham, S., & Hebert, P. D. N. (2007). BOLD: The Barcode of Life Data System: Barcoding.

956 *Molecular Ecology Notes*, 7(3), 355-364. https://doi.org/10.1111/j.1471-8286.2007.01678.x

957 Ratovonjato, J., Olive, M. M., Tantely, L. M., Andrianavolambo, L., Tata, E., Razainirina, J.,

958 Jeanmaire, E., Reynes, J. M., & Elissa, N. (2011). Detection, isolation, and genetic

959 characterization of Rift Valley fever virus from anopheles (Anopheles) coustani, anopheles

960 (Anopheles) squamosus, and culex (Culex) antennatus of the haute matsiatra region,

961 Madagascar. *Vector-Borne and Zoonotic Diseases*, 11(6), 753-759.

962 https://doi.org/10.1089/vbz.2010.0031

963 Ratsitorahina, M., Harisoa, J., Ratovonjato, J., Biacabe, S., Reynes, J. M., Zeller, H., Raoelina, Y.,

964 Talarmin, A., Richard, V., & Soares, J. L. (2008). Outbreak of dengue and chikungunya fevers,

965 Toamasina, Madagascar, 2006. *Emerging Infectious Diseases*, 14(7), 1135-1137.

966 https://doi.org/10.3201/EID1407.071521

967 Rattanarithikul, R., Harbach, R. E., Harrison, B. A., Panthusiri, P., & Coleman, R. E. (2007). Illustrated
968 keys to the mosquitoes of Thailand V. Genera Orthopodomyia, Kimia, Malaya, Topomyia,
969 Tripteroides, and Toxorhynchites. *Suppl* 1, 38(Suppl 2), 1–65.

970 Rattanarithikul, R., Harbach, R. E., Harrison, B. A., Panthusiri, P., Coleman, R. E., & Richardson, J. H.
971 (2010). Illustrated keys to the mosquitoes of Thailand. VI. Tribe Aedini. *Southeast Asian Journal
972 of Tropical Medicine and Public Health*, 41(Suppl 1), 1–225.

973 Rattanarithikul, R., Harbach, R. E., Harrison, B. A., Panthusiri, P., Jones, J. W., & Coleman, R. E.
974 (2005). Illustrated keys to the mosquitoes of Thailand. II. Genera Culex and Lutzia. *The
975 Southeast Asian Journal of Tropical Medicine and Public Health*.

976 Rattanarithikul, R., Harrison, B. A., Harbach, R. E., Panthusiri, P., & Coleman, R. E. (2006). Illustrated
977 keys to the mosquitoes of Thailand IV. Anopheles. *Southeast Asian Journal of Tropical Medicine
978 and Public Health*, 37(Suppl 2), 1–26.

979 Rattanarithikul, R., Harrison, B. A., Panthusiri, P., & Coleman, R. E. (2005). Illustrated keys to the
980 mosquitoes of Thailand I. Background; geographic distribution; lists of genera, subgenera, and
981 species; and a key to the genera. *Southeast Asian Journal of Tropical Medicine and Public
982 Health*, 36(Suppl 1), 1–80.

983 Rattanarithikul, R., Harrison, B. A., Panthusiri, P., Peyton, E. L., & Coleman, R. E. (2006). Illustrated
984 keys to the mosquitoes of Thailand: III. Genera Aedeomyia, Ficalbia, Mimomyia, Hodgesia,
985 Coquillettidia, Mansonia, and Uranotaenia. *Southeast Asian Journal of Tropical Medicine and
986 Public Health*, 37(Suppl 1), 1–10.

987 Rausch, T., Fritz, M. H. Y., Untergasser, A., & Benes, V. (2020). Tracy: Basecalling, alignment,
988 assembly and deconvolution of sanger chromatogram trace files. *BMC Genomics*, 21(1), 230.
989 https://doi.org/10.1186/s12864-020-6635-8

990 Rausch, T., Hsi-Yang Fritz, M., Korbel, J. O., & Benes, V. (2019). Alfred: Interactive multi-sample
991 BAM alignment statistics, feature counting and feature annotation for long- and short-read
992 sequencing. *Bioinformatics*, 35(14), 2489–2491. https://doi.org/10.1093/bioinformatics/bty1007

993 Reidenbach, K. R., Cook, S., Bertone, M. A., Harbach, R. E., Wiegmann, B. M., & Besansky, N. J.
994 (2009). Phylogenetic analysis and temporal diversification of mosquitoes (Diptera: Culicidae)
995 based on nuclear genes and morphology. *BMC Evolutionary Biology*, 9(1), 1–14.
996 https://doi.org/10.1186/1471-2148-9-298/FIGURES/4

997 Romero-Alvarez, D., & Escobar, L. E. (2018). Oropouche fever, an emergent disease from the
998 Americas. *Microbes and Infection*, 20(3), 135-146.

999 https://doi.org/10.1016/J.MICINF.2017.11.013

1000 Rueda, L. M. (2004). Pictorial keys for the identification of mosquitoes (Diptera: Culicidae) associated
1001 with Dengue Virus Transmission. *Zootaxa*. https://doi.org/10.11646/zootaxa.589.1.1

1002 Ruzzante, L., Reijnders, M. J. M. F., & Waterhouse, R. M. (2019). Of Genes and Genomes: Mosquito
1003 Evolution and Diversity. *Trends in Parasitology*, 35(1), 32-51.

1004 https://doi.org/10.1016/J.PT.2018.10.003/ATTACHMENT/B9BE6BC5-D73A-4FEF-A654-CC1374C59925/MMC1.MP4
1005

1006 Sallum, M. A. M., & Forattini, O. P. (1996). Revision of the Spissipes Section of Culex (Melanoconion)
1007 (diptera: Culicidae). *Journal of the American Mosquito Control Association*, 12(3), 517-600.

1008 Saluzzo, J. F., Evidera, T. V., Veas, F., & Gonzalez, J.-P. J. (2018). *Arbovirus Discovery in Central African Republic (1973-1993): Zika, Bozo, Bouboui, and More*.

1010 https://www.researchgate.net/publication/321587457

1011 Sirivanakarn, S. (1982). A review of the Systematics and a Proposed Scheme of Internal
1012 Classification of the New World Subgenus Melanoconion of Culex (Diptera, Culicidae). *Mosquito Systematics*, 14(4), 265-333.

1014 Stevenson, J. C., Simubali, L., Mbambara, S., Musonda, M., Mweetwa, S., Mudenda, T., Pringle, J.
1015 C., Jones, C. M., & Norris, D. E. (2016). Detection of plasmodium falciparum infection in
1016 anopheles squamosus (diptera: Culicidae) in an area targeted for malaria elimination, Southern
1017 Zambia. *Journal of Medical Entomology*, 53(6), 1482-1487. https://doi.org/10.1093/jme/tjw091

1018 Sun, L., Li, T. J., Fu, W. B., Yan, Z. T., Si, F. L., Zhang, Y. J., Mao, Q. M., Demari-Silva, B., & Chen,
1019 B. (2019). The complete mt genomes of Lutzia halifaxia, Lt. fuscanus and Culex pallidothorax
1020 (Diptera: Culicidae) and comparative analysis of 16 Culex and Lutzia mt genome sequences.
1021 *Parasites and Vectors*, 12, 368. https://doi.org/10.1186/s13071-019-3625-2

1022 Tabue, R. N., Awono-Ambene, P., Etang, J., Atangana, J., Antonio-Nkondjio, C., Toto, J. C.,
1023 Patchoke, S., Leke, R. G. F., Fondjo, E., Mnzava, A. P., Knox, T. B., Tougordi, A., Donnelly, M.
1024 J., & Bigoga, J. D. (2017). Role of Anopheles (Cellia) rufipes (Gough, 1910) and other local
1025 anophelines in human malaria transmission in the northern savannah of Cameroon: a cross-
1026 sectional survey. *Parasites and Vectors*, 10(1), 1-11. https://doi.org/10.1186/S13071-016-1933-3
1027 3/FIGURES/6
1028 Takhampunya, R., Kim, H. C., Tippayachai, B., Kengluecha, A., Klein, T. A., Lee, W. J., Grieco, J., & Evans, B. P. (2011). Emergence of Japanese encephalitis virus genotype v in the Republic of Korea. *Virology Journal*, 8, 449. https://doi.org/10.1186/1743-422X-8-449

1031 Talaga, S., Duchemin, J. B., Girod, R., & Dusfour, I. (2021). The Culex Mosquitoes (Diptera: Culicidae) of French Guiana: A Comprehensive Review With the Description of Three New Species. *Journal of Medical Entomology*, 58(1), 182-221. https://doi.org/10.1093/JME/TJAA205

1034 Thongsripong, P., Chandler, J. A., Kittayapong, P., Wilcox, B. A., Kapan, D. D., & Bennett, S. N. (2021). Metagenomic shotgun sequencing reveals host species as an important driver of virome composition in mosquitoes. *Scientific Reports*, 11(1), 8448. https://doi.org/10.1038/s41598-021-87122-0

1038 Torres-Gutierrez, C., Bergo, E. S., Emerson, K. J., de Oliveira, T. M. P., Greni, S., & Sallum, M. A. M. (2016). Mitochondrial COI gene as a tool in the taxonomy of mosquitoes Culex subgenus Melanoconion. *Acta Tropica*, 164, 137-149. https://doi.org/10.1016/j.actatropica.2016.09.007

1041 Torres-Gutierrez, C., De Oliveira, T. M. P., Emerson, K. J., Bergo, E. S., & Sallum, M. A. M. (2018). Molecular phylogeny of Culex subgenus Melanoconion (Diptera: Culicidae) based on nuclear and mitochondrial protein-coding genes. *Royal Society Open Science*, 5, 171900. https://doi.org/10.1098/rsos.171900

1045 Travassos Da Rosa, J. F., De Souza, W. M., De Paula Pinheiro, F., Figueiredo, M. L., Cardoso, J. F., Acrani, G. O., & Teixeira Nunes, M. R. (2017). Oropouche Virus: Clinical, Epidemiological, and Molecular Aspects of a Neglected Orthobunyavirus. *The American Journal of Tropical Medicine and Hygiene*, 96(5), 1019. https://doi.org/10.4269/AJTMH.16-0672

1049 Turell, M. J., O'guinn, M. L., Dohm, D., Zyzak, M., Watts, D., Fernandez, R., Calampa, C., Klein, T. A., & Jones, J. W. (2008). Susceptibility of Peruvian Mosquitoes to Eastern Equine Encephalitis Virus. *Journal of Medical Entomology*, 45(4), 720-725. https://doi.org/10.1093/JMEDENT/45.4.720

1053 Turell, Michael J. (1999). Vector competence of three Venezuelan mosquitoes (Diptera: Culicidae) for an epizootic IC strain of Venezuelan equine encephalitis virus. *Journal of Medical Entomology*, 36(4), 407-409. https://doi.org/10.1093/jmedent/36.4.407

1056 Ughasi, J., Bekard, H. E., Coulibaly, M., Adabie-Gomez, D., Gyapong, J., Appawu, M., Wilson, M. D., & Boakye, D. A. (2012). Mansonia africana and Mansonia uniformis are Vectors in the
1058 transmission of Wuchereria bancrofti lymphatic filariasis in Ghana. *Parasites and Vectors*, 5(1),
1–5. https://doi.org/10.1186/1756-3305-5-89

1060 Valentine, M. J., Murdock, C. C., & Kelly, P. J. (2019). Sylvatic cycles of arboviruses in non-human
1061 primates. *Parasites and Vectors*, 12(1), 1–18. https://doi.org/10.1186/S13071-019-3732-0/TABLES/4

1063 Vasconcelos, P. F. C., Costa, Z. G., Travassos da Rosa, E. S., Luna, E., Rodrigues, S. G., Barros, V.
1064 L. R. S., Dias, J. P., Monteiro, H. A. O., Oliva, O. F. P., Vasconcelos, H. B., Oliveira, R. C.,
1065 Sousa, M. R. S., Barbosa Da Silva, J., Cruz, A. C. R., Martins, E. C., & Travassos Da Rosa, J.
1066 F. S. (2001). Epidemic of jungle yellow fever in Brazil, 2000: Implications of climatic alterations in
disease spread. *Journal of Medical Virology*, 65(3), 598–604.
1067 https://doi.org/10.1002/jmv.2078 abs

1069 Vázquez González, A., Ruiz, S., Herrero, L., Moreno, J., Molero, F., Magallanes, A., Sánchez-Seco,
1070 M. P., Figuerola, J., & Tenorio, A. (2011). West Nile and Usutu viruses in mosquitoes in Spain,
2008-2009. *American Journal of Tropical Medicine and Hygiene*, 85(1), 178–181.
1071 https://doi.org/10.4269/ajtmh.2011.11-0042

1073 Vezenegho, S. B., Issaly, J., Carinci, R., Gaborit, P., Girod, R., Dusfour, I., & Briolant, S. (2022).
1074 Discrimination of 15 Amazonian Anopheline Mosquito Species by Polymerase Chain Reaction—
1075 Restriction Fragment Length Polymorphism. *Journal of Medical Entomology*, 59(3), 1060–1064.
1076 https://doi.org/10.1093/JME/TJAC008

1077 Weaver, S. C., Ferro, C., Barrera, R., Boshell, J., & Navarro, J. C. (2004). Venezuelan Equine
Encephalitis. *Annual Review of Entomology*, 49, 141–174.
1078 https://doi.org/10.1146/annurev.ento.49.061802.123422

1080 Webster, J. P., Gower, C. M., Knowles, S. C. L., Molyneux, D. H., & Fenton, A. (2016). One health -
an ecological and evolutionary framework for tackling Neglected Zoonotic Diseases.
1081 Evolutionary Applications, 9(2), 313–333. https://doi.org/10.1111/eva.12341

1083 Weedall, G. D., Irving, H., Hughes, M. A., & Wondji, C. S. (2015). Molecular tools for studying the
major malaria vector Anopheles funestus: Improving the utility of the genome using a
1084 comparative poly(A) and Ribo-Zero RNAseq analysis. BMC Genomics, 16(1), 931.
1085 https://doi.org/10.1186/s12864-015-2114-z

1087 WHO. (2017). Global vector control response 2017–2030. In *World Health Organization*.
1088 Zakrzewski, M., Rašić, G., Darbro, J., Krause, L., Poo, Y. S., Filipović, I., Parry, R., Asgari, S., Devine,

1089 G., & Suhrbier, A. (2018). Mapping the virome in wild-caught Aedes aegypti from Cairns and

1090 Bangkok. *Scientific Reports*, 8(1), 4690. https://doi.org/10.1038/s41598-018-22945-y

1091 Zeller, H., Van Bortel, W., & Sudre, B. (2016). Chikungunya: Its History in Africa and Asia and Its

1092 Spread to New Regions in 2013–2014. *The Journal of Infectious Diseases*, 214(suppl_5), S436–

1093 S440. https://doi.org/10.1093/INFDIS/JIW391

1094 Zittra, C., Flechl, E., Kothmayer, M., Vitecek, S., Rossiter, H., Zechmeister, T., & Fuehrer, H. P.

1095 (2016). Ecological characterization and molecular differentiation of Culex pipiens complex taxa

1096 and Culex torrentium in eastern Austria. *Parasites and Vectors*, 9, 197.

1097 https://doi.org/10.1186/s13071-016-1495-4

1098
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## Genus colour codes