Anopheles merus distribution and TEP1 genotypes along the Kenyan coast

doi:10.21203/rs.3.rs-1660930/v1

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Anopheles merus distribution and TEP1 genotypes along the Kenyan coast

https://doi.org/10.21203/rs.3.rs-1660930/v1

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Background

Malaria remains one of the most important infectious diseases in Sub-Saharan Africa, responsible for approximately 228 million cases and 602,000 deaths in 2020. Malaria transmission is mainly driven by mosquitoes of the Anopheles gambiae and more recently Anopheles funestus complex. The gains made in malaria control are threatened by insecticide resistance and behavioural plasticity among these vectors. This, therefore, calls for the development of alternative approaches such as malaria transmission-blocking using gene drive systems that can lead to population replacement of infection-susceptible mosquitoes with mosquitoes that are refractory to Plasmodium spp. infection. One such gene that can be utilised is the Thioester-containing protein 1 (TEP1) gene which mediates the killing of Plasmodium falciparum in the mosquito midgut. Here we investigated the frequencies and distribution of TEP1 alleles in wild-caught malaria vectors along the Kenyan coast.

Methods

Mosquitoes were collected using CDC light traps both indoors and outdoors from 20 houses in Garithe village, along the Kenyan coast. The mosquitoes were dissected, and the different parts were used to determine their species, blood-meal source, and sporozoite status. The data was analysed and visualised using the R (v 4.0.1) and STATA (v 17.0).

Results

A total of 18,802 mosquitoes were collected consisting of 77.8% (n = 14,631) Culex spp, 21.4% (n = 4,026) An. gambiae s.l, 0.4% (n = 67) An. funestus and 0.4% (n = 78) other Anopheles (An. coustanii, An. pharoensis and An. pretoriensis). A subset of randomly selected An. gambiae s.l (n = 518) was identified by polymerase chain reaction (PCR), of these 77.2% were An. merus, 22% were An. arabiensis and the rest were not detected. Mosquitoes collected were predominantly exophilic with the outdoor catches being higher across all the species: Culex spp 93% (IRR = 11.6, 95% Cl [5.9–22.9] p < 0.001), An. gambiae s.l 92% (IRR = 7.2, 95% Cl [3.6–14.5]; p < 0.001), An. funestus 91% (IRR = 10.3, 95% Cl [3.3–32.3]; p < 0.001). We identified the following genotypes among An. merus: *R2/R2, *R3/R3, *R3/S2, *S1/S1 and *S2/S2. Among An. arabiensis, we identified *R2/R2, *S1/S1, *S2/S2. Tests on haplotype diversity showed that the most diverse allele was TEP1*S1 followed by TEP1*R2. Tajima’s D values were positive for TEP1*S1 indicating that there is a balancing selection, negative for TEP1*R2 indicating there is a recent selective sweep and as for TEP1*R3 there was no evidence of selection. Phylogenetic analysis showed two distinct clades exist: Refractory and susceptible alleles.

Conclusion

We find that the malaria vectors An. gambiae s.l and An. funestus are predominantly exophilic. TEP1 genotyping for An. merus revealed 5 allelic combinations: *R2/R2, *R3/R3, *R3/S2, *S1/S1 and *S2/S2 while in An. arabiensis, we only identified 3 allelic combinations: *R2/R2, *S1/S1, *S2/S2. The TEP1*R3 allele was restricted to only An. merus among these sympatric mosquito species and we find that there is no evidence of recombination or selection in this allele.

Anopheles merus

Thioester-containing protein 1

allele

Kenya

Malaria remains one of the most important infectious diseases in Sub-Saharan Africa, responsible for approximately 228 million cases and 602,000 deaths in 2020 (1). Significant advances in malaria control have been achieved in the last two decades, these have been mostly by vector control interventions including long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). Between 2000 and 2019 we have seen a reduction of 28% and 44% in global malaria incidence and mortality(2,3). Unfortunately, this progress has plateaued in the last 5 years and the World Health Organization’s (WHO) 2016–2030 Global Technical Strategy for Malaria (GTS) (4) is off-track, with a global incidence reduction of less than 2% between 2015 and 2020 (1).

Malaria transmission in sub-Saharan Africa is mainly driven by mosquitoes of the Anopheles gambiae and more recently Anopheles funestus complex (5–7). Despite the scale-up of malaria control interventions, insecticide resistance (6,8) and increased vector behavioural plasticity allow these to preclude interventions by avoiding exposure to insecticide-based interventions (9–12) as well as blood-feeding on a variety of hosts (13) are threatening the gains made in malaria control. This, therefore, calls for the development of more effective novel interventions, e.g. transmission-blocking vaccines or gene drive systems that can lead to population replacement of infection-susceptible mosquitoes with those that are refractory to Plasmodium spp. infection (14).

Although there are 475 Anopheles species only 70 are known primary or secondary vectors of malaria(15). This is partly attributable to the mosquito’s innate immunity against invading parasites (16,17). One of such anti-parasitic immunity is mediated by Thioester-containing protein 1 (TEP1), a homologue of the mammalian complement factor 3. TEP1 recognizes and binds to ookinetes, mediating parasite lysis and melanisation (8). TEP1 is a highly polymorphic protein consisting of variants differentiated by amino acid sequence variation in the thioester domain (TED) region (18). The variants are classified into two main subclasses: refractory TEP1*R (*R1 and *R2) and susceptible TEP1*S (*S1 and *S2). Mosquitoes bearing the R alleles are more effective at parasite killing, with *R1/R1 and *S2/S2 mosquitoes being fully resistant and susceptible to infection respectively (16). The TEP1*S and TEP1*R alleles are found in all species of Anopheles gambiae s.l., albeit with marked variation in distribution both geographically and within the different species. Additionally, TEP1 alleles have been shown to affect male fitness in wild mosquito populations, due to their ability to clear off defective sperms, mosquitoes bearing the TEP1*S2 alleles have been shown to have higher fertility rates (19).

Recently, a novel resistance-encoding allele, termed TEP1*R3, was discovered in An. merus populations from the Kenyan coast (20). Its genetic diversity and functional role in controlling the development of malaria parasites within the mosquito has not been described. The present study aimed at characterising the TEP1 alleles of An. merus populations collected from coastal Kenya.

Study area

Entomological collections were conducted in Garithe village, Kilifi County along the Kenyan coast (Supplementary Figure 1). This region is highly diverse, made up of dense forests, dry thorny bushes, savannah vegetation and seasonal swamps of brackish water. There are two distinct rainy seasons: long rains which occur between April and July and short rains between October and November (21). The site was targeted for collections based on previous studies that described An. merus high density (22).

Mosquitoes were collected in 20 houses over six consecutive days during the month of November in 2019 (Supplementary Figure 1). Collections were carried out using Centers for Disease Control and Prevention light traps (CDC-LT) both indoors and outdoors for each house from dusk (1800hrs) to dawn (0600hrs), whilst coordinates were collected using eTrex® 10 (Garmin, Kansas, United States of America). The indoor traps were set in houses where at least one person spent the night during the collection period. The outdoor traps were strategically set next to the livestock shed and where livestock was absent, the trap was set approximately 5 metres from the household selected for indoor sampling. The collected mosquitoes were identified morphologically in the field laboratory(23) and sorted by physiological stage and sex. All the Anopheles spp were preserved individually in 1.5ml microcentrifuge tubes containing silica pellets and transported to the KEMRI Wellcome Trust Research Programme (KWTRP) laboratory and stored at -80˚C.

Mosquito processing

Using sterile scalpel and forceps, the female Anopheles mosquitoes were dissected and separated into distinct body parts for different assays. The legs and wings were used for Anopheles gambiae s.l sibling species identification, the head and thorax for the Plasmodium falciparum infection status, and the abdomen of blood-fed mosquitoes for trophic pattern and preference analysis as previously described(24)

Anopheles gambiae s.l sibling species speciation

Genomic deoxyribonucleic acid (DNA) was extracted from the legs and wings of mosquitoes as previously described with minor modifications (25). Briefly, the mosquito parts were transferred into 1.5 ml microcentrifuge tubes containing 50 µl of 20% chelex and crushed using polypropylene pestles. The lysate was incubated at 100˚C while shaking at 650 revolutions per minute (RPM) using a thermomixer (Eppendorf, Hamburg, Germany). The solution was then centrifuged at 10,000 x g for 2 minutes (mins) and the supernatant was transferred to a new 1.5 ml microcentrifuge tube, this was repeated twice, and the DNA was stored at -80˚C.

Anopheles gambiae s.l. sibling species were identified using a previously described method using primers that target the intergenic spacer (IGS) region of the ribosomal DNA (26). The species were distinguished by their band sizes after running agarose gel electrophoresis as follows: 153 bp for Anopheles quadrianulatus, 315 bp for Anopheles arabiensis, 390 bp for Anopheles gambiae s.s., 464 bp for Anopheles melas and 466 bp for Anopheles merus (26). An. funestus complex sibling species were also identified using PCR with primers targeting the internal transcribed Spacer Region 2 (ITS2) (27).

Bloodmeal analysis

The abdomens of the blood-fed female Anopheles mosquitoes were crushed in 50 µl of molecular grade water using sterile polypropylene pestles. 30 µl of the lysate was mixed with 500 µl of phosphate-buffered saline (PBS) and then used to determine the source of bloodmeal using direct enzyme-linked immunosorbent assays (ELISA) as previously described(28,29) with slight modifications. The samples were tested against the anti-host immunoglobulin gamma (IgG): human, goat, bovine and chicken. The results were read visually as previously described (30).

Plasmodium falciparum sporozoite analysis

The mosquito head and thorax were crushed in 100 µl of 1X phosphate buffered saline (PBS) in 1.5 ml microcentrifuge tubes. Thereafter, 10 µl of 10X saponin was added to the lysate. The solution was then incubated at room temperature for 20 mins, and then centrifuged at 20,000 x g for 2 mins and the supernatant was discarded. The pellet was then resuspended in 100 µl 1X PBS. The solution was then centrifuged at 2000 x g for 2 mins and the supernatant was discarded. The pellet was thereafter resuspended in 50 µl of 20% chelex and DNA extracted using the procedure described above. Thereafter, the extracted DNA was used for SYBR green real-time PCR assays using primers described in Hermsen et al., 2001(31). Briefly, the RT-PCR reaction consisted of 7.5 µl of QuantiTect SYBR Green PCR master mix (Qiagen, Hilden, Germany), 0.75 µl of forward primer: 5’-GTAATTGGAATGATAGGATTTACAAGGT-3’ and 0.75µl of the reverse primer: 5’-TCAACTACGAACGTTTTAACTGCAAC-3’, 2 µl of nuclease-free water and 4 µl of the DNA. RT-PCR conditions were as follows: 95°C for 10 mins for HotStarTaq DNA Polymerase activation, 40 cycles of 95°C for 30 seconds (secs), 60°C for 45 secs, 68°C for 45 secs and finally the melt curve phase: 95°C for 15 secs, 60°C for 1 min and 95°C for 30 secs.

TEP1 genotyping

The highly polymorphic thioester domain (TED) region of thioester-containing protein 1 (TEP1) was amplified using the primers VB229 5’-TCAACTTGGACATCAACAAGAAG-3’ and VB004 5’- ACATCAATTTGCTCCGAGTT-3’ as previously described (19,32). Thereafter the 1088 ± 1 base pairs amplicon were cleaned using Qiaquick PCR purification kit (Qiagen, Hilden, Germany) and eluted in 15 µl of DNase free water. The samples were subjected to both Sanger and Next Generation Sequencing (NGS).

For the Sanger sequencing, the PCR amplicons were sequenced using the primers (VB004 and VB229), Big dye terminator chemistry v3.1 (Applied Biosystems, UK) and the reaction was run on an ABI 3730xl capillary sequencer (Applied Biosystems, UK). The resulting sequences chromatograms were curated, edited and aligned using CLC Main Workbench 7 (CLC Bio, Qiagen, Aarhus, Denmark).

For NGS, 75 cycles of paired-end sequencing were done on the Miseq platform using the Nextera DNA Flex library preparation protocol and Miseq reagent kit V3 (Illumina, USA). The resulting reads were demultiplexed, then FastQC v0.11.9 was used to remove indexes, low-quality PhiX and adapter reads. Identity of the resultant reads was ascertained using both BLASTn v2.11.0 and mapping onto the TEP1 references.

Phylogenetic analysis

Multiple sequence alignment and sequence editing was conducted in AliView software v1.25, using the newly sequenced TEP1-TED sequences and those collated from GenBank(33). Maximum Likelihood phylogenies were reconstructed using the TIM+F+I substitution model with 4 gamma categories (TIM+F+I+G4) as the best fitting model as inferred by JmodelTest in iqtree v1.6.9(34) and visualized using FigTree v1.4.4.

Statistical analysis

Statistical analysis, visualisation and mapping were done using R software (v 4.1.0)(35). The data was further analysed using a generalised negative binomial regression model with a log link and robust errors whereby the dependent variable was the number of collected mosquitoes, and the independent variables were site (indicator variable), day and household using STATA v17.0 (StataCorp, College Station, TX, USA). The human blood index (HBI) was calculated as the proportion of mosquitoes that had fed on humans divided by total blood meals tested for each species

Haplotype statistics

Haplotype statistics such as the number of haplotypes, haplotype frequencies and haplotype configuration and tests of natural selection: Tajima’s D(36), Fu and Li's D and Fu & Li’s F(37) were calculated using the DNA sequence polymorphism software(DnaSP)(38).

Abundance and diversity of Anopheles spp mosquitoes

A total of 18,802 mosquitoes were collected. Using morphological cues, the mosquito consisted of 77.8% (n = 14,631) Culex spp, 21.4% (n = 4,026) An. gambiae s.l, 0.4% (n = 67) An. funestus and (0.4% n = 78) other Anopheles (An. coustanii, An. pharoensis, An. pretoriensis). A subset of the Anopheles gambiae s.l, (n=518) mosquitos were analysed for sibling species by PCR. Of these 77.2% (n=400) were identified as An. merus, 22% (n=114) as An. arabiensis and 0.8% (n=4) were not detected by PCR, this could be due to very low DNA concentrations, presence of PCR inhibitors or morphological misidentification (Figure 1). Anopheles funestus complex consisted of 42% (n=28) An. rivulorum, 25% (n=17) An. leesoni and 3%(n=2) An. parensis, and the rest could not be detected (30%, n=20).

All the mosquitoes collected had greater proportions of mosquitoes resting outdoors (Table 1). An funestus had 91% found outdoors (IRR = 10.3, 95% Cl [3.3 – 32.3]; p < 0.001). The same was observed for An. gambiae s.s at 92% (IRR = 7.2, 95% Cl [3.6 – 14.5]; p < 0.001) and Culex spp at 93% (IRR = 11.6, 95% Cl [5.9 – 22.9] p < 0.001).

Physiological state and source of bloodmeal

Generally, there’s a greater number of mosquitoes in all the physiologic states found outdoors (Supplementary Table 1). Among the blood-fed mosquitoes that were analysed for the blood meal sources, 38.5% (n=5) of outdoor An. arabiensis had fed on goats and 23.1% on humans (n=3, HBI = 0.27). Among outdoor An. merus a greater proportion fed on goats (77.1%, n=54) and 2.9% had fed on human (n=2.9%, HBI = 0.03). The HBI among the An. merus was slightly higher indoors.

Plasmodium infection in mosquitoes

Of the 4026 An. gambiae s.l., 280 mosquitoes (An. arabiensis [n=70] and An. merus [n=210]) were screened for sporozoite infection by PCR. From these 9.5% (20/210) An. merus and 8.5% (6/70) An. arabiensis were sporozoite positive. Additionally, the sporozoite rates were higher outdoors at 83% (5/6) for An. arabiensis and 95% (19/20) for An. merus.

TEP1 distribution

We genotyped 175 samples from both An. merus and An. arabiensis. *R2/R2 (84%, n=32)allele was predominant in An. arabiensis, followed by *S2/S2 (11%, n=4) (Figure 3). For An. merus, the predominant allele was *S1/S1 (66%, n = 91) followed by*R3/R3 (22%, n = 30) (Figure 3). Further analysis between TEP1 alleles and Plasmodium falciparum positivity, for samples tested for both, showed that An. merus and An. arabiensis with *R2/R2 and *S1/S1 allele were positive for sporozoites.

Haplotype statistics

Full-length TEP1-thioester domain sequences were used for haplotype statistics and test of neutrality (n=110, length=758bp) (Table 4). The number of haplotypes among the specific alleles was most abundant in TEP1*S1. Haplotype diversity ranged from 0 to 0.999, with TEP1*S1 having the highest value (0.999) and TEP1*R3 with the least (Table 3).

TEP1 phylogenetics

Generally, two major clades exist with the susceptible (TEP1*S) and resistant (TEP1*R) genotypes having evolved separately and independent of each other (Figure 4). The distribution of these alleles was not mosquito species-specific except for TEP1*R3 as the allele was only found among An. merus mosquitoes. Amongst the susceptible, two further subclades exist (TEP1*S1 and TEP1*S2) that are evolving independently of each other since last separating from their most recent common ancestor (MRCA). The TEP1*S1 allele is the most prevalent overall, and more diverse than TEP1*S2. Amongst the mosquito carrying the resistant allele, two subclades exist (TEP1*R1-TEP1*R2 and TEP1*R3). TEP1*R1 and TEP1*R2 are closely related, with the former forming a subclade nested within TEP1*R2.

An. merus was the predominant mosquito from the An. gambiae complex making 77.2% of those that were identified by PCR, this is consistent with previous studies in the same area (21,22,39). In contrast with the other studies, we did not find any An. gambiae s.s we assume that they may have been cleared by interventions such as indoor residual sprays and long-lasting insecticide-treated nets (9,40). We also found that the majority of the mosquitoes were outdoor feeders, this could also be due to pressures from insecticides as previously observed in Tanzania among Anopheles funestus mosquitoes (9). Additionally, An. arabiensis and An. merus have also been shown to be exophilic and zoophilic (22,39,41,42).

An. merus is an important vector of Plasmodium falciparum and Wuchereria bancrofti. This mosquito species is especially notorious due to its exophilic tendencies. From the 18s rRNA PCR, we identified P. falciparum infection rate of 9.5% (20/210) in An. merus and 8.6% (6/70) in An. arabiensis with a majority of these being outdoor feeders meaning they may be involved with outdoor malaria transmission. Noteworthy, the blood-fed mosquitoes identified either indoor or outdoor exhibited relaxed feeding preference by drawing blood meals from a variety of vertebrate hosts including humans, goats and bovines.

Of interest for the TEP1 alleles analysed the TEP1*S1/S1 was the most prevalent allele (66%) and the only allele positive for sporozoite infection (n = 6). We also found the TEP1*R3 allele exclusively in An. merus though being sympatric with An arabiensis which is consistent with previous findings (20,43). However, the function of the TEP1*R3, allele is yet to be elucidated although our data suggested they may associate with mosquito refractoriness to Plasmodium infection since none of the genotyped TEP1*R3 mosquitoes were positive for P. falciparum. The TEP1*R3 allele was fairly distributed in Garithe and found in about a third of sampled houses (Supplementary Fig. 2). The *S1/S1 and *R3/R3 genotypes were enriched in An. merus similar to previous findings(43,44). The *R1/R1 allele is more efficient in parasite clearance compared to *R2/R2, with *S2/S2 and *S1/S1 being fully susceptible.

The tests on haplotype diversity showed that the most diverse allele was TEP1*S1 followed by TEP1*R2 (Table 3). The low nucleotide diversity values for all the alleles indicates that there is a modest difference among the alleles. Tajima’s D values were positive for TEP1*S1 indicating that there is a balancing selection, negative for TEP1*R2 indicating there is a recent selective sweep and as for TEP1*R3 there is no evidence of selection. Lastly, the Fu and Li’s D and F statistics were calculated, where all the values were positive, ranging from 0 to 2.398 indicative of a few unique variants and an abundance of the present variants.

Phylogenetic analysis showed that the refractory and susceptible TEP1 alleles emerged independently of each other as shown in the phylogenetic tree (Fig. 4). They exist as separate clades each clade evolving uniquely and exhibiting high diversity. The separate emergence and evolution of the refractory and susceptible alleles is likely due to two different gene conversions in the TEP1 loci (18). Previously, the resistant and susceptible alleles have been reported to be recombinants and under positive selection (18).

An. merus a saltwater breeding mosquito vector of malaria and lymphatic filariasis was the most dominant species of An gambiae complex identified in Garithe village in coastal Kenya. Mosquitoes in the study area were predominantly exophilic and utilized a variety of vertebrate host for bloodmeal requirements. Given our observations TEP1*S1 allele had the highest frequency and positive for P. falciparum in An. merus. In addition, TEP1*S1 mosquitos were predominantly exophilic meaning indoor adult mosquito control strategies may be ineffective for their control. Thus, suggesting potential risk of these mosquitoes becoming major players in persistent outdoor malaria transmission. Mosquitoes bearing TEP1*R3 allele were second in frequency, although the role of this allele in malaria has not been established it’s unlikely they would support malaria given they were negative for P. falciparum infection and the fact they belong to family of refractory alleles. However, this needs further confirmation by experimental infections. Why this allele is mosquito species-specific remains a mystery and is subject to further investigation and potential for allele in applications for Plasmodium falciparum transmission control such as gene drive system.

DNA: Deoxyribonucleic acid

DNA SP: DNA sequence polymorphism software

ELISA: Enzyme-linked Immunosorbent Assay

GARD: Genetic algorithm for recombination detection

KWTRP: KEMRI Wellcome Trust Research Programme

IgG: Immunoglobulin gamma

IRS: Indoor residual spraying

ITS: Intergenic spacer

LLINs: Long-Lasting Insecticide-treated nets

NGS: Next-generation sequencing

PBS: Phosphate Buffered Saline

PCR: Polymerase chain reaction

TEP1*R: Refractory

RT-PCR: Real-time polymerase chain reaction

TEP1*S: Susceptible

TED: Thioester domain

TEP1: Thioester-containing protein 1

WHO: World Health Organization

Ethics approval and consent to participate

The study was approved by the KEMRI Scientific and Ethics Review Unit (SERU) (Protocol number: KEMRI/SERU/CGMR-C/024/3148). Oral consent was obtained from the household head before mosquito collections commenced.

Consent for publication

The authors declare no competing interests of financial or non-financial nature. Mention of trade names or commercial products in this publication is solely for providing specific information and does not imply recommendation or endorsement by any of the authors or respective affiliations.

Availability of data and material

The data and the analysis codes used in R (v 4.0.1) are available in Harvard Dataverse: https://doi.org/10.7910/DVN/CKMX29. The sequences have been deposited at GenBank using the accession number: OM237458 - OM237611.

Competing interests

The authors declare that they have no competing interests.

Funding

The work is supported by The Royal Society FLAIR fellowship grant FLR_R1_190497 (awarded to MKR). The funding bodies did not have any role in the design, data collection, data analysis and interpretation and writing of the manuscript.

Author’s contributions

BB, JK, KO identified the mosquitoes, BB, DO carried out the bioinformatics analysis, BB, MM, MK carried out statistical analysis, CM, JM, MM, MK revised and provided critical reviews on the manuscript.MK designed, supervised, and provided funding for the study.

Acknowledgements

We are grateful to the Garithe community for allowing us to carry out the study in their homesteads. Many thanks to the technical and field staff: Festus Yaa, and Gabriel Nzai who devoted their time to assist in the field collection of mosquito samples.

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Table 1. Comparisons of outdoor and indoor mosquitoes using negative binomial generalised linear models, where the dependent variable was the number of collected mosquitoes, independent variables were site (indicator variable), day of collection and household. Generally, An. funestus, An. gambiae and Culex spp are exophilic.

Species	Site	Number of sampling days	Number of households	n	Median	IQR	IRR	95% Conf. Intervals IRR	p-value
An. funestus	Indoors	5	20	6	0	[0-0]	1	-	-
	Outdoors	5	20	61	0	[0-2]	10.3	[3.3 – 32.3]	<0.001
An. gambiae	Indoors	5	20	333	1	[0-6]	1	-	-
	Outdoors	5	20	3’696	5	[0-51]	7.2	[3.6 – 14.5]	<0.001
Culex spp	Indoors	5	20	1’076	13.5	[7-25]	1	-	-
	Outdoors	5	20	13’555	33.5	[10-200]	11.6	[5.9 – 22.9]	<0.001

Table 2. Bloodmeals source identification both indoors and outdoors and estimates of the human blood index.

species	location	Human	Bovine	Goat	Bovine_Goat	not_detected	HBI
An. arabiensis	Indoor	0	0	1 (7.7%)	0	1 (7.7%)	N/A
An. arabiensis	Outdoor	3 (23.1%)	1 (7.7%)	5 (38.5%)	1 (7.7%)	1 (7.7%)	0.27
An. merus	Indoor	1 (1.4%)	3 (4.3%)	2 (2.9%)	1 (1.4%)	0	0.14
An. merus	Outdoor	2 (2.9%)	2 (2.9%)	54 (77.1%)	1 (1.4%)	4 (5.7%)	0.03

Table 3. Haplotype statistics and tests of neutrality from full TEP1-Thioester Domain sequences. In terms of haplotype diversity, TEP1*S1 was the most diverse allele. The low nucleotide diversity values indicate that there is a modest difference within the alleles.

	*TEP1* alleles
Test	All alleles	**TEP1S1***	**TEP1R2***	**TEP1R3***
Sequences no.	110	48	54	8
Length(bp)	758	758	758	758
Haplotypes no.	66	47	18	1
Haplotype diversity	0.890	0.999	0.583	0.000
Variable sites no.	116	92	83	0
Mutations no.	121	92	83	0
Nucleotide diversity	0.056	0.050	0.020	0.000
Tajima's D	2.747	2.996	-0.555	NA
Fu and Li's D	1.426	1.297	0.861	0.000
Fu and Li's F	2.398	2.298	0.392	0.000

No competing interests reported.

SupplementaryFigure1.docx
Supplementary Figure 1. The respective houses in Garithe village where mosquitoes were sampled.
SupplementaryFigure2.png
SupplementaryTable1.docx
Supplementary Table 1. The mosquito counts in each physiologic state: blood-fed, gravid, half gravid and unfed. Generally, there is a greater number of mosquitoes found outdoors across all the physiologic states

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Anopheles merus distribution and TEP1 genotypes along the Kenyan coast

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