Molecular Characterization and Genotype Distribution of Thioester-containing Protein 1 Gene, A Key Regulator of Malaria Transmission in An. Gambiae Mosquitoes in Western Kenya


 BackgroundEvolutionary pressures lead to the selection of efficient malaria vectors either resistant or susceptible to Plasmodiumparasites.These forcesmay elevate the introduction of new species genotypes that adapt to new breeding habitats which could have serious implications on malaria transmission.Thioester-containing protein 1 (TEP1) of Anopheles gambiaeplays an important role in innate immune defenses against parasites. This study aims to characterize the distribution pattern of TEP1 polymorphisms determining vector competence and subsequently malaria transmission in western Kenya. MethodsAnopheles gambiaeadult and larvae were collected using pyrethrum spray catches (PSC) and plastic dippers respectivelyfrom Homa Bay, Kakamega, Bungoma, and Kisumu countiesbetween 2017 and 2020.Collected adults and larvae reared to the adult stage were morphologically identified and then identified to sibling species by PCR.TEP1 alleles were determined using restriction fragment length polymorphisms-polymerase chain reaction (RFLP-PCR) and to validate the TEP1 genotyping results, a representative sample of alleles was sequenced.ResultsTwo TEP1 alleles (TEP1*S1 and TEP1*R2)and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2)were identified. TEP1*S1 and TEP1*R2 with their corresponding genotypes, homozygous *S1/S1 and heterozygous *R2/S1 were widely distributed across all sites with allele frequencies of approximately 80% and 20%, respectively bothin An. gambiaeand An. arabiensis. There was no significant difference detected among the population and between the two mosquito species in TEP1 allele frequency and genotype frequency. The overall low levels in population structure (FST= 0.019) across all sites corresponded to an effective migration index (Nm= 12.571) and lowNei’s genetic distance values (<0.500) among the subpopulation.The comparative fixation index values revealed minimal genetic differentiation between speciesand high levels of gene flow among populations.ConclusionThere is a low genetic diversity and population structure in western Kenya. TEP1* R2 and TEP1*S1 were the most common alleles in both species which may have been maintained through generations in time, However, the TEP1*R2 allele was in low frequencies and may be used to estimatemalaria prevalence. Continued surveillance of the distribution of TEP1 is essential for monitoring the population dynamics of local vectors and their implications on malaria transmission and hence designing targeted vector interventions.


Abstract Background
Evolutionary pressures lead to the selection of e cient malaria vectors either resistant or susceptible to Plasmodiumparasites.These forcesmay elevate the introduction of new species genotypes that adapt to new breeding habitats which could have serious implications on malaria transmission.Thioester-containing protein 1 (TEP1) of Anopheles gambiaeplays an important role in innate immune defenses against parasites. This study aims to characterize the distribution pattern of TEP1 polymorphisms determining vector competence and subsequently malaria transmission in western Kenya.

Methods
Anopheles gambiaeadult and larvae were collected using pyrethrum spray catches (PSC) and plastic dippers respectivelyfrom Homa Bay, Kakamega, Bungoma, and Kisumu countiesbetween 2017 and 2020.Collected adults and larvae reared to the adult stage were morphologically identi ed and then identi ed to sibling species by PCR.TEP1 alleles were determined using restriction fragment length polymorphisms-polymerase chain reaction (RFLP-PCR) and to validate the TEP1 genotyping results, a representative sample of alleles was sequenced.

Results
Two TEP1 alleles (TEP1*S1 and TEP1*R2)and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2)were identi ed. TEP1*S1 and TEP1*R2 with their corresponding genotypes, homozygous *S1/S1 and heterozygous *R2/S1 were widely distributed across all sites with allele frequencies of approximately 80% and 20%, respectively bothin An. gambiaeand An. arabiensis. There was no signi cant difference detected among the population and between the two mosquito species in TEP1 allele frequency and genotype frequency. The overall low levels in population structure (F ST = 0.019) across all sites corresponded to an effective migration index (Nm= 12.571) and lowNei's genetic distance values (<0.500) among the subpopulation.The comparative xation index values revealed minimal genetic differentiation between speciesand high levels of gene ow among populations.

Conclusion
There is a low genetic diversity and population structure in western Kenya. TEP1* R2 and TEP1*S1 were the most common alleles in both species which may have been maintained through generations in time, However, the TEP1*R2 allele was in low frequencies and may be used to estimatemalaria prevalence. Continued surveillance of the distribution of TEP1 is essential for monitoring the population dynamics of local vectors and their implications on malaria transmission and hence designing targeted vector interventions.

Background
Anopheles gambiae mosquitoes are competent vectors for malaria in sub-Saharan Africa (1,2). Ongoing vector control interventions (3, 4), climate change (5-9), and environmental modi cations modulate the mosquito abundance promoting the abundance of e cient malaria vectors either refractory or susceptible to Plasmodium parasites (10). These factors may cause vectorial rearrangement exacting selection pressure that could change TEP1 allele frequencies and subsequently have serious implications on malaria transmission.
Despite the increased vector densities, malaria transmission is dependent on infectious parasites and competent vectors to in uence susceptibility to infections in local vector populations. A vector's susceptibility and/or resistance to Plasmodium parasites is a determining factor for vector competence and is in part in uenced by the thioester containing protein 1 (TEP1).
In Anopheles gambiae, TEP1 exhibits allelic variations that alter vector competence and subsequently in uence malaria infectivity (11,12). These variations may be as a result of selective pressures such as climate change and vector control interventions acting on the TEP1 gene that eventually in uence the vector's ability to transmit the Plasmodium parasite (12). The TEP1 gene was reported to target the Plasmodium parasite in the early stages of infection in the mosquito host mostly the ookinetes (13,14) either by melanization or lysis (15,16) effectively reducing oocysts and sporozoite numbers in the vector. However, there is a lack of knowledge on how these allelic polymorphisms in vector competence affect malaria transmission (17,18). Therefore, understanding molecular mechanisms underlying mosquito genotypes and Plasmodium adaptations to different Anopheles species is essential and can be used to monitor infection trends in vectors that directly have an impact on malaria transmission.
The complement-like thioester-containing protein 1 (TEP1) plays a key role in immunity against pathogens (18-21). TEP1 is a highly polymorphic protein (22)(23)(24) located in the thioester domain (TED) on chromosome 3L coding for 1338 amino acids long protein contributing to phenotypic divergence and demonstrates genetic variations associated with distinct genotypes in its refractoriness to Plasmodium parasites. Six allelic classes; TEP1*S1, TEP1*S2, TEP1*S3, TEP1*R1, TEP1*R2, and TEP1*R3 have recently been characterized in the An. gambiae complex in Africa (14,15,25). TEP1*S1 and TEP1*R2 are the most common TEP1 alleles identi ed across Africa. The TEP1*S1 however lacks a de ned geographical structure. The TEP1*S2 allele identi ed in the 4Arr strain is speci c to An. coluzzii (25) and gets rid of the damaged sperm cells in the male mosquitoes (26) bringing forth varying Anopheles population abundance. TEP1*S3 allele closely related to TEP1*S1 is xed in the G3 strain associated with susceptibility of infection to P. berghei (14). TEP1*R1 identi ed in the L3-5 strain depicts the highest level of resistance to Plasmodium associated with melanization (14,26,27) and documented in An. coluzzii in West Africa (25). A newly identi ed allele TEP1*R3 is speci c to the saline water mosquito, An. merus found at the Kenyan coast. Selective pressures in uence these variations in the genetic structure of the natural An. gambiae populations in different ecological settings and differences in their refractoriness to Plasmodium parasites are not clear. Genotyping TEP1 in local vector populations is therefore critical for monitoring changes in abundance that explain sporozoite rates and potential malaria prevalence in varying levels of endemicities and is a potential tool for developing vector control interventions. This study was designed to elucidate the characterization and distribution of TEP1 alleles circulating in An. gambiae vectors in malaria-endemic regions in western Kenya.

Study sites and design
This study was conducted in four counties in western Kenya namely, Bungoma, Kakamega, Kisumu, and Homa Bay (Fig. 1). Two malaria epidemic-prone highland sites including Kimaeti (00.6029° N, 034.4073° E; altitude 1,430-1545 m above sea level) in Bungoma, and Iguhu (34°45′E, 0°10′N; 1,430-1,580 m above sea level) in Kakamega, and two lowland sites located around Lake Victoria; Kombewa (34°30′E, 0°07′N; 1,150-1,300 m above sea level) in Kisumu and Kendu Bay (34.64190°E-0.38000°S; 1134-1330 m above sea level) in Homa Bay. The climate in western Kenya consists of long and short rainy seasons that malaria transmission peaks between March to May and October to November respectively. Temperature ranges from a minimum of 14-18 0 C to a maximum of 30-36 0 C and average rainfall ranges between 1740mm and 1940mm annually. Plasmodium falciparum is the most common cause of malaria and is transmitted by An. arabiensis, An. gambiae and An. funestus (28, 29). The key vector control interventions are long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS) (30). Indoor residual spray was conducted in Homa Bay County once a year in 2017 and 2018, unlike the other sampling sites.

Adult Sampling
Anopheles mosquitoes were collected in a cross-sectional study design using Pyrethrum Spray catch (PSC) from 30 randomly selected houses per site between 2017 and 2020 during the dry and rainy seasons. Collections were conducted between 0630 and 1000hrs in the morning and transported to the Sub-Saharan Africa International Center of Excellence for Malaria Research (ICEMR), Homa Bay, Kenya. Samples were stored at -20 o C in 1.5 ml Eppendorf tubes containing silica gel and assigned a unique code for further molecular processing.

Larval Sampling
Larval sampling was conducted using 350 ml standard dippers and hand pipettes (31). A maximum of 10 dips was taken at each habitat and the presence or absence of larvae was recorded. To avoid collecting siblings from the same pool, larvae were randomly sampled from different breeding habitats. Collected larvae were labeled by habitat type and identi ed morphologically using the referenced keys (32). Only Anopheles larvae were sorted and transported to the ICEMR insectary. The larvae were reared to adults using standardized rearing methods (33). Emerged adults were anesthetized using chloroform and identi ed using the morphological key in the laboratory as described by Gillies and Coetzee to species (34,35).

Molecular Identi cation Of Mosquito Species
Genomic DNA was extracted from randomly selected single An. gambiae female adult using the Chelex resin (chelex® -100) method following a protocol by Musapa et al (36). Brie y, deionized water was added into single mosquito sample tubes and ground into a uniform suspension. Phosphate buffer saline 1X and 10% Saponin was then added to sample homogenates, mixed gently, and incubated at room temperature for 20 minutes. The suspension was then centrifuged and the supernatant discarded. The pellets were then resuspended in PBS 1X and centrifuged, supernatant discarded, and gently vortexed. The pellets were then suspended in sterile deionized water and 20% Chelex-resin suspension in deionized water. The samples were incubated at 85 o C for 10 minutes, centrifuged at 20,000 x g for a minute, and DNA transferred into prelabelled storage vials. Anopheles gambiae was identi ed to sibling species using polymerase chain reaction (PCR) as described by Scott et al. (37).
Genotyping and DNA sequencing of TEP1 alleles in Anopheles gambiae mosquitoes Genotyping of TEP1 alleles was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method as described by Gildenhard et al (25). Brie y, the initial PCR was conducted using Nest 1 primers -VB3 and VB4-targeting 892 base pairs, followed by a second PCR performed on 5µl of the resulting product from Nest 1 with Nest 2 primers VB1 and VB2 producing a nal fragment length of 758 base pairs. Both PCR reaction conditions were set as denaturation at 95°C for 3 minutes, 35 cycles of 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and a nal step at 72°C for 6 min using DreamTaq Green Master Mix (Thermo Fisher Scienti c). PCR products were digested by restriction enzymes Bam HI, Hind III, or Bse NI (New England Biolabs Inc) (S1 Table) according to the manufacturer's instructions and analyzed the result with 2.5% agarose gel electrophoresis. The TEP 1 allelic classes were then determined by fragment size of restriction enzyme digestion (S1 Table). A subset of samples with identi ed TEP1 alleles was further con rmed through sequencing of respective Nested II amplicons. Sequencing was done using 3700/3730 BigDye® Terminator v3.

Evolutionary Relationship Based On Tep1 Gene
The phylogenetic analysis of TEP1 sequences showed that alleles were clustered into susceptible and resistant groups with high bootstrap values, ranging from 72-100%. Out of the sequences retrieved from the gene bank, TEP1*S1 alleles identi ed in western Kenya have a common lineage with TEP1*S1 (AF291654) from Suakoko, Liberia. TEP1*S1 evolved as a result of a mutation on the mosquito strain G3 with TEP1*S3  (Table 2).
A deviation was observed among An. gambiae from Kisumu and An. arabiensis from Kakamega and Homa Bay which displayed slightly higher expected heterozygosity than observed signifying the presence of inbreeding among these populations (Table 2).    The AMOVA results revealed that 99% of the observed variations in allele frequency were among individuals within respective populations, and a 1% variation was observed among populations and within individuals (Table 4). These results show that the level of genetic differentiation among populations was very low.

Discussion
Plasmodium falciparum triggers an immune response in An. gambiae mosquitoes (42). Following infections with P. falciparum in An. gambiae, the midgut mounts speci c and nonspeci c immune responses to minimize epithelial damage (43) A key component of the immune system is the thioester containing protein 1 (TEP1) that displays allelic variations associated with distinct genotypes in their ability to eliminate Plasmodium parasites. Originally, TEP1 variations have been characterized into ve allelic classes including *R1, *R2, *R3 *S1, and *S2 (14,23). This study identi ed two alleles (TEP1*R2 and, TEP1*S1) in An. gambiae s.l from western Kenya. These alleles were characterized from western Kenya regions with different malaria endemicities. A high similarity index was observed among sequenced alleles that were initially identi ed by RFLP-PCR and sequences retrieved from NCBI. Consistent with previous reports, TEP1*R2 and TEP1*S1 were the most common identi ed alleles (25,44) circulating in western Kenya and did not display a de ned distribution in sampled regions implying that they are conserved and may represent ancestral alleles maintained over generations in time. Furthermore, why these alleles have been maintained in the local populations and their roles and signi cance in vector competence is still not clear (14,22,23,45).
Low TEP1*R allele frequencies observed in these malaria-endemic areas in our study sites may be a product of selective pressures in the TEP1 gene resulting in functional variations that select for susceptible mosquitoes to Plasmodium infection (12,44,46) as well as encourage evolutionary processes in the TEP1 loci (22

Conclusion
This study reveals minimal genetic differentiation and a low population structure in the highland and lowland regions of western Kenya with different malaria transmission patterns. TEP1*R2 and TEP1*S1 were the most common alleles across all regions indicating that An. gambiae and An. arabiensis may have had these speci c alleles before inhabiting new ecological niches. However, TEP1*R allele frequencies observed especially in the highlands may be used to estimate malaria prevalence as compared to the lowlands. Therefore, continued Availability of data and materials The data sets generated and analyzed during this study are available from the corresponding authors on reasonable requests

Con ict of interest
All authors declare that they have no con ict of interest.

Funding
The study was supported by grants from the National Institutes of Health (U19 AI129326, D43 TW001505, R01 AI050243, and R01 A1123074).