Vector Competence for Dengue-2 Virus and Genetic Diversity of Permethrin Resistant/ Susceptible Aedes Aegypti Sub-populations From Lamu County, Kenya

Vector Competence for Dengue-2 Virus and Genetic Diversity of Permethrin Resistant/ Susceptible Aedes Aegypti Sub-populations From Lamu County, Kenya | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Vector Competence for Dengue-2 Virus and Genetic Diversity of Permethrin Resistant/ Susceptible Aedes Aegypti Sub-populations From Lamu County, Kenya Kulthoom Suhailah Qureish, Francis Mulwa, Richard Odinga, Santos Yalwala, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8339335/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Dengue virus, primarily transmitted by Aedes aegypti mosquitoes, is endemic in Lamu County with the most recent outbreak in 2021. There is currently no definitive treatment for dengue; therefore, vector control remains the most effective method to curb transmission. Prolonged exposure of Ae. aegypti to insecticides may confer resistance. This study aimed to determine the levels of resistance to permethrin, a commonly used insecticide, and to compare the vectorial competence and genetic differences of resistant versus susceptible populations for dengue-2 virus. Methods Ae. aegypti mosquito eggs were collected from Lamu (Shela, Hindi, and Mokowe) using ovitraps and hatched in the insectary. The emerging adults were assayed for permethrin resistance/ susceptibility. WHO insecticide resistance assays (n = 145) were conducted utilizing 3,525 emerged mosquitoes; 1,575 each from Hindi and Mokowe, and 375 from Shela. The mosquitoes (F 2 ) were exposed to dengue-2 infectious bloodmeals and fully engorged mosquitoes selected, incubated for up to 21 days, and tested for infection and dissemination by plaque assay. Proportion test of differences was used to determine significant differences between sub-populations and incubation periods at 0.05 level of significance. Individual legs were dissected from 31 female (F 0 ) mosquitoes for isolation of genomic DNA. Polymerase Chain Reaction was used to amplify the target mitochondrial DNA using HCO2198 and LCO1490 primers. The amplicons were Sanger sequenced to determine genetic diversity. Results The Shela sub-population displayed lower resistance (1%) compared to the Hindi and Mokowe sub-populations at 53% and 62.4% respectively. Of the mosquitoes from Hindi (n = 60); Mokowe (n = 90); and Shela (n = 130), n = 14 (23%), n = 24 (27%), and n = 40 (31%) respectively, had midgut infections with dengue-2; while n = 12 (20%), n = 11 (12%), and n = 21 (16%) respectively had disseminated infection. Genetic analysis showed that all the samples were Ae. aegypti formosus sub-species. Haplotype diversity was high at 0.989 while nucleotide diversity was low at 0.01184. Conclusion These findings suggest that Ae. aegypti formosus sub-species is prevalent in Lamu County. The study also demonstrated high infection and dissemination rates among the Ae. aegypti sub- populations. This confirms the risk of DENV-2 transmission and highlights the need for regular entomological surveillance for early detection of virus and initiation of preventive strategies such as vector control. Ae. aegypti dengue-2 Lamu permethrin resistant susceptible vector competence Figures Figure 1 Figure 2 Figure 3 Figure 4 BACKGROUND Aedes aegypti mosquito is the primary vector for dengue virus ( 1 ). It is found in tropical, sub-tropical, and temperate regions such as sub-Saharan Africa, the South American continent and South Asian countries ( 2 ). Dengue fever is caused by four dengue virus serotypes: DENV-1, DENV-2, DENV-3 and DENV-4 ( 3 ). Dengue virus serotype 2 (DENV-2) is the most prevalent in Kenya, particularly along the coastal region ( 4 ). Globally, about 390 million people are infected by DENV annually ( 5 ) which has devastating effects both on public health and the economy. Currently, there is neither a specific drug treatment for dengue nor a suitable approved vaccine for the general population ( 6 ) leaving vector control as the only way to combat this disease. Different types of insecticides are used for vector control including organophosphates such as malathion and P-methyl which inhibit the enzyme acetylcholinesterase (AChE), resulting in overabundance of the neurotransmitter acetylcholine ( 7 ). Organochlorines such as dieldrin and dichloro-diphenyl-trichloroethane (DDT) interact with the neuron membrane and alter the transmission of the nerve impulses ( 8 ) while carbamates like bendiocarb interfere with acetylcholinesterase therefore disrupting the nervous system ( 9 ). These groups of insecticides are gradually being replaced by pyrethrins due to their significant and persistent toxic effects on humans and the environment ( 10 ). One such pyrethrin is permethrin, which the present study focuses on. Extensive use of insecticides and agricultural pesticides has led to development of resistance in many insects species ( 12 , 13 , 14 ). Permethrin resistance has been observed in some Ae. aegypti populations ( 14 ) probably due to the differences in their genetic makeup ( 15 ). The Ae. aegypti populations have shown varying degrees of resistance to pyrethroids, particularly permethrin ( 16 ). In Lamu County, located on the Kenyan coast, there have been outbreaks of arboviral diseases such as dengue and chikungunya ( 17 ), as recently as 2025. It is believed that this is due to ineffective vector control due to insecticide resistance among vector populations ( 18 ). Knockdown resistance (kdr), also known as altered target – site resistance ( 19 ) due to the mutation that alters the target- site, reduces or removes the toxic effect of the insecticide ( 20 ). These kdr mutations are passed down through generations. The mode of inheritance of insecticide resistance shows homozygosity of the resistance and susceptible genes involved ( 21 ). Some studies have shown that insecticide resistance may affect the vector competence of Ae. aegypti mosquitoes by increasing dissemination rates and altering their gut microbiota ( 15 , 25 ). Vector competence is the intrinsic ability of a vector to be infected by a pathogen, amplify, disseminate, and transmit it ( 23 ). This is affected by extrinsic and intrinsic factors ( 24 ). Extrinsic factors include temperature and gut microbiota; higher temperatures increase susceptibility of mosquitoes to viruses ( 25 ). The development of many pathogens is temperature sensitive with higher temperatures decreasing the extrinsic incubation period for the viruses ( 26 ). Microbiota found in the gut of insect vectors also decrease viral and parasitic infections in mosquito vectors by activating immune responses or directly inhibiting pathogen development ( 27 ). Internal factors include the immune responses and barriers such as: midgut infection barrier (MIB) which prevents the virus from attaching to the receptors present on midgut epithelial cells; midgut escape barrier (MEB) which prevents the release of viral particles from midgut epithelial cells; salivary gland infection barrier (SIB) which prevents the virus from infecting acinar cells; and salivary gland escape barrier (SEB) which prevents releasing of viral particles from salivary gland acinar cells ( 28 ). To determine the genetic differences between sub-populations, the Cytochrome c oxidase subunit 1 (CO1) gene has proven very useful ( 29 ). The CO1 gene is considered ideal for barcoding due to its presence in most living eukaryotes ( 30 ). There have been recurring outbreaks of dengue, mostly associated with DENV-2, in most of coastal Kenya since 2011, with limited data on insecticide resistance of Ae. aegypti in Kenya, and how this impacts arbovirus transmission. Therefore, this study aimed to investigate the insecticide resistance among Ae. aegypti sub-populations in Lamu County and its effect on vector ability to acquire, amplify and transmit DENV-2, as well as entomological factors that contribute to dengue transmission. The findings of this study will help to understand the epidemiology of dengue in the county. MATERIALS AND METHODS Study sites This study was conducted in Lamu County, along the Kenyan Coast (Fig. 1 ) where three sites were selected for sample collection: Shela, Hindi, and Mokowe. Lamu County (latitude 2.2696° S, longitude 40.9006° E) generally experiences a warm, humid tropical climate with temperatures ranging from 24°C to 30°C throughout the year making it conducive for mosquito breeding. The county has a bimodal pattern of rainfall with the long rains occurring between April and July, with the highest rainfall occurring in the month of May and short rains in November and December. Collection and identification of mosquito samples Adult mosquitoes were sampled using BG-Sentinel 2 Traps and CDC light traps baited with carbon dioxide (CO 2 ). Mosquito eggs were collected using ovitraps that consisted of black ovicups lined with oviposition paper and half-filled with water. The mosquitoes were collected during periods of higher population densities which usually coincide with rainy seasons (April – June). The traps were set between 6.30pm and 7:00pm in the evening and collected the following morning between 6:00am and 6.30am. The trapped mosquitoes were knocked down using and triethylamine transported to the KEMRI laboratories for morphological identification to species level using taxonomic keys (Edwards 1941 and Peter G. Jupp 1986). Genetic diversity determination DNA extraction Field collected (n = 31) female mosquitoes (F 0 ) were sampled from various collection sites in Shela, Hindi, and Mokowe. Each mosquito was from a different site to avoid analyzing siblings. Individual mosquito legs were dissected using sterile forceps and placed in a sterile RNase/DNase-free Eppendorf tube containing a sterile copper bead, 200ml of Phosphate buffered saline was added into each Eppendorf tube, and the sample homogenized in an Omni Bead Rapture 24 (OMNI International) set to run at the speed of 2.10 mm for 20 seconds. Qiagen™ DNeasy Blood and Tissue Extraction Kit ( 31 ) was then used to extract DNA from the homogenates following the manufacturers directives. Polymerase Chain Reaction Polymerase Chain Reaction (PCR) was used to amplify the target mitochondrial DNA (COI) in mosquito samples using the universal reverse primer HCO2198 and forward primer LCO1490. The PCR master mix was prepared in a Biosafety level II molecular laboratory and contained 12.5 µl Amplitaq solution, 1 µl forward primer, 1 µl reverse primer, and 7.5 µl sterile nuclease free water. Next, 22µl of the master mix was transferred into well-labelled RNase-free, 0.2 ml (8-strip format, Invitrogen) PCR tubes and 3 µl of sample DNA added into each tube. The tubes were tightly sealed, vortexed, and placed in a Thermocycler set to run 35 reaction cycles of 94°C for 30 secs, 55°C for 30s, and 72°C for 60s. The amplicons were visualized in a 2% agarose gel with an expected band size of approximately 650 bp, stained with SYBR Green. The amplicons underwent clean up to remove leftover primers and deoxyribonucleotide triphosphates (dNTPs) from PCR. This was done using the Exo-CIP™ Rapid PCR Cleanup protocol ( 32 ). The products were then quantified to determine the concentration of DNA using a nanodrop spectrophotometer and Sanger sequenced ( 33 ). Bioassays for determination of susceptibility and resistance of Ae. aegypti to permethrin Ae. aegypti mosquitoes were subjected to World Health Organisation (WHO) tube bioassays for susceptibility to permethrin ( 34 ). Twenty-five mosquitoes were aspirated at a time, from each site and transferred into a holding tube to ensure that damaged specimens were not included in the test. The holding tubes were set upright, mesh-screen end uppermost for approximately 30 minutes after which, any mosquitoes that may have been damaged during aspiration were removed. A sheet of paper impregnated with 0.75% permethrin insecticide was introduced into each of the exposure tubes then rolled into a cylinder and fastened into position with a copper clip. The mosquitoes were introduced into the exposure tube by attaching it to a vacant screw-top in the slide. The mosquitoes were gently blown down into the exposure tube. The slide was closed and the holding tube detached. The exposure tubes were left standing upright with mesh-screen end uppermost for 1 hour. The behavior of the mosquitoes was monitored and the number of those knocked down recorded after every 10 minutes for 1 hour. A mosquito was considered to be knocked down if it was unable to stand or fly in a coordinated way. At the end of the 1hour exposure period, the mosquitoes were transferred to the holding tubes set on the slide and a pad of cotton wool soaked in sugar solution placed on the mesh-screen. The holding tubes were kept for 24 hours (recovery period) in a secluded, shady place where the temperatures did not exceed 30˚C. At the end of recovery period, (24-hour post-exposure), the number of dead mosquitoes was counted and recorded. An adult mosquito is considered to be alive if it is able to fly regardless of the number of legs remaining. Affected specimens that are unable to walk should be considered as dead. Calculation of mortality rates After 24-hour post-exposure, the number of dead mosquitoes in the holding tube was counted. The mortality is calculated by summing the number of dead mosquitoes per site (i.e., out of a total of 100 mosquitoes from four replicates of 25 mosquitoes each per site) expressed this as a percentage of the number of exposed mosquitoes using the formula: Vector competence for dengue-2 determination Mosquito rearing Mosquitoes were reared in the KEMRI insectary, maintained at a temperature of 28℃ − 32℃, 70% − 80% relative humidity (RH) and 12:12 hour light: dark (L: D) photoperiod. The batches of F 1 eggs were dispensed in water on larval trays for hatching in a level 2 insectary. The larvae were fed on Tetramin® fish food until they pupated, then pupae were collected every morning and put in a holding cup containing water. The cups with pupae were placed in a plastic cage with a netting material on top and allowed to develop into adults. The emerging adults were inactivated by placing them in a -20°C freezer for one minute, then morphologically identified under a dissecting microscope using taxonomic keys of Edwards ( 35 ) and Jupp ( 36 ) to ensure they are Ae. aegypti mosquitoes. The mosquitoes were fed on a diet of 10% glucose solution for 3 days. On the fifth day, they were fed on mice after being starved for 12 hours. Albino mice were anaesthetized using Pentobarbital sodium injection at a dosage of 75 mg/kg ( 37 ). The mice were injected intraperitoneally then placed in mosquito cages for 30 minutes. Ovicups half-filled with water and lined with ovipapers were placed in the cages for the mosquitoes to lay F 2 eggs which were then hatched and reared into adulthood. Dengue virus amplification and quantification Live preserved viral isolates (DENV-2 008/01/2012) from Mombasa County were amplified in a confluent monolayer of Vero E6 cells in a T-25 flask containing maintenance media and incubated at 37℃ for 7 days. Upon showing signs of cytopathic effect (CPE), the virus was harvested, aliquoted into cryovials and stored at -80℃ for further use. Quantification of dengue virus was performed by plaque assay, i.e. tenfold serial dilutions of the amplified DENV ( 38 ). 900µl of maintenance media were added into nine Eppendorf tubes, 100µl of the amplified DENV-2 were transferred to the first Eppendorf tube and pipette mixed with the media, then 100µl of the mixture was picked and transferred to the next Eppendorf tube and pipette mixed. This step was repeated until the virus had been diluted ten times. The first well of a 12-well plate was inoculated with 100µl of virus over the confluent monolayers of Vero E6 cells, with 100µl of the subsequent dilutions inoculated into the other wells, leaving two wells for the negative controls. The plate was incubated for 1 hour at 37℃ with rocking every 15 minutes. Methylcellulose was added onto the inoculated cells and the plate observed over the next 14 days. The plate was then fixed for 2 hours with 10% formalin then stained overnight with 0.5% crystal violet. The plaques were counted and calculated to quantify the virus using the formula ( 39 ): where d is the dilution factor and V is the volume of diluted virus added to the well. Preparation of blood and virus mixture (infectious bloodmeal) Sheep blood was acquired from a farm in Kabete, collected into 50ml falcon tubes and transported to the laboratory. Cryovials of the amplified and quantified DENV-2 virus were thawed on ice. Two mice were sacrificed by injecting them intraperitoneally with Ketamine Hydrochloride at a dosage of 150mg/kg. The mice were skinned carefully with the fatty tissues being removed from the skin. The skins were fitted onto Hemotek columns and secured with bands. The DENV-2 was mixed with the blood by pipetting in a ratio of 1:1. The mixture was then transferred to the Hemotek feeding column. Mosquito exposure to infectious bloodmeal Three experimental replicates were carried out in which mosquitoes (n = 280) were starved of glucose for 12 hours prior to exposure to DENV-2 infectious bloodmeal. They were allowed to feed for 45 minutes. Fully engorged mosquitoes were aspirated into separate cages, counted and incubated for 7, 14 and 21 days post exposure (dpe). They were later fed on 10% glucose solution ad libitum. The three periods of extrinsic incubation (7, 14 and 21 dpe) were chosen to represent early, mid and late phases of virus dissemination kinetics ( 40 ). Three vector competence indices: (i) the proportion of mosquitoes exposed to infectious bloodmeal that developed midgut infection, (ii) the proportion of mosquitoes with midgut infection that developed a disseminated infection and (iii) the infectious titre of disseminated virus. The last two indices represent two successive aspects of the infection process in mosquitoes that lead to their ability to transmit the virus. Dissection of mosquitoes After 7, 14 and 21 dpe, a third of the mosquitoes were sampled from the cage into a container and cold-anaesthetized at -20℃ for 45 seconds and maintained on wet ice (+ 4℃). Scalpels were used to pick the mosquitoes by their wings and to hold them in place while separating the legs from the body. The dissected bodies and legs were placed into separate 1.5 mL microfuge tubes (Eppendorf) containing 500 µl and 200µl respectively, of homogenization media (HM), made of MEM, supplemented with 15% FBS, 2% L-glutamine, and 2% antibiotic/antimycotic, then stored in -80℃ freezer to await testing. Test for infection and dissemination rates Mosquito bodies were homogenized using a mini bead beater (BioSpec Products Inc, Bartlesville, OK 74005 USA) with the aid of a copper bead (BB-caliber airgun shot). They were centrifuged at 8000rpm for 10 minutes at 4℃. The supernatants were then inoculated onto a 70% confluent monolayer of Vero cells on 24- well plates. One hundred microliters of the abdominal homogenates were added to each of ten wells of the 12-well plate to infect the cells with the remaining two wells used for negative control. The plates were incubated at 37℃ in a 5% CO 2 incubator with frequent agitation after every 15 minutes for 1 hour to allow for virus adsorption. The infected cell monolayers were then overlaid with 1ml of maintenance media. The plates were incubated at 37℃ and 5% carbon dioxide (CO2) for 14 days and observed for signs of cytopathic effect (CPE). On observation of CPE, the plates were fixed for 1 hour with 10% formalin, and stained for 2 hours with 0.5% crystal violet, washed on running tap water, dried overnight and the plaques observed on a light box. The DENV-2 positive bodies were used to determine the infection rates. The legs of the corresponding mosquito bodies that showed signs of CPE underwent the same procedure. Plaques were counted and calculated to determine the viral titer. If the virus was detected in the mosquito’s body but not in the legs, the mosquito was considered to have a non-disseminated infection, limited to the midgut. Detection of virus in the body and legs was considered evidence of successful infection and dissemination, respectively ( 41 ). Data management and analysis Genetic diversity analysis The raw sequences were viewed using Chromas v2.6.6 (Technelysium), edited by trimming and deleting poor-quality sections and converted into FASTA format sequences. Basic Local Alignment Search Tool (BLAST) was used to compare the sequences against the GenBank database to confirm species identity. The sequences were aligned using Molecular Evolutionary Genetics Analysis (MEGA) software via the Muscle algorithm. For genetic diversity analysis, DnaSP v6.12 was used to determine nucleotide variation within the populations and provide information on nucleotide diversity (π), the number of haplotypes (H), and haplotype diversity (Hd). Neutrality tests (Tajima’s and Fu and Li’s tests) were also conducted. The aligned sequences were used to carry out phylogenetic analysis. Sequences belonging to Ae. aegypti aegypti (AF390098, AY432106), Ae. aegypti formosus (AY056597) and Ae. albopictus (MF148303) were downloaded from GenBank and used as references. The combined sequences were aligned in MEGA and used to infer a maximum likelihood phylogeny in IQTREE. The phylogenetic tree was visualized in FigTree v1.4.4. Vector competence Data was stored in Microsoft Excel spreadsheet and checked for errors and inconsistencies prior to analysis. Key rates were defined as follows: i) Infection rate (IR) which was determined as the proportion of mosquitoes whose bodies were infected with dengue virus, and ii) Dissemination rate (DR) as the proportion of mosquitoes whose legs were infected with dengue virus. The proportions (IR and DR) were calculated and the 95% confidence intervals (CIs) determined using binomial regression model. Proportion test of differences was used to determine significant differences between sites and incubation periods, and the associated p-values determined at 0.05 level of significance. Further, a comparative bar graph was used to compare the rates between the three sites. All the analyses were carried out using STATA version 15.1. All visualizations were done in RStudio version 2023.06.1. RESULTS Genetic diversity There was a high level of genetic diversity between Hindi, Mokowe and Shela towns of Lamu County. Overall, 14 sequences of Ae. aegypti mosquitoes showed 14 unique haplotypes with a high haplotype diversity (Hd) of 1.0 (Hd > 0.5). For the individual sites: Hindi sub-population showed 6 haplotypes with Hd of 1.0; Mokowe sub-population showed 6 haplotypes with Hd of 1.0; and Shela sub-population showed 2 haplotypes with Hd of 1.0. Overall, nucleotide diversity (π) was low (π < 0.5) at 0.01184 with 0.00982 observed for Hindi sub-population and 0.01266 for Mokowe (Table 1 ). However, this could not be calculated for the Shela sub-population due to the limited number (n = 2) of sequences against the required minimum of three. Neutrality analysis Tajima’s D and Fu’s F neutrality tests were carried out to analyse evolutionary history of the Ae. aegypti sub-populations. Tajima’s D values were negative overall (-1.09398), in Hindi (-0.65366), and Mokowe (-0.76939). Fu’s F values were also negative: -1.734 in Hindi, -1.298 in Mokowe, and − 5.526 for the combined sub-populations (Table 1 ). These observations align with Tajima’s D test. Table 1 Genetic diversity of Ae. aegypti mosquitoes from Lamu County. Sites n Hn Hd π D Fu’s F Hindi 6 6 1.0 0.00982 -0.65366 -1.734 Mokowe 6 6 1.0 0.01266 -0.76939 -1.298 Shela 2 2 1.0 - - - Combined 14 14 1.0 0.01184 -1.09398 -5.526 n = Number of sequences, Hn = Number of haplotypes, Hd = Haplotype diversity, π = Nucleotide diversity, D = Tajima’s D, Fu’s F = Fu’s selection test. Phylogenetic analysis Phylogenetic analysis of the mosquito sequences showed that all the Ae. aegypti sub-populations were Ae. aegypti formosus (Aaf) (Fig. 2 ). Bioassay for determining susceptibility or resistance of Ae. aegypti to permethrin The Ae. aegypti mosquitoes were divided into two categories: susceptible (≥ 98% mortality), and resistant (< 90% mortality). The Hindi sub-population showed a mortality rate of 46.98% (47%) which is 53% resistance against permethrin; Mokowe sub-population showed a mortality rate of 37.59% (37.6%) which is 62.4% resistance; and the Shela sub-population showed a mortality rate of 98.93% (99%) which is a 1% resistance. The Shela sub-population displayed significantly lower resistance (1%) compared to the Hindi and Mokowe sub-populations at 53% and 62.4%, respectively (Chi-square = χ² = 458.47, df = 2, p < 0.001; pairwise comparisons, p < 0.05 for all pairs). Quantification of dengue virus The amplified DENV-2 virus had a titre of 6.7 X 10 4 Pfu/ml while the infectious blood meal’s titre was 6.3 X 10 4 Pfu/ml. These two titres were fairly comparable. Vector competence A total of 280 (1st replicate [n = 90], 2nd replicate [n = 100], 3rd replicate [n = 90]) Ae. aegypti mosquitoes were analyzed for vector competence. Infection and dissemination rates for DENV-2 were calculated. 130 (1st replicate [n = 35], 2nd replicate [n = 50], 3rd replicate [n = 45]) were obtained from Shela, while 90 (1st replicate [n = 30], 2nd replicate [n = 30], 3rd replicate [n = 30]) were from Mokowe; and 60 (1st replicate [n = 25], 2nd replicate [n = 20], 3rd replicate [n = 15]) from Hindi sites, respectively. Infection rate The overall infection rate of all three sites was 27.9% (95% CI: 23.0-33.6) and increased with the incubation period from day 7 (5.6%; 95% CI: 2.4–13.0), day 14 (17.0%; 95% CI: 11.0-26.2) and peaking at day 21 (62.2%; 95% CI: 53.0-73.1). The proportion test of difference revealed that the infection rates significantly increased by 56.6% at day 21 (Diff = 0.566, p < 0.001) and by 11.4% at day 14 (Diff = 0.114, p = 0.014), compared to day 7 (Tables 2 and 3 ). Infection rates were highest in Shela 30.8% (95% CI: 23.8–39.8), followed by Mokowe 26.7% (95% CI: 18.9–37.6) and least in Hindi 23.3% (95% CI: 14.7–36.9). On day 7, the infection rate was 8.0% (95% CI: 2.1–30.2), 6.7% (95% CI: 1.7–25.4), and 2.9% (95% CI: 4.1–19.7) in Hindi, Mokowe, and Shela sites respectively. At day 14, the infection rates increased to 10.0% (95% CI: 2.7–37.2), 26.7% (95% CI: 14.7–48.3), and 14.0% (95% CI: 7.0-27.8), in Hindi, Mokowe, and Shela sites respectively. At day 21, the infection rates further increased to 66.7% (95% CI: 46.6–95.3), 46.7% (95% CI: 31.8–68.4), and 71.1% (95% CI: 59.0-89.7) in Hindi, Mokowe and Shela sites respectively. A proportion test of difference for each site indicated that the infection rates significantly increased by 59.0% (Diff = 0.59, p < 0.001), 40.0% (Diff = 0.4, p < 0.001) and 68.0% (Diff = 0.68, p < 0.001) in Hindi, Mokowe and Shela sites respectively at day 21 compared to day 7. On day 14, only Mokowe site showed a significant increase in the infection rate of 20.0% (Diff = 0.2, p = 0.039) compared to day 7 (Tables 2 , 3 , and Fig. 3 ). Of the mosquitoes that were exposed to DENV-2, Hindi (n = 60); Mokowe (n = 90); and Shela (n = 130), n = 14 (23%), n = 24 (27%) and n = 40 (31%) from Hindi, Mokowe and Shela respectively, had midgut infections. However, the observed differences were not significant: (Chi-square χ² = 1.22, df = 2, p = 0.542). Table 2 Comparison of the infection and dissemination rates in the three study sites. Proportion of mosquitoes infected with Dengue virus % (95%CI); n = 280 Body (Infection) Leg (Dissemination) Overall 27.9 (23.0-33.6); n = 78 15.7 (12.0-20.6); n = 44 Site Hindi (n = 60) 23.3 (14.7–36.9); n = 14 20.0 (12.1–33.2); n = 12 Mokowe (n = 90) 26.7 (18.9–37.6); n = 24 12.2 (7.0-21.3); n = 11 Shela (n = 130) 30.8 (23.8–39.8); n = 40 16.2 (10.9–23.9); n = 21 Proportion test of the difference between Hindi and Mokowe (diff \(\:,\:p\:value\) ) Diff=-0.034, p = 0.639 Diff = 0.078, p = 0.194 Proportion test of the difference between Hindi and Shela (diff \(\:,\:p\:value\) ) Diff=-0.075, p = 0.287 Diff = 0.038, p = 0.521 Proportion test of the difference between Mokowe and Shela (diff \(\:,\:p\:value\) ) Diff=-0.041, p = 0.511 Diff=-0.04, p = 0.408 Table 3 Comparison of infection and dissemination rates in the three study sites by incubation periods. Site Day Body (Infection) Leg (Dissemination) Overall Day 7 5.6 (2.4–13.0); n = 5/90 4.4 (1.7–11.6); n = 4/90 Day 14 17.0 (11.0-26.2); n = 17/100 10.0 (5.6–18.0); n = 10/100 Day 21 62.2 (53.0-73.1); n = 56/90 33.3 (24.9–44.6); n = 30/90 Proportion test of difference at day 14 (diff \(\:,\:p\:value\) ) Diff=-0.114, p = 0.014* Diff=-0.056, p = 0.140 Proportion test of difference at day 21 (diff \(\:,\:p\:value\) ) Diff=-0.566, p < 0.001* Diff=-0.289, p < 0.001* Hindi Day 7 8.0 (2.1–30.2); n = 2/25 8.0 (2.1–30.2); n = 2/25 Day 14 10.0 (2.7–37.2); n = 2/20 5.0 (0.7–33.8); n = 1/20 Day 21 66.7 (46.6–95.3); n = 10/15 60.0 (39.7–90.7); 9/15 Proportion test of difference at day 14 (diff \(\:,\:p\:value\) ) Diff=-0.02, p = 0.815 Diff = 0.03, p = 0.689 Proportion test of difference at day 21 (diff \(\:,\:p\:value\) ) Diff=-0.59, p < 0.001* Diff=-0.52, p < 0.001* Mokowe Day 7 6.7 (1.7–25.4); n = 2/30 3.3 (0.4–22.9); n = 1/30 Day 14 26.7 (14.7–48.3); n = 8/30 13.3 (5.4–33.2); n = 4/30 Day 21 46.7 (31.8–68.4); 14/30 20.0 (9.8–40.9); n = 6/30 Proportion test of difference at day 14 (diff \(\:,\:p\:value\) ) Diff=-0.2, p = 0.039* Diff=-0.1, p = 0.153 Proportion test of difference at day 21 (diff \(\:,\:p\:value\) ) Diff=-0.4, p < 0.001* Diff=-0.17, p = 0.039* Shela Day 7 2.9 (4.1–19.7); n = 1/35 2.9 (0.4–19.7); n = 1/35 Day 14 14.0 (7.0-27.8); n = 7/50 10.0 (4.4–23.0); n = 5/50 Day 21 71.1 (59.0-89.7); n = 32/45 33.3 (22.1–50.4); n = 15/45 Proportion test of difference at day 14 (diff \(\:,\:p\:value\) ) Diff=-0.11, p = 0.088 Diff=-0.07, p = 0.217 Proportion test of difference at day 21 (diff \(\:,\:p\:value\) ) Diff=-0.68, p < 0.001* Diff=-0.3, p < 0.001* Dissemination rate Overall, results showed mosquito dissemination rate of 15.7% (95% CI: 12.0-20.6). The overall dissemination rate increased with the incubation period, the lowest rate observed on day 7 (4.4%, 95% CI: 1.7–11.6), increasing to 10.0% (95% CI: 5.6–18.0) on day 14 and peaking on day 21 (33.3%, 95% CI: 24.9–44.6) post exposure. A proportion test of difference revealed that the dissemination rates significantly increased by 28.9% at day 21 (Diff = 0.289, p < 0.001), while no significant increase was observed at day 14. By sites, the mosquito dissemination rate was highest in Hindi at 20.0% (95% CI: 12.1–33.2), followed by Shela at 16.2% (95% CI: 10.9–23.9) and the least in Mokowe at 12.2% (95% CI: 7.0-21.3). The dissemination rates increased consistently with the incubation period in two sites. On day 7, the dissemination rate was 8.0% (95% CI: 2.1–30.2), 3.3% (95% CI: 0.4–22.9), 2.9% (95% CI: 4.1–19.7) in Hindi, Mokowe and Shela sites respectively. On day 14, the dissemination rates increased to 13.3% (95% CI: 5.4–33.2) and 10.0% (95% CI: 4.4–23.0) in Mokowe and Shela sites respectively while in Hindi site, the dissemination rate decreased to 5.0% (95% CI: 0.7–33.8). At day 21, dissemination rates increased to 60.0% (95% CI: 39.7–90.7), 33.3% (95% CI: 22.1–50.4), and 20.0% (95% CI: 9.8–40.9) in Hindi, Shela, and Mokowe sites respectively. A proportion test of difference for each site indicated that the dissemination rates significantly increased by 52.0% (Diff = 0.52, p < 0.001), 30.0% (Diff = 0.30, p < 0.001), and 17.0% (Diff = 0.17, p = 0.039) in Hindi, Shela, and Mokowe sites respectively at day 21 compared to day 7. There was no significant increase in dissemination rates in any of the sites at day 14 compared to day 7 (Tables 2 , 3 , and Fig. 4 ). Of the mosquitoes, from Hindi (n = 60); Mokowe (n = 90); and Shela (n = 130) that were exposed to DENV-2, n = 12 (20%), n = 11 (12%), and n = 21 (16%) respectively had disseminated infections. The observed differences were not significant: (Chi-square χ² = 1.68, df = 2, p = 0.432). Dissemination was used as proxy for transmission rates as saliva collection via capillary method tends to underestimate virus transmission. The results for the transmission rate were therefore the same as those of the dissemination rate. The plaque assay performed on the positive mosquito bodies and legs confirmed the presence of live viral particles. The mean body and leg titres for all three sites on 7, 14 and 21 dpe are visualized on Tables 4 and 5 . Table 4 Mean DENV-2 titres (Pfu/ml) in mosquito bodies for all three sites 7, 14 and 21 dpe Site Day 7 Day 14 Day 21 Hindi 3.2 X 10 4 3.2 X 10 4 3.8 X 10 4 Mokowe 3.2 X 10 4 3.3 X 10 4 3.5 X 10 4 Shela 3.0 X 10 4 3.1 X 10 4 3.8 X 10 4 Table 5 Mean DENV-2 titres (Pfu/ml) in mosquito legs for all three sites 7, 14 and 21 dpe Site Day 7 Day 14 Day 21 Hindi 2.9 X 10 4 2.9 X 10 4 3.2 X 10 4 Mokowe 2.7 X 10 4 2.9 X 10 4 2.9 X 10 4 Shela 2.5 X 10 4 2.6 X 10 4 2.9 X 10 4 DISCUSSION The genetic diversity analysis carried out in this study has informed of high haplotype diversity for the individual sub-populations from different sites in Lamu County (1.0 for Shela, Hindi and Mokowe), as well as the combined sub-populations (0.989). The nucleotide diversity observed was 0.01184 for the combined sub-populations. According to Grant and Bowen (1998), values greater than 0.5 are considered high while those lesser than 0.5 are considered low when classifying the genetic diversity of populations ( 41 ). The findings of this study therefore fall within the high haplotype diversity (Hd) and low nucleotide diversity (π) classification which indicate a recent population growth from a small one with few, related ancestors ( 42 ). The new individuals inherited the existing haplotypes in increasing numbers, leading to high haplotype diversity ( 43 ). Gene flow from populations that are genetically close to the population they are flowing to can also cause an increase in haplotype diversity ( 44 ). This increase happened in a short time, however, and hindered the mutation rate and divergence of the haplotypes at nucleotide level, leading to low nucleotide diversity ( 45 ). Two neutrality tests were also carried out: Tajima’s D and Fu’s F tests. Tajima’s D test determines whether a DNA sequence has evolved randomly (neutrally) or non-randomly (selectively). It is done by getting the difference between the mean of pairwise differences and the number of segregating sites ( 46 ). The two values are expected to be the same in a population that has evolved neutrally and maintained a constant size. Deviation from this neutrality and the difference in the values informs us of the population’s history. A negative Tajima’s D value indicates an abundance of rare alleles, most likely caused by an expanding population after a recent bottleneck and selective sweep ( 47 ). Fu’s F test works hand in hand with Tajima’s D test. It also tests for deviation from neutrality by investigating non-random recombination. It is determined by calculating the distribution of alleles and haplotypes in a DNA sequence ( 48 ). Negative Fu’s F values also indicate a recent population expansion and positive selection ( 49 ). The evolutionary history was inferred using the Maximum Likelihood method and Tamura-Nei model ( 50 ). The tree with the highest log likelihood (-3082.37) was shown. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model and then selecting the topology with superior log likelihood value. The evolutionary analyses were determined in MEGA11 ( 51 ). The phylogenetic analysis revealed that all the samples belonged to the Ae. aegypti formosus (Aaf) sub-species which is native to Kenya ( 52 ). Ae. aegypti formosus is primarily sylvan (forest- dwelling mosquito), though it has spread into urban areas ( 53 ) and become domesticated ( 54 ). Ae. aegypti formosus is also a known zoophilic mosquito, although this may be changing with its spread into areas densely-populated by humans ( 55 ). This adaptability is a cause for concern as it was previously considered less of a threat than its counterpart Ae. aegypti aegypti . Ae. aegypti mosquitoes from Shela were 99% susceptible to permethrin insecticide while the Hindi and Mokowe populations were 53% and 62.4% resistant, respectively. This suggests that permethrin is an effective insecticide in Shela, but ineffective in controlling Ae. aegypti populations in Hindi and Mokowe. Therefore, these areas need a more effective product to replace permethrin in the control of Ae. aegypti vectors. The midgut infection rate (MIR) for all three sub-populations gradually increased over the three-week duration indicating that the midgut infection barrier (MIB) gradually weakened to allow for infection of DENV-2. For instance, the Shela mosquito sub-population which was 99% susceptible to permethrin, had a MIR of only 3% at 7dpe, suggesting a strong MIB at this point. However, this increased to 14% at 14 dpe and > 71% at 21 dpe. This gradual increase in MIR was observed in all the sub-populations, with the highly resistant Mokowe having a MIR of 7% and the moderately resistant Hindi having 8% at 7 dpe; increasing to 27% and 10% at 14 dpe and then > 46% and > 66% at 21 dpe for Mokowe and Hindi sub-populations respectively. The MIB had greatly been overcome by the virus at this point with the results showing an overall increase in viral titre over the incubation period. This suggests that life expectancy of an infected mosquito vector is important for transmission of DENV-2 to occur, since the longer it lives the more the MIB is weakened. There was no significant difference in infection rates between the different populations (Chi-square χ² = 1.22, df = 2, p = 0.542) suggesting that susceptibility/ resistance of Ae. aegypti to permethrin has no effect on mosquito susceptibility to DENV-2 infection. The dissemination rate (DIR) 7dpe for Hindi and Shela sub-populations was 100%, and 50% for Mokowe. This indicates that the midgut escape barrier (MEB) was very vulnerable to virus penetration which was therefore easily released from the midgut epithelial cells. At 14dpe, the DIR for both Hindi and Mokowe was 50% while that for Shela was 71%. The MEB was therefore more penetrable for the susceptible mosquito populations. The DIR greatly increased for all three sub-populations 21dpe: 90%, 42.9% and 46.9% for Hindi, Mokowe and Shela respectively. The MEB seems to be unaffected by permethrin resistance or susceptibility as it varied indiscriminately among the three sub-populations and the days post exposure. Dissemination was used as proxy for transmission. Saliva collection via capillary method requires forced salivation which tends to underestimate virus transmission rates. The transmission rates in a natural setting are more likely to be higher than in a laboratory setting. Dissemination is considered adequate in predicting the vector’s transmission abilities ( 56 ). The p-values for the infection rate (p = 0.542) and the dissemination rate (p = 0.432) for the three sites; as well as those of the proportion tests of difference between the three sites when compared to each other (p = 0.639, p = 0.287, p = 0.511); were above the 0.05 level of significance. This is statistically not significant and leads to the conclusion that there is no relationship between permethrin resistance/ susceptibility and vector competence. CONCLUSION The analysis showed that Ae. aegypti mosquito sub-populations from Lamu County have high infection and dissemination rates with dengue virus. Although the rates increased significantly with the incubation periods, they varied insignificantly by mosquito collection sites. The genetic diversity tests confirmed the presence of Ae. aegypti formosus in Lamu County. The negative Tajima’s D and Fu’s F values combined with the high haplotype diversity and low nucleotide diversity indicate a recent population expansion with a selective sweep. The spread of Ae. aegypti formosus into urban areas most likely contributed to the expansion. This elevates the risk of transmission, and possibly more outbreaks, of diseases such as dengue fever. Even though Ae. aegypti formosus is considered a weaker vector of arboviruses, its changing habits such as moving into urban areas makes it crucial for regular entomological surveillance so as to monitor it extensively and to implement interventions aimed at controlling the spread of dengue virus in the coastal region of Kenya with the goal of preventing more outbreaks from occurring. Abbreviations Ae. : Aedes ; CO1: Cytochrome Oxidase 1; CO 2 : Carbon dioxide; CPE: Cytopathic effect; DENV: Dengue virus; DIR: Dissemination rate; MIB: Midgut infection barrier; MEB: Midgut escape barrier. Hd: Haplotype diversity; mtDNA: mitochondrial DNA; PCR: Polymerase Chain Reaction; SIB: salivary gland infection barrier; SEB: salivary gland escape barrier. Declarations Ethics approval This study was approved by the Scientific Ethics Review Unit (SERU) of the Kenya Medical Research Institute (KEMRI) under approval number, SERU 4768. Approval to use mice in the study was granted via the Animal Care and Use Committee (ACUC) (KEMRI/ACUC/02.09.23). Informed consent was not required owing to this study not involving human participants. Consent for publication Not applicable Availability of data and materials All data generated or analysed during this study are included in this article. Competing interests The authors declare that they have no competing interests. Funding This work was funded by the Armed Forces Health Surveillance Branch (AFHSB) and its Global Emerging Infections Surveillance (GEIS) Section, FY2022 ProMIS ID: P0116_22_KY and FY2023 ProMIS ID P0094_23_KY. The funders had a role in validation of the project, administration and decision to publish. This Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presenta­tion and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Authors' contributions KS did data curation, investigation, methodology, analysis, and writing and editing of this study. FM facilitated the methodology, visualization and review of the write up. RO assisted with data curation. SY also assisted with data curation and validation. JL took part in the methodology of this study, project administration, review and editing of the write up and supervision of the study. JB supervised the study. GK and JE handled funding acquisition and validation of the study. 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Olson MF, Ndeffo-Mbah ML, Juarez JG, Garcia-Luna S, Martin E, Borucki MK, et al. High rate of non-human feeding by aedes aegypti reduces zika virus transmission in South Texas. Viruses. 2020;12(4):1–20. Gloria-Soria A, Brackney DE, Armstrong PM. Saliva collection via capillary method may underestimate arboviral transmission by mosquitoes. Parasit Vectors. 2022;15(1):103. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8339335","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":559058152,"identity":"a0389cd3-547a-4a65-91d6-f49583969d0a","order_by":0,"name":"Kulthoom Suhailah Qureish","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACCQbmBmYIk4fxAYjkI6yFsbEZqoXZAESykaKFTQJEEdQiOSOx/XFBhU20/IzcY5Vfc+xk2BiYHz66gUeLtERiY/OMM2m5G27kpd2W3ZYMdBibsXEOHi1yIC28bYdzN0jkmN2W3MYM1MLDJk2UlvkzcsyKJbfVE9YiDdPScCPHjPHjtsOEtUj2PGyczQPyy5k3xtKM247zsDET8IvE8eQDn3kqbHLnt+cYfvy5rdqen7354WN8WlAAMw+YJFY5CDD+IEX1KBgFo2AUjBgAAPyhRfPR3J0hAAAAAElFTkSuQmCC","orcid":"","institution":"Walter Reed Army Institute of Research-Africa/Kenya Medical Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Kulthoom","middleName":"Suhailah","lastName":"Qureish","suffix":""},{"id":559058154,"identity":"d97fbd6a-b9a7-4b01-a8c1-1fdec725fb66","order_by":1,"name":"Francis Mulwa","email":"","orcid":"","institution":"Kenya Medical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Francis","middleName":"","lastName":"Mulwa","suffix":""},{"id":559058156,"identity":"95d7c194-0666-4287-9702-900198c5b8cf","order_by":2,"name":"Richard Odinga","email":"","orcid":"","institution":"Walter Reed Army Institute of Research-Africa/Kenya Medical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Odinga","suffix":""},{"id":559058158,"identity":"24724bf8-5802-4dea-93b8-ff061891815d","order_by":3,"name":"Santos Yalwala","email":"","orcid":"","institution":"Kenya Medical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Santos","middleName":"","lastName":"Yalwala","suffix":""},{"id":559058160,"identity":"d62253a1-1724-4aff-9b30-bc6994d8696a","order_by":4,"name":"Joel Lutomiah","email":"","orcid":"","institution":"Kenya Medical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Joel","middleName":"","lastName":"Lutomiah","suffix":""},{"id":559058162,"identity":"6f9b1585-5596-482f-932c-ee8a99c18b9a","order_by":5,"name":"Joel L. Bargul","email":"","orcid":"","institution":"Jomo Kenyatta University of Agriculture and Technology (JKUAT)","correspondingAuthor":false,"prefix":"","firstName":"Joel","middleName":"L.","lastName":"Bargul","suffix":""},{"id":559058164,"identity":"b2612df9-ab4c-45b7-8dd1-3594a2f1e78b","order_by":6,"name":"Gerald G. Kellar","email":"","orcid":"","institution":"Walter Reed Army Institute of Research-Africa","correspondingAuthor":false,"prefix":"","firstName":"Gerald","middleName":"G.","lastName":"Kellar","suffix":""},{"id":559058166,"identity":"44f17a72-8527-4ac7-8b1c-ddc472ee0d40","order_by":7,"name":"John Eads","email":"","orcid":"","institution":"Walter Reed Army Institute of Research-Africa","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Eads","suffix":""},{"id":559058168,"identity":"81807c90-6151-4d69-a8ca-f4bb778f6b47","order_by":8,"name":"Fredrick Eyase","email":"","orcid":"","institution":"Walter Reed Army Institute of Research-Africa/Kenya Medical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Fredrick","middleName":"","lastName":"Eyase","suffix":""}],"badges":[],"createdAt":"2025-12-11 18:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8339335/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8339335/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98050937,"identity":"1c9890e0-6fea-420d-81a3-9a6cfb4cb0f9","added_by":"auto","created_at":"2025-12-12 08:54:58","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":187839,"visible":true,"origin":"","legend":"","description":"","filename":"MANUSCRIPTGENETICDIVERSITYANDVECTORCOMPETENCEBMCKulthoom.docx","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/afa7634a8336c8fa73ffb252.docx"},{"id":98050924,"identity":"7700b87a-a4e4-408c-b1b2-3358e1d1c8fe","added_by":"auto","created_at":"2025-12-12 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08:54:53","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19268,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/41301f000cab563f9325e18b.png"},{"id":98050931,"identity":"f5b9f7a6-4310-4968-8628-0cde2d0efe9d","added_by":"auto","created_at":"2025-12-12 08:54:54","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7653,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/36d1096780b4b802fb2b76fb.png"},{"id":98050929,"identity":"8e4b71a1-49ec-44e1-b71b-a0480e334850","added_by":"auto","created_at":"2025-12-12 08:54:53","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":149414,"visible":true,"origin":"","legend":"","description":"","filename":"bd51a93df8674ecfb365cb227b6656ff1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/df2028fe2f65832c706f900c.xml"},{"id":98050935,"identity":"787a5171-1093-4581-bcdc-845561c3116d","added_by":"auto","created_at":"2025-12-12 08:54:58","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":164027,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/e811cb9578be73004b8a8873.html"},{"id":98050930,"identity":"6ada1d27-bf3d-470f-bac3-e2062d0ecd99","added_by":"auto","created_at":"2025-12-12 08:54:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":328106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe map of Kenya and, inset, Lamu County showing the sampling sites\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/f44293a5d88db29445881501.png"},{"id":98050925,"identity":"65a46dec-d7cd-46b5-8e0b-db457fbed30b","added_by":"auto","created_at":"2025-12-12 08:54:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":332383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum likelihood tree of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAe. aegypti \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emosquitoes collected from Lamu County.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/dc2502c054bcb3eeb34316d8.png"},{"id":98050923,"identity":"1496777e-6ed9-4467-ad5d-d34c3b14bc02","added_by":"auto","created_at":"2025-12-12 08:54:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71649,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfection percentages by site and incubation periods\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/1a1640bdb052498eb6b21c09.png"},{"id":98050927,"identity":"0a5232ab-f743-403f-a314-5d766631bea7","added_by":"auto","created_at":"2025-12-12 08:54:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDissemination percentages by site and incubation periods\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/87ff64a59dfd85f24cb0104e.png"},{"id":98775141,"identity":"5c65795c-72d7-4832-bb23-e27a45fd55d4","added_by":"auto","created_at":"2025-12-22 12:18:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2106694,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8339335/v1/745020a0-0bc4-4ebc-a92e-a41119bca780.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eVector Competence for Dengue-2 Virus and Genetic Diversity of Permethrin Resistant/ Susceptible Aedes Aegypti Sub-populations From Lamu County, Kenya\u003c/p\u003e","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003e \u003cem\u003eAedes aegypti\u003c/em\u003e mosquito is the primary vector for dengue virus (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is found in tropical, sub-tropical, and temperate regions such as sub-Saharan Africa, the South American continent and South Asian countries (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Dengue fever is caused by four dengue virus serotypes: DENV-1, DENV-2, DENV-3 and DENV-4 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Dengue virus serotype 2 (DENV-2) is the most prevalent in Kenya, particularly along the coastal region (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Globally, about 390\u0026nbsp;million people are infected by DENV annually (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) which has devastating effects both on public health and the economy.\u003c/p\u003e \u003cp\u003eCurrently, there is neither a specific drug treatment for dengue nor a suitable approved vaccine for the general population (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) leaving vector control as the only way to combat this disease.\u003c/p\u003e \u003cp\u003eDifferent types of insecticides are used for vector control including organophosphates such as malathion and P-methyl which inhibit the enzyme acetylcholinesterase (AChE), resulting in overabundance of the neurotransmitter acetylcholine (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Organochlorines such as dieldrin and dichloro-diphenyl-trichloroethane (DDT) interact with the neuron membrane and alter the transmission of the nerve impulses (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) while carbamates like bendiocarb interfere with acetylcholinesterase therefore disrupting the nervous system (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). These groups of insecticides are gradually being replaced by pyrethrins due to their significant and persistent toxic effects on humans and the environment (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). One such pyrethrin is permethrin, which the present study focuses on.\u003c/p\u003e \u003cp\u003eExtensive use of insecticides and agricultural pesticides has led to development of resistance in many insects species (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePermethrin resistance has been observed in some \u003cem\u003eAe. aegypti\u003c/em\u003e populations (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) probably due to the differences in their genetic makeup (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAe. aegypti\u003c/em\u003e populations have shown varying degrees of resistance to pyrethroids, particularly permethrin (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In Lamu County, located on the Kenyan coast, there have been outbreaks of arboviral diseases such as dengue and chikungunya (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), as recently as 2025. It is believed that this is due to ineffective vector control due to insecticide resistance among vector populations (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eKnockdown resistance (kdr), also known as altered target \u0026ndash; site resistance (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) due to the mutation that alters the target- site, reduces or removes the toxic effect of the insecticide (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). These kdr mutations are passed down through generations. The mode of inheritance of insecticide resistance shows homozygosity of the resistance and susceptible genes involved (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome studies have shown that insecticide resistance may affect the vector competence of \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes by increasing dissemination rates and altering their gut microbiota (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Vector competence is the intrinsic ability of a vector to be infected by a pathogen, amplify, disseminate, and transmit it (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This is affected by extrinsic and intrinsic factors (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Extrinsic factors include temperature and gut microbiota; higher temperatures increase susceptibility of mosquitoes to viruses (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The development of many pathogens is temperature sensitive with higher temperatures decreasing the extrinsic incubation period for the viruses (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Microbiota found in the gut of insect vectors also decrease viral and parasitic infections in mosquito vectors by activating immune responses or directly inhibiting pathogen development (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInternal factors include the immune responses and barriers such as: midgut infection barrier (MIB) which prevents the virus from attaching to the receptors present on midgut epithelial cells; midgut escape barrier (MEB) which prevents the release of viral particles from midgut epithelial cells; salivary gland infection barrier (SIB) which prevents the virus from infecting acinar cells; and salivary gland escape barrier (SEB) which prevents releasing of viral particles from salivary gland acinar cells (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo determine the genetic differences between sub-populations, the Cytochrome c oxidase subunit 1 (CO1) gene has proven very useful (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The CO1 gene is considered ideal for barcoding due to its presence in most living eukaryotes (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere have been recurring outbreaks of dengue, mostly associated with DENV-2, in most of coastal Kenya since 2011, with limited data on insecticide resistance of \u003cem\u003eAe. aegypti\u003c/em\u003e in Kenya, and how this impacts arbovirus transmission. Therefore, this study aimed to investigate the insecticide resistance among \u003cem\u003eAe. aegypti\u003c/em\u003e sub-populations in Lamu County and its effect on vector ability to acquire, amplify and transmit DENV-2, as well as entomological factors that contribute to dengue transmission. The findings of this study will help to understand the epidemiology of dengue in the county.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy sites\u003c/h2\u003e \u003cp\u003eThis study was conducted in Lamu County, along the Kenyan Coast (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) where three sites were selected for sample collection: Shela, Hindi, and Mokowe. Lamu County (latitude 2.2696\u0026deg; S, longitude 40.9006\u0026deg; E) generally experiences a warm, humid tropical climate with temperatures ranging from 24\u0026deg;C to 30\u0026deg;C throughout the year making it conducive for mosquito breeding. The county has a bimodal pattern of rainfall with the long rains occurring between April and July, with the highest rainfall occurring in the month of May and short rains in November and December.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCollection and identification of mosquito samples\u003c/h3\u003e\n\u003cp\u003eAdult mosquitoes were sampled using BG-Sentinel 2 Traps and CDC light traps baited with carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e). Mosquito eggs were collected using ovitraps that consisted of black ovicups lined with oviposition paper and half-filled with water.\u003c/p\u003e \u003cp\u003eThe mosquitoes were collected during periods of higher population densities which usually coincide with rainy seasons (April \u0026ndash; June). The traps were set between 6.30pm and 7:00pm in the evening and collected the following morning between 6:00am and 6.30am. The trapped mosquitoes were knocked down using and triethylamine transported to the KEMRI laboratories for morphological identification to species level using taxonomic keys (Edwards 1941 and Peter G. Jupp 1986).\u003c/p\u003e\n\u003ch3\u003eGenetic diversity determination\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction\u003c/h2\u003e \u003cp\u003eField collected (n\u0026thinsp;=\u0026thinsp;31) female mosquitoes (F\u003csub\u003e0\u003c/sub\u003e) were sampled from various collection sites in Shela, Hindi, and Mokowe. Each mosquito was from a different site to avoid analyzing siblings. Individual mosquito legs were dissected using sterile forceps and placed in a sterile RNase/DNase-free Eppendorf tube containing a sterile copper bead, 200ml of Phosphate buffered saline was added into each Eppendorf tube, and the sample homogenized in an Omni Bead Rapture 24 (OMNI International) set to run at the speed of 2.10 mm for 20 seconds. Qiagen\u0026trade; DNeasy Blood and Tissue Extraction Kit (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) was then used to extract DNA from the homogenates following the manufacturers directives.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePolymerase Chain Reaction\u003c/h3\u003e\n\u003cp\u003ePolymerase Chain Reaction (PCR) was used to amplify the target mitochondrial DNA (COI) in mosquito samples using the universal reverse primer HCO2198 and forward primer LCO1490. The PCR master mix was prepared in a Biosafety level II molecular laboratory and contained 12.5 \u0026micro;l Amplitaq solution, 1 \u0026micro;l forward primer, 1 \u0026micro;l reverse primer, and 7.5 \u0026micro;l sterile nuclease free water. Next, 22\u0026micro;l of the master mix was transferred into well-labelled RNase-free, 0.2 ml (8-strip format, Invitrogen) PCR tubes and 3 \u0026micro;l of sample DNA added into each tube. The tubes were tightly sealed, vortexed, and placed in a Thermocycler set to run 35 reaction cycles of 94\u0026deg;C for 30 secs, 55\u0026deg;C for 30s, and 72\u0026deg;C for 60s. The amplicons were visualized in a 2% agarose gel with an expected band size of approximately 650 bp, stained with SYBR Green. The amplicons underwent clean up to remove leftover primers and deoxyribonucleotide triphosphates (dNTPs) from PCR. This was done using the Exo-CIP\u0026trade; Rapid PCR Cleanup protocol (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The products were then quantified to determine the concentration of DNA using a nanodrop spectrophotometer and Sanger sequenced (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBioassays for determination of susceptibility and resistance of\u003c/b\u003e \u003cb\u003eAe. aegypti\u003c/b\u003e \u003cb\u003eto permethrin\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes were subjected to World Health Organisation (WHO) tube bioassays for susceptibility to permethrin (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwenty-five mosquitoes were aspirated at a time, from each site and transferred into a holding tube to ensure that damaged specimens were not included in the test. The holding tubes were set upright, mesh-screen end uppermost for approximately 30 minutes after which, any mosquitoes that may have been damaged during aspiration were removed. A sheet of paper impregnated with 0.75% permethrin insecticide was introduced into each of the exposure tubes then rolled into a cylinder and fastened into position with a copper clip.\u003c/p\u003e \u003cp\u003eThe mosquitoes were introduced into the exposure tube by attaching it to a vacant screw-top in the slide. The mosquitoes were gently blown down into the exposure tube. The slide was closed and the holding tube detached. The exposure tubes were left standing upright with mesh-screen end uppermost for 1 hour. The behavior of the mosquitoes was monitored and the number of those knocked down recorded after every 10 minutes for 1 hour. A mosquito was considered to be knocked down if it was unable to stand or fly in a coordinated way.\u003c/p\u003e \u003cp\u003eAt the end of the 1hour exposure period, the mosquitoes were transferred to the holding tubes set on the slide and a pad of cotton wool soaked in sugar solution placed on the mesh-screen. The holding tubes were kept for 24 hours (recovery period) in a secluded, shady place where the temperatures did not exceed 30˚C. At the end of recovery period, (24-hour post-exposure), the number of dead mosquitoes was counted and recorded. An adult mosquito is considered to be alive if it is able to fly regardless of the number of legs remaining. Affected specimens that are unable to walk should be considered as dead.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCalculation of mortality rates\u003c/h2\u003e \u003cp\u003eAfter 24-hour post-exposure, the number of dead mosquitoes in the holding tube was counted. The mortality is calculated by summing the number of dead mosquitoes per site (i.e., out of a total of 100 mosquitoes from four replicates of 25 mosquitoes each per site) expressed this as a percentage of the number of exposed mosquitoes using the formula:\u003c/p\u003e \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003ch3\u003eVector competence for dengue-2 determination\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eMosquito rearing\u003c/h2\u003e\n \u003cp\u003eMosquitoes were reared in the KEMRI insectary, maintained at a temperature of 28℃ \u0026minus;\u0026thinsp;32℃, 70% \u0026minus;\u0026thinsp;80% relative humidity (RH) and 12:12 hour light: dark (L: D) photoperiod. The batches of F\u003csub\u003e1\u003c/sub\u003e eggs were dispensed in water on larval trays for hatching in a level 2 insectary. The larvae were fed on Tetramin\u0026reg; fish food until they pupated, then pupae were collected every morning and put in a holding cup containing water. The cups with pupae were placed in a plastic cage with a netting material on top and allowed to develop into adults.\u003c/p\u003e\n \u003cp\u003eThe emerging adults were inactivated by placing them in a -20\u0026deg;C freezer for one minute, then morphologically identified under a dissecting microscope using taxonomic keys of Edwards (\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e) and Jupp (\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e) to ensure they are \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes. The mosquitoes were fed on a diet of 10% glucose solution for 3 days. On the fifth day, they were fed on mice after being starved for 12 hours. Albino mice were anaesthetized using Pentobarbital sodium injection at a dosage of 75 mg/kg (\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e). The mice were injected intraperitoneally then placed in mosquito cages for 30 minutes. Ovicups half-filled with water and lined with ovipapers were placed in the cages for the mosquitoes to lay F\u003csub\u003e2\u003c/sub\u003e eggs which were then hatched and reared into adulthood.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eDengue virus amplification and quantification\u003c/h2\u003e\n \u003cp\u003eLive preserved viral isolates (DENV-2 008/01/2012) from Mombasa County were amplified in a confluent monolayer of Vero E6 cells in a T-25 flask containing maintenance media and incubated at 37℃ for 7 days. Upon showing signs of cytopathic effect (CPE), the virus was harvested, aliquoted into cryovials and stored at -80℃ for further use.\u003c/p\u003e\n \u003cp\u003eQuantification of dengue virus was performed by plaque assay, i.e. tenfold serial dilutions of the amplified DENV (\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e). 900\u0026micro;l of maintenance media were added into nine Eppendorf tubes, 100\u0026micro;l of the amplified DENV-2 were transferred to the first Eppendorf tube and pipette mixed with the media, then 100\u0026micro;l of the mixture was picked and transferred to the next Eppendorf tube and pipette mixed. This step was repeated until the virus had been diluted ten times.\u003c/p\u003e\n \u003cp\u003eThe first well of a 12-well plate was inoculated with 100\u0026micro;l of virus over the confluent monolayers of Vero E6 cells, with 100\u0026micro;l of the subsequent dilutions inoculated into the other wells, leaving two wells for the negative controls. The plate was incubated for 1 hour at 37℃ with rocking every 15 minutes. Methylcellulose was added onto the inoculated cells and the plate observed over the next 14 days.\u003c/p\u003e\n \u003cp\u003eThe plate was then fixed for 2 hours with 10% formalin then stained overnight with 0.5% crystal violet. The plaques were counted and calculated to quantify the virus using the formula (\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e):\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n \u003cp\u003ewhere d is the dilution factor and V is the volume of diluted virus added to the well.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of blood and virus mixture (infectious bloodmeal)\u003c/h2\u003e\n \u003cp\u003eSheep blood was acquired from a farm in Kabete, collected into 50ml falcon tubes and transported to the laboratory. Cryovials of the amplified and quantified DENV-2 virus were thawed on ice. Two mice were sacrificed by injecting them intraperitoneally with Ketamine Hydrochloride at a dosage of 150mg/kg. The mice were skinned carefully with the fatty tissues being removed from the skin. The skins were fitted onto Hemotek columns and secured with bands.\u003c/p\u003e\n \u003cp\u003eThe DENV-2 was mixed with the blood by pipetting in a ratio of 1:1. The mixture was then transferred to the Hemotek feeding column.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eMosquito exposure to infectious bloodmeal\u003c/h2\u003e\n \u003cp\u003eThree experimental replicates were carried out in which mosquitoes (n\u0026thinsp;=\u0026thinsp;280) were starved of glucose for 12 hours prior to exposure to DENV-2 infectious bloodmeal. They were allowed to feed for 45 minutes. Fully engorged mosquitoes were aspirated into separate cages, counted and incubated for 7, 14 and 21 days post exposure (dpe). They were later fed on 10% glucose solution ad libitum. The three periods of extrinsic incubation (7, 14 and 21 dpe) were chosen to represent early, mid and late phases of virus dissemination kinetics (\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThree vector competence indices: (i) the proportion of mosquitoes exposed to infectious bloodmeal that developed midgut infection, (ii) the proportion of mosquitoes with midgut infection that developed a disseminated infection and (iii) the infectious titre of disseminated virus. The last two indices represent two successive aspects of the infection process in mosquitoes that lead to their ability to transmit the virus.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eDissection of mosquitoes\u003c/h2\u003e\n \u003cp\u003eAfter 7, 14 and 21 dpe, a third of the mosquitoes were sampled from the cage into a container and cold-anaesthetized at -20℃ for 45 seconds and maintained on wet ice (+\u0026thinsp;4℃). Scalpels were used to pick the mosquitoes by their wings and to hold them in place while separating the legs from the body.\u003c/p\u003e\n \u003cp\u003eThe dissected bodies and legs were placed into separate 1.5 mL microfuge tubes (Eppendorf) containing 500 \u0026micro;l and 200\u0026micro;l respectively, of homogenization media (HM), made of MEM, supplemented with 15% FBS, 2% L-glutamine, and 2% antibiotic/antimycotic, then stored in -80℃ freezer to await testing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eTest for infection and dissemination rates\u003c/h2\u003e\n \u003cp\u003eMosquito bodies were homogenized using a mini bead beater (BioSpec Products Inc, Bartlesville, OK 74005 USA) with the aid of a copper bead (BB-caliber airgun shot). They were centrifuged at 8000rpm for 10 minutes at 4℃. The supernatants were then inoculated onto a 70% confluent monolayer of Vero cells on 24- well plates. One hundred microliters of the abdominal homogenates were added to each of ten wells of the 12-well plate to infect the cells with the remaining two wells used for negative control.\u003c/p\u003e\n \u003cp\u003eThe plates were incubated at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator with frequent agitation after every 15 minutes for 1 hour to allow for virus adsorption. The infected cell monolayers were then overlaid with 1ml of maintenance media. The plates were incubated at 37℃ and 5% carbon dioxide (CO2) for 14 days and observed for signs of cytopathic effect (CPE). On observation of CPE, the plates were fixed for 1 hour with 10% formalin, and stained for 2 hours with 0.5% crystal violet, washed on running tap water, dried overnight and the plaques observed on a light box.\u003c/p\u003e\n \u003cp\u003eThe DENV-2 positive bodies were used to determine the infection rates. The legs of the corresponding mosquito bodies that showed signs of CPE underwent the same procedure. Plaques were counted and calculated to determine the viral titer. If the virus was detected in the mosquito\u0026rsquo;s body but not in the legs, the mosquito was considered to have a non-disseminated infection, limited to the midgut. Detection of virus in the body and legs was considered evidence of successful infection and dissemination, respectively (\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eData management and analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003eGenetic diversity analysis\u003c/h2\u003e\n \u003cp\u003eThe raw sequences were viewed using Chromas v2.6.6 (Technelysium), edited by trimming and deleting poor-quality sections and converted into FASTA format sequences. Basic Local Alignment Search Tool (BLAST) was used to compare the sequences against the GenBank database to confirm species identity. The sequences were aligned using Molecular Evolutionary Genetics Analysis (MEGA) software via the Muscle algorithm.\u003c/p\u003e\n \u003cp\u003eFor genetic diversity analysis, DnaSP v6.12 was used to determine nucleotide variation within the populations and provide information on nucleotide diversity (\u0026pi;), the number of haplotypes (H), and haplotype diversity (Hd). Neutrality tests (Tajima\u0026rsquo;s and Fu and Li\u0026rsquo;s tests) were also conducted. The aligned sequences were used to carry out phylogenetic analysis. Sequences belonging to \u003cem\u003eAe. aegypti aegypti\u003c/em\u003e (AF390098, AY432106), \u003cem\u003eAe. aegypti formosus\u003c/em\u003e (AY056597) and \u003cem\u003eAe. albopictus\u003c/em\u003e (MF148303) were downloaded from GenBank and used as references. The combined sequences were aligned in MEGA and used to infer a maximum likelihood phylogeny in IQTREE. The phylogenetic tree was visualized in FigTree v1.4.4.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eVector competence\u003c/h2\u003e\n \u003cp\u003eData was stored in Microsoft Excel spreadsheet and checked for errors and inconsistencies prior to analysis. Key rates were defined as follows: i) Infection rate (IR) which was determined as the proportion of mosquitoes whose bodies were infected with dengue virus, and ii) Dissemination rate (DR) as the proportion of mosquitoes whose legs were infected with dengue virus. The proportions (IR and DR) were calculated and the 95% confidence intervals (CIs) determined using binomial regression model. Proportion test of differences was used to determine significant differences between sites and incubation periods, and the associated p-values determined at 0.05 level of significance. Further, a comparative bar graph was used to compare the rates between the three sites. All the analyses were carried out using STATA version 15.1. All visualizations were done in RStudio version 2023.06.1.\u003c/p\u003e\n\u003c/div\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eGenetic diversity\u003c/h2\u003e \u003cp\u003eThere was a high level of genetic diversity between Hindi, Mokowe and Shela towns of Lamu County. Overall, 14 sequences of \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes showed 14 unique haplotypes with a high haplotype diversity (Hd) of 1.0 (Hd\u0026thinsp;\u0026gt;\u0026thinsp;0.5). For the individual sites: Hindi sub-population showed 6 haplotypes with Hd of 1.0; Mokowe sub-population showed 6 haplotypes with Hd of 1.0; and Shela sub-population showed 2 haplotypes with Hd of 1.0.\u003c/p\u003e \u003cp\u003eOverall, nucleotide diversity (π) was low (π\u0026thinsp;\u0026lt;\u0026thinsp;0.5) at 0.01184 with 0.00982 observed for Hindi sub-population and 0.01266 for Mokowe (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, this could not be calculated for the Shela sub-population due to the limited number (n\u0026thinsp;=\u0026thinsp;2) of sequences against the required minimum of three.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eNeutrality analysis\u003c/h2\u003e \u003cp\u003eTajima\u0026rsquo;s D and Fu\u0026rsquo;s F neutrality tests were carried out to analyse evolutionary history of the \u003cem\u003eAe. aegypti\u003c/em\u003e sub-populations. Tajima\u0026rsquo;s D values were negative overall (-1.09398), in Hindi (-0.65366), and Mokowe (-0.76939). Fu\u0026rsquo;s F values were also negative: -1.734 in Hindi, -1.298 in Mokowe, and \u0026minus;\u0026thinsp;5.526 for the combined sub-populations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These observations align with Tajima\u0026rsquo;s D test.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenetic diversity of \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes from Lamu County.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSites\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHn\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eπ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFu\u0026rsquo;s F\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHindi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.65366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1.734\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMokowe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.76939\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1.298\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShela\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCombined\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.09398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-5.526\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;Number of sequences, Hn\u0026thinsp;=\u0026thinsp;Number of haplotypes, Hd\u0026thinsp;=\u0026thinsp;Haplotype diversity, \u003cb\u003eπ\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Nucleotide diversity, D\u0026thinsp;=\u0026thinsp;Tajima\u0026rsquo;s D, Fu\u0026rsquo;s F\u0026thinsp;=\u0026thinsp;Fu\u0026rsquo;s selection test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003ePhylogenetic analysis of the mosquito sequences showed that all the \u003cem\u003eAe. aegypti\u003c/em\u003e sub-populations were \u003cem\u003eAe. aegypti formosus (Aaf)\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBioassay for determining susceptibility or resistance of\u003c/b\u003e \u003cb\u003eAe. aegypti\u003c/b\u003e \u003cb\u003eto permethrin\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes were divided into two categories: susceptible (\u0026ge;\u0026thinsp;98% mortality), and resistant (\u0026lt;\u0026thinsp;90% mortality).\u003c/p\u003e \u003cp\u003eThe Hindi sub-population showed a mortality rate of 46.98% (47%) which is 53% resistance against permethrin; Mokowe sub-population showed a mortality rate of 37.59% (37.6%) which is 62.4% resistance; and the Shela sub-population showed a mortality rate of 98.93% (99%) which is a 1% resistance.\u003c/p\u003e \u003cp\u003eThe Shela sub-population displayed significantly lower resistance (1%) compared to the Hindi and Mokowe sub-populations at 53% and 62.4%, respectively (Chi-square\u0026thinsp;=\u0026thinsp;χ\u0026sup2; = 458.47, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; pairwise comparisons, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all pairs).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eQuantification of dengue virus\u003c/h2\u003e \u003cp\u003eThe amplified DENV-2 virus had a titre of 6.7 X 10\u003csup\u003e4\u003c/sup\u003e Pfu/ml while the infectious blood meal\u0026rsquo;s titre was 6.3 X 10\u003csup\u003e4\u003c/sup\u003e Pfu/ml. These two titres were fairly comparable.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eVector competence\u003c/h2\u003e \u003cp\u003eA total of 280 (1st replicate [n\u0026thinsp;=\u0026thinsp;90], 2nd replicate [n\u0026thinsp;=\u0026thinsp;100], 3rd replicate [n\u0026thinsp;=\u0026thinsp;90]) \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes were analyzed for vector competence. Infection and dissemination rates for DENV-2 were calculated. 130 (1st replicate [n\u0026thinsp;=\u0026thinsp;35], 2nd replicate [n\u0026thinsp;=\u0026thinsp;50], 3rd replicate [n\u0026thinsp;=\u0026thinsp;45]) were obtained from Shela, while 90 (1st replicate [n\u0026thinsp;=\u0026thinsp;30], 2nd replicate [n\u0026thinsp;=\u0026thinsp;30], 3rd replicate [n\u0026thinsp;=\u0026thinsp;30]) were from Mokowe; and 60 (1st replicate [n\u0026thinsp;=\u0026thinsp;25], 2nd replicate [n\u0026thinsp;=\u0026thinsp;20], 3rd replicate [n\u0026thinsp;=\u0026thinsp;15]) from Hindi sites, respectively.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eInfection rate\u003c/h2\u003e \u003cp\u003eThe overall infection rate of all three sites was 27.9% (95% CI: 23.0-33.6) and increased with the incubation period from day 7 (5.6%; 95% CI: 2.4\u0026ndash;13.0), day 14 (17.0%; 95% CI: 11.0-26.2) and peaking at day 21 (62.2%; 95% CI: 53.0-73.1). The proportion test of difference revealed that the infection rates significantly increased by 56.6% at day 21 (Diff\u0026thinsp;=\u0026thinsp;0.566, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and by 11.4% at day 14 (Diff\u0026thinsp;=\u0026thinsp;0.114, p\u0026thinsp;=\u0026thinsp;0.014), compared to day 7 (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInfection rates were highest in Shela 30.8% (95% CI: 23.8\u0026ndash;39.8), followed by Mokowe 26.7% (95% CI: 18.9\u0026ndash;37.6) and least in Hindi 23.3% (95% CI: 14.7\u0026ndash;36.9). On day 7, the infection rate was 8.0% (95% CI: 2.1\u0026ndash;30.2), 6.7% (95% CI: 1.7\u0026ndash;25.4), and 2.9% (95% CI: 4.1\u0026ndash;19.7) in Hindi, Mokowe, and Shela sites respectively. At day 14, the infection rates increased to 10.0% (95% CI: 2.7\u0026ndash;37.2), 26.7% (95% CI: 14.7\u0026ndash;48.3), and 14.0% (95% CI: 7.0-27.8), in Hindi, Mokowe, and Shela sites respectively. At day 21, the infection rates further increased to 66.7% (95% CI: 46.6\u0026ndash;95.3), 46.7% (95% CI: 31.8\u0026ndash;68.4), and 71.1% (95% CI: 59.0-89.7) in Hindi, Mokowe and Shela sites respectively. A proportion test of difference for each site indicated that the infection rates significantly increased by 59.0% (Diff\u0026thinsp;=\u0026thinsp;0.59, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 40.0% (Diff\u0026thinsp;=\u0026thinsp;0.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 68.0% (Diff\u0026thinsp;=\u0026thinsp;0.68, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in Hindi, Mokowe and Shela sites respectively at day 21 compared to day 7. On day 14, only Mokowe site showed a significant increase in the infection rate of 20.0% (Diff\u0026thinsp;=\u0026thinsp;0.2, p\u0026thinsp;=\u0026thinsp;0.039) compared to day 7 (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOf the mosquitoes that were exposed to DENV-2, Hindi (n\u0026thinsp;=\u0026thinsp;60); Mokowe (n\u0026thinsp;=\u0026thinsp;90); and Shela (n\u0026thinsp;=\u0026thinsp;130), n\u0026thinsp;=\u0026thinsp;14 (23%), n\u0026thinsp;=\u0026thinsp;24 (27%) and n\u0026thinsp;=\u0026thinsp;40 (31%) from Hindi, Mokowe and Shela respectively, had midgut infections. However, the observed differences were not significant: (Chi-square χ\u0026sup2; = 1.22, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;=\u0026thinsp;0.542).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the infection and dissemination rates in the three study sites.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eProportion of mosquitoes infected with Dengue virus\u003c/p\u003e \u003cp\u003e% (95%CI); n\u0026thinsp;=\u0026thinsp;280\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBody (Infection)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLeg (Dissemination)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOverall\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.9 (23.0-33.6); n\u0026thinsp;=\u0026thinsp;78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.7 (12.0-20.6); n\u0026thinsp;=\u0026thinsp;44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSite\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHindi (n\u0026thinsp;=\u0026thinsp;60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.3 (14.7\u0026ndash;36.9); n\u0026thinsp;=\u0026thinsp;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.0 (12.1\u0026ndash;33.2); n\u0026thinsp;=\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMokowe (n\u0026thinsp;=\u0026thinsp;90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.7 (18.9\u0026ndash;37.6); n\u0026thinsp;=\u0026thinsp;24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.2 (7.0-21.3); n\u0026thinsp;=\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShela (n\u0026thinsp;=\u0026thinsp;130)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.8 (23.8\u0026ndash;39.8); n\u0026thinsp;=\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.2 (10.9\u0026ndash;23.9); n\u0026thinsp;=\u0026thinsp;21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProportion test of the difference between Hindi and Mokowe (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiff=-0.034, p\u0026thinsp;=\u0026thinsp;0.639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff\u0026thinsp;=\u0026thinsp;0.078, p\u0026thinsp;=\u0026thinsp;0.194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProportion test of the difference between Hindi and Shela (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiff=-0.075, p\u0026thinsp;=\u0026thinsp;0.287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff\u0026thinsp;=\u0026thinsp;0.038, p\u0026thinsp;=\u0026thinsp;0.521\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProportion test of the difference between Mokowe and Shela (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiff=-0.041, p\u0026thinsp;=\u0026thinsp;0.511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff=-0.04, p\u0026thinsp;=\u0026thinsp;0.408\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of infection and dissemination rates in the three study sites by incubation periods.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBody (Infection)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLeg (Dissemination)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOverall\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.6 (2.4\u0026ndash;13.0); n\u0026thinsp;=\u0026thinsp;5/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.4 (1.7\u0026ndash;11.6); n\u0026thinsp;=\u0026thinsp;4/90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.0 (11.0-26.2); n\u0026thinsp;=\u0026thinsp;17/100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.0 (5.6\u0026ndash;18.0); n\u0026thinsp;=\u0026thinsp;10/100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.2 (53.0-73.1); n\u0026thinsp;=\u0026thinsp;56/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.3 (24.9\u0026ndash;44.6); n\u0026thinsp;=\u0026thinsp;30/90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProportion test of difference at day 14 (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff=-0.114, p\u0026thinsp;=\u0026thinsp;0.014*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiff=-0.056, p\u0026thinsp;=\u0026thinsp;0.140\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProportion test of difference at day 21 (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff=-0.566, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiff=-0.289, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHindi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.0 (2.1\u0026ndash;30.2); n\u0026thinsp;=\u0026thinsp;2/25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.0 (2.1\u0026ndash;30.2); n\u0026thinsp;=\u0026thinsp;2/25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.0 (2.7\u0026ndash;37.2); n\u0026thinsp;=\u0026thinsp;2/20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0 (0.7\u0026ndash;33.8); n\u0026thinsp;=\u0026thinsp;1/20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66.7 (46.6\u0026ndash;95.3); n\u0026thinsp;=\u0026thinsp;10/15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.0 (39.7\u0026ndash;90.7); 9/15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProportion test of difference at day 14 (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff=-0.02, p\u0026thinsp;=\u0026thinsp;0.815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiff\u0026thinsp;=\u0026thinsp;0.03, p\u0026thinsp;=\u0026thinsp;0.689\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProportion test of difference at day 21 (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff=-0.59, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiff=-0.52, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMokowe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.7 (1.7\u0026ndash;25.4); n\u0026thinsp;=\u0026thinsp;2/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.3 (0.4\u0026ndash;22.9); n\u0026thinsp;=\u0026thinsp;1/30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.7 (14.7\u0026ndash;48.3); n\u0026thinsp;=\u0026thinsp;8/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.3 (5.4\u0026ndash;33.2); n\u0026thinsp;=\u0026thinsp;4/30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.7 (31.8\u0026ndash;68.4); 14/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.0 (9.8\u0026ndash;40.9); n\u0026thinsp;=\u0026thinsp;6/30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProportion test of difference at day 14 (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff=-0.2, p\u0026thinsp;=\u0026thinsp;0.039*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiff=-0.1, p\u0026thinsp;=\u0026thinsp;0.153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProportion test of difference at day 21 (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff=-0.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiff=-0.17, p\u0026thinsp;=\u0026thinsp;0.039*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShela\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9 (4.1\u0026ndash;19.7); n\u0026thinsp;=\u0026thinsp;1/35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.9 (0.4\u0026ndash;19.7); n\u0026thinsp;=\u0026thinsp;1/35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.0 (7.0-27.8); n\u0026thinsp;=\u0026thinsp;7/50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.0 (4.4\u0026ndash;23.0); n\u0026thinsp;=\u0026thinsp;5/50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.1 (59.0-89.7); n\u0026thinsp;=\u0026thinsp;32/45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.3 (22.1\u0026ndash;50.4); n\u0026thinsp;=\u0026thinsp;15/45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProportion test of difference at day 14 (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff=-0.11, p\u0026thinsp;=\u0026thinsp;0.088\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiff=-0.07, p\u0026thinsp;=\u0026thinsp;0.217\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProportion test of difference at day 21 (diff\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:,\\:p\\:value\\)\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiff=-0.68, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiff=-0.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eDissemination rate\u003c/h2\u003e \u003cp\u003eOverall, results showed mosquito dissemination rate of 15.7% (95% CI: 12.0-20.6). The overall dissemination rate increased with the incubation period, the lowest rate observed on day 7 (4.4%, 95% CI: 1.7\u0026ndash;11.6), increasing to 10.0% (95% CI: 5.6\u0026ndash;18.0) on day 14 and peaking on day 21 (33.3%, 95% CI: 24.9\u0026ndash;44.6) post exposure. A proportion test of difference revealed that the dissemination rates significantly increased by 28.9% at day 21 (Diff\u0026thinsp;=\u0026thinsp;0.289, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while no significant increase was observed at day 14.\u003c/p\u003e \u003cp\u003eBy sites, the mosquito dissemination rate was highest in Hindi at 20.0% (95% CI: 12.1\u0026ndash;33.2), followed by Shela at 16.2% (95% CI: 10.9\u0026ndash;23.9) and the least in Mokowe at 12.2% (95% CI: 7.0-21.3). The dissemination rates increased consistently with the incubation period in two sites. On day 7, the dissemination rate was 8.0% (95% CI: 2.1\u0026ndash;30.2), 3.3% (95% CI: 0.4\u0026ndash;22.9), 2.9% (95% CI: 4.1\u0026ndash;19.7) in Hindi, Mokowe and Shela sites respectively. On day 14, the dissemination rates increased to 13.3% (95% CI: 5.4\u0026ndash;33.2) and 10.0% (95% CI: 4.4\u0026ndash;23.0) in Mokowe and Shela sites respectively while in Hindi site, the dissemination rate decreased to 5.0% (95% CI: 0.7\u0026ndash;33.8). At day 21, dissemination rates increased to 60.0% (95% CI: 39.7\u0026ndash;90.7), 33.3% (95% CI: 22.1\u0026ndash;50.4), and 20.0% (95% CI: 9.8\u0026ndash;40.9) in Hindi, Shela, and Mokowe sites respectively. A proportion test of difference for each site indicated that the dissemination rates significantly increased by 52.0% (Diff\u0026thinsp;=\u0026thinsp;0.52, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 30.0% (Diff\u0026thinsp;=\u0026thinsp;0.30, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 17.0% (Diff\u0026thinsp;=\u0026thinsp;0.17, p\u0026thinsp;=\u0026thinsp;0.039) in Hindi, Shela, and Mokowe sites respectively at day 21 compared to day 7. There was no significant increase in dissemination rates in any of the sites at day 14 compared to day 7 (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf the mosquitoes, from Hindi (n\u0026thinsp;=\u0026thinsp;60); Mokowe (n\u0026thinsp;=\u0026thinsp;90); and Shela (n\u0026thinsp;=\u0026thinsp;130) that were exposed to DENV-2, n\u0026thinsp;=\u0026thinsp;12 (20%), n\u0026thinsp;=\u0026thinsp;11 (12%), and n\u0026thinsp;=\u0026thinsp;21 (16%) respectively had disseminated infections. The observed differences were not significant: (Chi-square χ\u0026sup2; = 1.68, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;=\u0026thinsp;0.432).\u003c/p\u003e \u003cp\u003eDissemination was used as proxy for transmission rates as saliva collection via capillary method tends to underestimate virus transmission. The results for the transmission rate were therefore the same as those of the dissemination rate.\u003c/p\u003e \u003cp\u003eThe plaque assay performed on the positive mosquito bodies and legs confirmed the presence of live viral particles. The mean body and leg titres for all three sites on 7, 14 and 21 dpe are visualized on Tables\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean DENV-2 titres (Pfu/ml) in mosquito bodies for all three sites 7, 14 and 21 dpe\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 21\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHindi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.2 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.2 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMokowe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.2 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.3 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.5 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShela\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.0 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.1 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean DENV-2 titres (Pfu/ml) in mosquito legs for all three sites 7, 14 and 21 dpe\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 21\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHindi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.9 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.2 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMokowe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.7 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.9 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShela\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.6 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.9 X 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe genetic diversity analysis carried out in this study has informed of high haplotype diversity for the individual sub-populations from different sites in Lamu County (1.0 for Shela, Hindi and Mokowe), as well as the combined sub-populations (0.989). The nucleotide diversity observed was 0.01184 for the combined sub-populations. According to Grant and Bowen (1998), values greater than 0.5 are considered high while those lesser than 0.5 are considered low when classifying the genetic diversity of populations (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe findings of this study therefore fall within the high haplotype diversity (Hd) and low nucleotide diversity (π) classification which indicate a recent population growth from a small one with few, related ancestors (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The new individuals inherited the existing haplotypes in increasing numbers, leading to high haplotype diversity (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Gene flow from populations that are genetically close to the population they are flowing to can also cause an increase in haplotype diversity (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). This increase happened in a short time, however, and hindered the mutation rate and divergence of the haplotypes at nucleotide level, leading to low nucleotide diversity (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwo neutrality tests were also carried out: Tajima\u0026rsquo;s D and Fu\u0026rsquo;s F tests. Tajima\u0026rsquo;s D test determines whether a DNA sequence has evolved randomly (neutrally) or non-randomly (selectively). It is done by getting the difference between the mean of pairwise differences and the number of segregating sites (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The two values are expected to be the same in a population that has evolved neutrally and maintained a constant size. Deviation from this neutrality and the difference in the values informs us of the population\u0026rsquo;s history. A negative Tajima\u0026rsquo;s D value indicates an abundance of rare alleles, most likely caused by an expanding population after a recent bottleneck and selective sweep (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFu\u0026rsquo;s F test works hand in hand with Tajima\u0026rsquo;s D test. It also tests for deviation from neutrality by investigating non-random recombination. It is determined by calculating the distribution of alleles and haplotypes in a DNA sequence (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Negative Fu\u0026rsquo;s F values also indicate a recent population expansion and positive selection (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe evolutionary history was inferred using the Maximum Likelihood method and Tamura-Nei model (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). The tree with the highest log likelihood (-3082.37) was shown. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model and then selecting the topology with superior log likelihood value. The evolutionary analyses were determined in MEGA11 (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe phylogenetic analysis revealed that all the samples belonged to the \u003cem\u003eAe. aegypti formosus (Aaf)\u003c/em\u003e sub-species which is native to Kenya (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). \u003cem\u003eAe. aegypti formosus\u003c/em\u003e is primarily sylvan (forest- dwelling mosquito), though it has spread into urban areas (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) and become domesticated (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). \u003cem\u003eAe. aegypti formosus\u003c/em\u003e is also a known zoophilic mosquito, although this may be changing with its spread into areas densely-populated by humans (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). This adaptability is a cause for concern as it was previously considered less of a threat than its counterpart \u003cem\u003eAe. aegypti aegypti\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes from Shela were 99% susceptible to permethrin insecticide while the Hindi and Mokowe populations were 53% and 62.4% resistant, respectively. This suggests that permethrin is an effective insecticide in Shela, but ineffective in controlling \u003cem\u003eAe. aegypti\u003c/em\u003e populations in Hindi and Mokowe. Therefore, these areas need a more effective product to replace permethrin in the control of \u003cem\u003eAe. aegypti\u003c/em\u003e vectors.\u003c/p\u003e \u003cp\u003eThe midgut infection rate (MIR) for all three sub-populations gradually increased over the three-week duration indicating that the midgut infection barrier (MIB) gradually weakened to allow for infection of DENV-2. For instance, the Shela mosquito sub-population which was 99% susceptible to permethrin, had a MIR of only 3% at 7dpe, suggesting a strong MIB at this point. However, this increased to 14% at 14 dpe and \u0026gt;\u0026thinsp;71% at 21 dpe. This gradual increase in MIR was observed in all the sub-populations, with the highly resistant Mokowe having a MIR of 7% and the moderately resistant Hindi having 8% at 7 dpe; increasing to 27% and 10% at 14 dpe and then \u0026gt;\u0026thinsp;46% and \u0026gt;\u0026thinsp;66% at 21 dpe for Mokowe and Hindi sub-populations respectively. The MIB had greatly been overcome by the virus at this point with the results showing an overall increase in viral titre over the incubation period. This suggests that life expectancy of an infected mosquito vector is important for transmission of DENV-2 to occur, since the longer it lives the more the MIB is weakened. There was no significant difference in infection rates between the different populations (Chi-square χ\u0026sup2; = 1.22, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;=\u0026thinsp;0.542) suggesting that susceptibility/ resistance of \u003cem\u003eAe. aegypti\u003c/em\u003e to permethrin has no effect on mosquito susceptibility to DENV-2 infection.\u003c/p\u003e \u003cp\u003eThe dissemination rate (DIR) 7dpe for Hindi and Shela sub-populations was 100%, and 50% for Mokowe. This indicates that the midgut escape barrier (MEB) was very vulnerable to virus penetration which was therefore easily released from the midgut epithelial cells. At 14dpe, the DIR for both Hindi and Mokowe was 50% while that for Shela was 71%. The MEB was therefore more penetrable for the susceptible mosquito populations. The DIR greatly increased for all three sub-populations 21dpe: 90%, 42.9% and 46.9% for Hindi, Mokowe and Shela respectively. The MEB seems to be unaffected by permethrin resistance or susceptibility as it varied indiscriminately among the three sub-populations and the days post exposure.\u003c/p\u003e \u003cp\u003eDissemination was used as proxy for transmission. Saliva collection via capillary method requires forced salivation which tends to underestimate virus transmission rates. The transmission rates in a natural setting are more likely to be higher than in a laboratory setting. Dissemination is considered adequate in predicting the vector\u0026rsquo;s transmission abilities (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe p-values for the infection rate (p\u0026thinsp;=\u0026thinsp;0.542) and the dissemination rate (p\u0026thinsp;=\u0026thinsp;0.432) for the three sites; as well as those of the proportion tests of difference between the three sites when compared to each other (p\u0026thinsp;=\u0026thinsp;0.639, p\u0026thinsp;=\u0026thinsp;0.287, p\u0026thinsp;=\u0026thinsp;0.511); were above the 0.05 level of significance. This is statistically not significant and leads to the conclusion that there is no relationship between permethrin resistance/ susceptibility and vector competence.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe analysis showed that \u003cem\u003eAe. aegypti\u003c/em\u003e mosquito sub-populations from Lamu County have high infection and dissemination rates with dengue virus. Although the rates increased significantly with the incubation periods, they varied insignificantly by mosquito collection sites.\u003c/p\u003e \u003cp\u003eThe genetic diversity tests confirmed the presence of \u003cem\u003eAe. aegypti formosus\u003c/em\u003e in Lamu County. The negative Tajima\u0026rsquo;s D and Fu\u0026rsquo;s F values combined with the high haplotype diversity and low nucleotide diversity indicate a recent population expansion with a selective sweep. The spread of \u003cem\u003eAe. aegypti formosus\u003c/em\u003e into urban areas most likely contributed to the expansion. This elevates the risk of transmission, and possibly more outbreaks, of diseases such as dengue fever. Even though \u003cem\u003eAe. aegypti formosus\u003c/em\u003e is considered a weaker vector of arboviruses, its changing habits such as moving into urban areas makes it crucial for regular entomological surveillance so as to monitor it extensively and to implement interventions aimed at controlling the spread of dengue virus in the coastal region of Kenya with the goal of preventing more outbreaks from occurring.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eAe.\u003c/em\u003e: \u003cem\u003eAedes\u003c/em\u003e; CO1: Cytochrome Oxidase 1; CO\u003csub\u003e2\u003c/sub\u003e: Carbon dioxide; CPE: Cytopathic effect; DENV: Dengue virus; DIR: Dissemination rate; MIB: Midgut infection barrier; MEB: Midgut escape barrier. Hd: Haplotype diversity; mtDNA: mitochondrial DNA; PCR: Polymerase Chain Reaction; SIB: salivary gland infection barrier; SEB: salivary gland escape barrier.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003ch3\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThis study was approved by the Scientific Ethics Review Unit (SERU) of the Kenya Medical Research Institute (KEMRI) under approval number, SERU 4768. Approval to use mice in the study was granted via the Animal Care and Use Committee (ACUC) (KEMRI/ACUC/02.09.23). Informed consent was not required owing to this study not involving human participants.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThis work was funded by the Armed Forces Health Surveillance Branch (AFHSB) and its Global Emerging Infections Surveillance (GEIS) Section, FY2022 ProMIS ID: P0116_22_KY and FY2023 ProMIS ID P0094_23_KY. The funders had a role in validation of the project, administration and decision to publish.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presenta\u0026shy;tion and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eKS did data curation, investigation, methodology, analysis, and writing and editing of this study. FM facilitated the methodology, visualization and review of the write up. RO assisted with data curation. SY also assisted with data curation and validation. JL took part in the methodology of this study, project administration, review and editing of the write up and supervision of the study. JB supervised the study. GK and JE handled funding acquisition and validation of the study. FE took part in conceptualization of the study, methodology, project administration, funding and resources acquisition, review and editing of the write up as well as supervision of this study.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the Director General of KEMRI for ensuring that the necessary research infrastructure such as well-equipped laboratories, was available to conduct the study. We thank Hellen Koka, George Kisoi, Paul Kyallo, and Solomon Lang\u0026rsquo;at for their assistance in extraction of genetic material; Jane Thiiru for assisting in data analysis and Samuel Owaka for generating the map of Kenya showing the sampling sites.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLozano-Fuentes S, Hayden MH, Welsh-Rodriguez C, Ochoa-Martinez C, Tapia-Santos B, Kobylinski KC, et al. The dengue virus mosquito vector Aedes aegypti at high elevation in M\u0026eacute;xico. Am J Trop Med Hyg. 2012;87(5):902\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisen L, Moore CG. Aedes (stegomyia) aegypti in the continental united states: A vector at the cool margin of its geographic range. J Med Entomol. 2013;50(3):467\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJagtap S, Pattabiraman C, Sankaradoss A, Krishna S, Roy R. Evolutionary dynamics of dengue virus in India. 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Parasit Vectors. 2022;15(1):103.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ae. aegypti, dengue-2, Lamu, permethrin, resistant, susceptible, vector competence","lastPublishedDoi":"10.21203/rs.3.rs-8339335/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8339335/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDengue virus, primarily transmitted by \u003cem\u003eAedes aegypti\u003c/em\u003e mosquitoes, is endemic in Lamu County with the most recent outbreak in 2021. There is currently no definitive treatment for dengue; therefore, vector control remains the most effective method to curb transmission. Prolonged exposure of \u003cem\u003eAe. aegypti\u003c/em\u003e to insecticides may confer resistance. This study aimed to determine the levels of resistance to permethrin, a commonly used insecticide, and to compare the vectorial competence and genetic differences of resistant versus susceptible populations for dengue-2 virus.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAe. aegypti\u003c/em\u003e mosquito eggs were collected from Lamu (Shela, Hindi, and Mokowe) using ovitraps and hatched in the insectary. The emerging adults were assayed for permethrin resistance/ susceptibility. WHO insecticide resistance assays (n\u0026thinsp;=\u0026thinsp;145) were conducted utilizing 3,525 emerged mosquitoes; 1,575 each from Hindi and Mokowe, and 375 from Shela. The mosquitoes (F\u003csub\u003e2\u003c/sub\u003e) were exposed to dengue-2 infectious bloodmeals and fully engorged mosquitoes selected, incubated for up to 21 days, and tested for infection and dissemination by plaque assay. Proportion test of differences was used to determine significant differences between sub-populations and incubation periods at 0.05 level of significance. Individual legs were dissected from 31 female (F\u003csub\u003e0\u003c/sub\u003e) mosquitoes for isolation of genomic DNA. Polymerase Chain Reaction was used to amplify the target mitochondrial DNA using HCO2198 and LCO1490 primers. The amplicons were Sanger sequenced to determine genetic diversity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe Shela sub-population displayed lower resistance (1%) compared to the Hindi and Mokowe sub-populations at 53% and 62.4% respectively. Of the mosquitoes from Hindi (n\u0026thinsp;=\u0026thinsp;60); Mokowe (n\u0026thinsp;=\u0026thinsp;90); and Shela (n\u0026thinsp;=\u0026thinsp;130), n\u0026thinsp;=\u0026thinsp;14 (23%), n\u0026thinsp;=\u0026thinsp;24 (27%), and n\u0026thinsp;=\u0026thinsp;40 (31%) respectively, had midgut infections with dengue-2; while n\u0026thinsp;=\u0026thinsp;12 (20%), n\u0026thinsp;=\u0026thinsp;11 (12%), and n\u0026thinsp;=\u0026thinsp;21 (16%) respectively had disseminated infection. Genetic analysis showed that all the samples were \u003cem\u003eAe. aegypti formosus\u003c/em\u003e sub-species. Haplotype diversity was high at 0.989 while nucleotide diversity was low at 0.01184.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings suggest that \u003cem\u003eAe. aegypti formosus\u003c/em\u003e sub-species is prevalent in Lamu County. The study also demonstrated high infection and dissemination rates among the \u003cem\u003eAe. aegypti\u003c/em\u003e sub- populations. This confirms the risk of DENV-2 transmission and highlights the need for regular entomological surveillance for early detection of virus and initiation of preventive strategies such as vector control.\u003c/p\u003e","manuscriptTitle":"Vector Competence for Dengue-2 Virus and Genetic Diversity of Permethrin Resistant/ Susceptible Aedes Aegypti Sub-populations From Lamu County, Kenya","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-12 08:54:47","doi":"10.21203/rs.3.rs-8339335/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94ab08a1-d7c2-4b17-a2c4-f6cb4c80e11f","owner":[],"postedDate":"December 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-17T16:41:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-12 08:54:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8339335","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8339335","identity":"rs-8339335","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}