Mitochondrial and microbial diversity of the invasive mosquito vector species Culex tritaeniorhynchus across its extensive inter-continental geographic range.

Background: Culex (Cx.) tritaeniorhynchus is an invasive mosquito species with an extensive and expanding inter-continental distribution, currently reported across Asia, Africa, the Middle East, Europe and now Australia. It is an important vector of medical and veterinary pathogens which cause significant morbidity and mortality in human and animal populations. Across regions endemic for Japanese encephalitis virus (JEV), Cx. tritaeniorhynchus is considered the major vector and has also been shown to contribute to the transmission of several other zoonotic arboviruses including Rift Valley fever virus (RVFV) and West Nile virus (WNV). Methods: In this study, we used laboratory vector competence experiments to determine if Cx. tritaeniorhynchus from a Southern European population were competent JEV vectors. We also obtained samples from multiple geographically dispersed Cx. tritaeniorhynchus populations from countries within Europe, Africa, Eurasia and Asia to perform phylogenetic analysis to measure the level of mitochondrial divergence using the cytochrome oxidase subunit 1 ( CO1) gene. We also undertook bacterial 16S rRNA gene amplicon sequencing to determine microbial diversity and used multi-locus sequence typing (MLST) to determine any evidence for the presence of strains of the naturally occurring endosymbiotic bacterium Wolbachia. Results: Cx. tritaeniorhynchus from a Greek population were shown be be competent vectors of JEV with high levels of virus present in saliva. We found a signficant level of mitochondrial genetic diversity using the mosquito CO1 gene between geographically dispersed populations. Furthermore, we report diverse microbiomes identified by 16S rRNA gene amplicon sequencing within and between geographical populations. Evidence for the detection of the endosymbiotic bacteria Wolbachia was confirmed using Wolbachia-specific PCR and MLST. Conclusions: This study enhances our understanding of the diversity of Cx. tritaeniorhynchus and the associated microbiome across its inter-continental range and highlights the need for greater surveillance of this invasive vector species in Europe.

The mosquito species Culex (Cx.) tritaeniorhynchus is expanding its range and is now present in over 50 countries across Asia, Africa, the Middle East, Europe and now Australasia. It can transmit human and animal pathogens, resulting in significant morbidity and mortality. This species transmits Japanese encephalitis virus in endemic areas of Asia, and it has also been shown to contribute to the transmission of several other viruses that can infect humans, including Rift Valley fever virus and West Nile virus. In this study, we firstly undertook some lab experiments to show that Cx. tritaeniorhynchus from a Southern European population are competent vectors of Japanese encephalitis virus. We also obtained field mosquitoes from countries within Europe, Africa, Eurasia and Asia and used phylogenetic analysis to demonstrate a high level of mitochondrial divergence within and between populations. In addition, we analysed the bacteria present within mosquitoes and found a high level of microbial diversity. Finally, we present evidence for the presence of Wolbachia endosymbiotic bacteria in some populations of this mosquito species. This study highlights the need for greater surveillance of this invasive vector species ­ particularly in Europe.

Non-Coding RNAs Potentially Involved in Pyrethroid Resistance of Anopheles funestus Population in Western Kenya.

Backgrounds: The resurgence of Anopheles funestus, a dominant vector of human malaria in western Kenya was partly attributed to insecticide resistance. However, evidence on the molecular basis of pyrethroid resistance in western Kenya is limited. Noncoding RNAs (ncRNAs) form a vast class of RNAs that do not code for proteins and are ubiquitous in the insect genome. Here, we demonstrated that multiple ncRNAs could play a potential role in An. funestusresistance to pyrethroid in western Kenya. Materials and Methods: Anopheles funestus mosquitoes were sampled by aspiration methods in Bungoma, Teso, Siaya, Port Victoria and Kombewa in western Kenya. The F1 progenies were exposed to deltamethrin (0.05%), permethrin (0.75%), DDT (4%) and pirimiphos-methyl (0.25%) following WHO test guidelines. A synergist assay using piperonyl butoxide (PBO) (4%) was conducted to determine cytochrome P450s' role in pyrethroid resistance. RNA-seq was conducted on a combined pool of specimens that were resistant and unexposed, and the results were compared with those of the FANG susceptible strain. This approach aimed to uncover the molecular mechanisms underlying pyrethroid resistance. Results: Pyrethroid resistance was observed in all the sites with an average mortality rate of 57.6%. Port Victoria had the highest level of resistance to permethrin (MR=53%) and deltamethrin (MR=11%) pyrethroids. Teso had the lowest level of resistance to permethrin (MR=70%) and deltamethrin (MR=87%). Resistance to DDT was observed only in Kombewa (MR=89%) and Port Victoria (MR=85%). A full susceptibility to P-methyl (0.25%) was observed in all the sites. PBO synergist assay revealed high susceptibility (>98%) to the pyrethroids in all the sites except for Port Victoria (MR=96%, n=100). Whole transcriptomic analysis showed that most of the gene families associated with pyrethroid resistance comprised non-coding RNAs (67%), followed by imipenemase (10%), cytochrome P450s (6%), cuticular proteins (5%), olfactory proteins (4%), glutathione S-transferases (3%), UDP-glycosyltransferases (2%), ATP-binding cassettes (2%) and carboxylesterases(1%). Conclusions: This study unveils the molecular basis of insecticide resistance in An. funestus in western Kenya, highlighting for the first time the potential role of non-coding RNAs in pyrethroid resistance. Targeting non-coding RNAs for intervention development could help in insecticide resistance management.

COVID-19 in patients presenting with malaria-like symptoms at a primary healthcare facility in Accra, Ghana.

BACKGROUND: Malaria is a common and severe public health problem in Ghana and largely responsible for febrile symptoms presented at health facilities in the country. Other infectious diseases, including COVID-19, may mimic malaria due to their shared non-specific symptoms such as fever and headache thus leading to misdiagnosis. This study therefore investigated COVID-19 among patients presenting with malaria-like symptoms at Korle-Bu Polyclinic, Accra, Ghana. METHODS: This study enrolled 300 patients presenting with malaria-like symptoms aged ≥18yrs. After consent was obtained from study patients, two to three millilitres of whole blood, nasopharyngeal and oropharyngeal swab samples, were collected for screening of Plasmodium falciparum using malaria rapid diagnostic test, microscopy and nested PCR, and SARS-CoV-2 using SARS-CoV-2 antigen test and Real-time PCR, respectively. The plasma and whole blood were also used for COVID-19 antibody testing and full blood counts using hematological analyser. SARS-CoV-2 whole genome sequencing was performed using MinIon sequencing. RESULTS: The prevalence of malaria by microscopy, RDT and nested PCR were 2.3%, 2.3% and 2.7% respectively. The detection of SARS-CoV-2 by COVID-19 Rapid Antigen Test and Real-time PCR were 8.7% and 20% respectively. The Delta variant was reported in 23 of 25 SARS-CoV-2 positives with CT values below 30. Headache was the most common symptom presented by study participants (95%). Comorbidities reported were hypertension, asthma and diabetes. One hundred and thirteen (37.8%) of the study participants had prior exposure to SARS CoV-2 and (34/51) 66.7% of Astrazeneca vaccinated patients had no IgG antibody. CONCLUSION: It may be difficult to use clinical characteristics to distinguish between patients with COVID-19 having malaria-like symptoms. Detection of IgM using RDTs may be useful in predicting CT values for SARS-CoV-2 real-time PCR and therefore transmission.

Detection of Invasive Anopheles stephensi Mosquitoes through Molecular Surveillance, Ghana.

The invasive Anopheles stephensi mosquito has rapidly expanded in range in Africa over the past decade. Consistent with World Health Organization guidelines, routine entomologic surveillance of malaria vectors in Accra, Ghana, now includes morphologic and molecular surveillance of An. stephensi mosquitoes. We report detection of An. stephensi mosquitoes in Ghana.

Baseline susceptibility of Anopheles gambiae to clothianidin in northern Ghana.

BACKGROUND: Clothianidin, an insecticide with a novel mode of action, has been deployed in the annual indoor residual spraying programme in northern Ghana since March 2021. To inform pragmatic management strategies and guide future studies, baseline data on local Anopheles gambiae sensu lato (s.l.) susceptibility to the clothianidin insecticide were collected in Kpalsogu, a village in the Northern region, Ghana. METHODS: Phenotypic susceptibility of An. gambiae mosquitoes to clothianidin was assessed using the World Health Organization (WHO) insecticide resistance monitoring bioassay. The WHO cone bioassays were conducted on mud and cement walls sprayed with Sumishield 50 wettable granules (WG) (with clothianidin active ingredient). Daily mortalities were recorded for up to 7 days to observe for delayed mortalities. Polymerase chain reaction (PCR) technique was used to differentiate the sibling species of the An. gambiae complex and also for the detection of knock down resistance genes (kdr) and the insensitive acetylcholinesterase mutation (ace-1). RESULTS: The WHO susceptibility bioassay revealed a delayed killing effect of clothianidin. Mosquitoes exposed to the cone bioassays for 5 min died 120 h after exposure. Slightly higher mortalities were observed in mosquitoes exposed to clothianidin-treated cement wall surfaces than mosquitoes exposed to mud wall surfaces. The kdr target-site mutation L1014F occurred at very high frequencies (0.89-0.94) across all vector species identified whereas the ace-1 mutation occurred at moderate levels (0.32-0.44). Anopheles gambiae sensu stricto was the most abundant species observed at 63%, whereas Anopheles arabiensis was the least observed at 9%. CONCLUSIONS: Anopheles gambiae s.l. mosquitoes in northern Ghana were susceptible to clothianidin. They harboured kdr mutations at high frequencies. The ace-1 mutation occurred in moderation. The results of this study confirm that clothianidin is an effective active ingredient and should be utilized in malaria vector control interventions.

Biting behaviour, spatio-temporal dynamics, and the insecticide resistance status of malaria vectors in different ecological zones in Ghana.

BACKGROUND: A significant decrease in malaria morbidity and mortality has been attained using long-lasting insecticide-treated nets and indoor residual spraying. Selective pressure from these control methods influences changes in vector bionomics and behavioural pattern. There is a need to understand how insecticide resistance drives behavioural changes within vector species. This study aimed to determine the spatio-temporal dynamics and biting behaviour of malaria vectors in different ecological zones in Ghana in an era of high insecticide use for public health vector control. METHODS: Adult mosquitoes were collected during the dry and rainy seasons in 2017 and 2018 from five study sites in Ghana in different ecological zones. Indoor- and outdoor-biting mosquitoes were collected per hour from 18:00 to 06:00 h employing the human landing catch (HLC) technique. Morphological and molecular species identifications of vectors were done using identification keys and PCR respectively. Genotyping of insecticide-resistant markers was done using the TaqMan SNP genotyping probe-based assays. Detection of Plasmodium falciparum sporozoites was determined using PCR. RESULTS: A total of 50,322 mosquitoes belonging to four different genera were collected from all the study sites during the sampling seasons in 2017 and 2018. Among the Anophelines were Anopheles gambiae s.l. 93.2%, (31,055/33,334), An. funestus 2.1%, (690/33,334), An. pharoensis 4.6%, (1545/33,334), and An. rufipes 0.1% (44/33,334). Overall, 76.4%, (25,468/33,334) of Anopheles mosquitoes were collected in the rainy season and 23.6%, (7866/33,334) in the dry season. There was a significant difference (Z = 2.410; P = 0.0160) between indoor-biting (51.1%; 15,866/31,055) and outdoor-biting An. gambiae s.l. (48.9%; 15,189/31,055). The frequency of the Vgsc-1014F mutation was slightly higher in indoor-biting mosquitoes (54.9%) than outdoors (45.1%). Overall, 44 pools of samples were positive for P. falciparum CSP giving an overall sporozoite rate of 0.1%. CONCLUSION: Anopheles gambiae s.l. were more abundant indoors across all ecological zones of Ghana. The frequency of G119S was higher indoors than outdoors from all the study sites, but with higher sporozoite rates in outdoor mosquitoes in Dodowa and Kpalsogu. There is, therefore, an urgent need for a supplementary malaria control intervention to control outdoor-biting mosquitoes.

First detection of Anopheles stephensi in Ghana using molecular surveillance.

The invasive Anopheles stephensi mosquito has been rapidly expanding in range in Africa over the last decade, spreading from the Indian sub-continent to several East African countries (Djibouti, Ethiopia, Sudan, Somalia and Kenya) and now in West Africa, Nigeria. The rapid expansion of this invasive vector poses a major threat to current malaria control and elimination efforts. In line with the WHO's strategy to stop the spread of this invasive species by enhancing surveillance and control measures in Africa, we incorporated morphological and molecular surveillance of An. stephensi into routine entomological surveillance of malaria vectors in the city of Accra, Ghana. Here, we report on the first detection of An. stephensi in Ghana. An. stephensi mosquitoes were confirmed using PCR and sequencing of the ITS2 regions. These findings highlight the urgent need for increased surveillance and response strategies to mitigate the spread of An. stephensi in Ghana.

Differences in malaria vector biting behavior and changing vulnerability to malaria transmission in contrasting ecosystems of western Kenya.

BACKGROUND: Designing, implementing, and upscaling of effective malaria vector control strategies necessitates an understanding of when and where transmission occurs. This study assessed the biting patterns of potentially infectious malaria vectors at various hours, locations, and associated human behaviors in different ecological settings in western Kenya. METHODS: Hourly indoor and outdoor catches of human-biting mosquitoes were sampled from 19:00 to 07:00 for four consecutive nights in four houses per village. The human behavior study was conducted via questionnaire surveys and observations. Species within the Anopheles gambiae complex and Anopheles funestus group were distinguished by polymerase chain reaction (PCR) and the presence of Plasmodium falciparum circumsporozoite proteins (CSP) determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Altogether, 2037 adult female anophelines were collected comprising the An. funestus group (76.7%), An. gambiae sensu lato (22.8%), and Anopheles coustani (0.5%). PCR results revealed that Anopheles arabiensis constituted 80.5% and 79% of the An. gambiae s.l. samples analyzed from the lowland sites (Ahero and Kisian, respectively). Anopheles gambiae sensu stricto (hereafter An. gambiae) (98.1%) was the dominant species in the highland site (Kimaeti). All the An. funestus s.l. analyzed belonged to An. funestus s.s. (hereafter An. funestus). Indoor biting densities of An. gambiae s.l. and An. funestus exceeded the outdoor biting densities in all sites. The peak biting occurred in early morning between 04:30 and 06:30 in the lowlands for An. funestus both indoors and outdoors. In the highlands, the peak biting of An. gambiae occurred between 01:00 and 02:00 indoors. Over 50% of the study population stayed outdoors from 18:00 to 22:00 and woke up at 05:00, coinciding with the times when the highest numbers of vectors were collected. The sporozoite rate was higher in vectors collected outdoors, with An. funestus being the main malaria vector in the lowlands and An. gambiae in the highlands. CONCLUSION: This study shows heterogeneity of anopheline distribution, high outdoor malaria transmission, and early morning peak biting activity of An. funestus when humans are not protected by bednets in the lowland sites. Additional vector control efforts targeting the behaviors of these vectors, such as the use of non-pyrethroids for indoor residual spraying and spatial repellents outdoors, are needed.

The Role of Car Tyres in the Ecology of Aedes aegypti Mosquitoes in Ghana.

Background: The Aedes aegyptimosquito is an important vector of arboviral diseases including dengue and yellow fever. Despite the wide distribution of the Aedes aegypti mosquito, there is limited data on the ecology of Aedes aegypti mosquitoes in Ghana. In this study, we report on the oviposition preference and the larval life table of Aedes aegypti mosquitoes in Accra, Ghana. Methods: The oviposition preference of Aedesmosquitoes to three habitat types (tyres, drums and bowls) was measured by setting up ovitraps. Ovitraps were checked for the presence of Aedes larvae every 3 days. The presence and number of larvae were recorded for each habitat type. Two-hour-old Aedes aegypti larvae were introduced into and raised in these three habitat types to undertake larval life tables. The number of surviving larvae at each developmental stage was recorded daily until they emerge as adults. Results: Car tyres showed a high abundance of Aedeslarvae (52.33%) than drums (32.49%) and bowls (15.18%) (ANOVA, F _ 18.79, df _ 2, 159, P < 0.001). The mean development time of Ae. aegypti larvae was significantly lower in car tyres (7 ± 1 days) compared to that of bowls (9 ± 0.0 days) and drums (12.6 ± 1.5 days) (H (2) = 7.448, P = 0.024). The differences in pupation rates and emergence rates were not significant across the habitat types, however, the highest pupation rate was observed in bowls (0.92) and the emergence rate was highest in tyres (0.84). The proportion of first-instar larvae that survived to adults was significantly higher in tyres with a shorter survival time (0.84; 9 days) compared to that of bowls (0.72; 10 days) and drums (0.62 ± 0.2; 13 days) (H (2) = 2.822, P= 0.009). Conclusion: The results confirm that discarded car tyres were the preferred habitat choice for the oviposition of gravid female Aedes aegypti mosquitoes and provide the best habitat condition for larval development and survival. These findings are necessary for understanding the ecology of Aedes mosquitoes to develop appropriate strategies for their control in Ghana.

Current observations on shifts in malaria vector biting behavior and changing vulnerability to malaria transmission in contrasting ecosystems in Western Kenya.

Background: Designing, implementing, and upscaling effective malaria vector control strategies necessitates understanding of when and where transmission occurs. This study assessed the biting patterns of potentially infectious malaria vectors at various hours, locations, and human behavior in different ecological settings in western Kenya. Methods: Hourly indoor and outdoor catches of human-biting mosquitoes were sampled from 1900 to 0700 hours for four consecutive nights in four houses per village using human landing collection method. The nocturnal biting activities of each Anopheles species were expressed as the mean number of mosquitoes landing per person per hour. The human behavior study was conducted via observations and questionnaire surveys. Species within Anopheles gambiae and Anopheles funestus complexes were differentiated by polymerase chain reaction (PCR) and the presence of Plasmodium falciparumcircumsporozoite proteins (CSP) determined by enzyme-linked immunosorbent assay (ELISA). Results: Altogether, a total of 2,037 adult female Anophelines were collected comprising of An. funestus s.l. (76.7%), An.gambiae s.l.(22.8%) and Anopheles coustani (0.5%). Overall, Anopheles funestus was the predominant species collected in Ahero (96.7%) while An. gambiae s.l was dominant in Kisian (86.6%) and Kimaeti (100%) collections. PCR results revealed that An. arabiensis constituted 80.5% and 79% of the An.gambiae s.l samples analysed from Ahero and Kisian respectively. An. gambiae s.s (hereafter An.gambiae) (98.1%) was the dominant species collected in Kimaeti. All the An. funestus s.l samples analysed belonged to An. funestus s.s (hereafter An. funestus). Indoor biting densities of Anopheles gambiae and An. funestus exceeded the outdoor biting densities in all sites. The peak biting occurred early morning between 0430-0630 hours in the lowlands for An. funestus both indoors and outdoors. In the highlands (Kimaeti), the peak biting of An.gambiae occurred between 0100-0200 hours indoors. Over 50% of the study population stayed outdoors from 1800 to 2200 hours and woke up at 0500 hours coinciding with the times highest numbers of vectors were collected. The sporozoite rate was higher in vectors collected outdoors, with An. funestus being the main malaria vector in the lowlands and An. gambiaein the highland. Conclusion: The study shows heterogeneity of Anophelines distribution, high outdoor malaria transmission, and peak biting activity by An. funestus (early morning) when humans are not protected by bed nets in the lowland sites. Additional vector control efforts targeting the behaviors of these vectors i.e using non-pyrethroids-based indoor residual spraying and spatial repellents outdoors are needed.

Larval habitat stability and productivity in two sites in Southern Ghana.

BACKGROUND: Mosquito larval source management (LSM) is a valuable additional tool for malaria vector control. Understanding the characteristics of mosquito larval habitats and its ecology in different land use types can give valuable insight for an effective larval control strategy. This study determined the stability and productivity of potential anopheline larval habitats in two different ecological sites: Anyakpor and Dodowa in southern Ghana. METHODS: A total of 59 aquatic habitats positive for anopheline larvae were identified, and sampled every two weeks for a period of 30 weeks using a standard dipping method. Larvae were collected using standard dippers and were raised in the insectary for identification. Sibling species of the Anopheles gambiae sensu lato (s.l.) were further identified by polymerase chain reaction. The presence of larval habitats, their stability and larvae positive habitats were compared between the two sites using Mann-Whitney U and the Kruskal-Wallis test. Factors affecting the presence of An. gambiae larvae and physicochemical properties at the sites were determined using multiple logistic regression analysis and Spearman's correlation. RESULTS: Out of a total of 13,681 mosquito immatures collected, 22.6% (3095) were anophelines and 77.38% (10,586) were culicines. Out of the 3095 anophelines collected, An. gambiae s.l. was predominant (99.48%, n = 3079), followed by Anopheles rufipes (0.45%, n = 14), and Anopheles pharoensis (0.064%, n = 2). Sibling species of the An. gambiae consisted of Anopheles coluzzii (71%), followed by An. gambiae s.s. (23%), and Anopheles melas (6%). Anopheles mean larval density was highest in wells [6.44 (95% CI 5.0-8.31) larvae/dip], lowest in furrows [4.18 (95% CI 2.75-6.36) larvae/dip] and man-made ponds [1.20 (95% CI 0.671-2.131) larvae/dip].The results also revealed habitat stability was highly dependent on rainfall intensity, and Anopheles larval densities were also dependent on elevated levels of pH, conductivity and TDS. CONCLUSION: The presence of larvae in the habitats was dependent on rainfall intensity and proximity to human settlements. To optimize the vector control measures of malaria interventions in southern Ghana, larval control should be focused on larval habitats that are fed by underground water, as these are more productive habitats.

Genetic Diversity and Population Structure of Anopheles funestus in Western Kenya Based on Mitochondrial DNA Marker COII.

The mitochondrial marker, COII, was employed to assess the genetic structure and diversity of Anopheles funestus, a very important malaria vector in Africa that adapt and colonize different ecological niches in western Kenya. Mosquitoes were collected using mechanical aspirators in four areas (Bungoma, Port Victoria, Kombewa, and Migori) in western Kenya. Following morphological identification, PCR was used to confirm the species. The COII gene was amplified, sequenced, and analyzed to determine genetic diversity and population structure. A total of 126 (Port Victoria-38, Migori-38, Bungoma-22, and Kombewa-28) sequences of COII were used for population genetic analysis. Anopheles funestus had a high haplotype diversity (Hd = 0.97 to 0.98) but low nucleotide diversity (Π = 0.004 to 0.005). The neutrality test revealed negative Tajima's D and Fs values indicating an excess of low-frequency variation. This could be attributed to either population expansion or negative selection pressure across all the populations. No genetic or structural differentiation (Fst = -0.01) and a high level of gene flow (Gamma St, Nm = 17.99 to 35.22) were observed among the populations. Population expansion suggests the high adaptability of this species to various ecological requirements, hence sustaining its vectorial capacity and malaria transmission.

Molecular characterization and genotype distribution of thioester-containing protein 1 gene in Anopheles gambiae mosquitoes in western Kenya.

BACKGROUND: Evolutionary pressures lead to the selection of efficient malaria vectors either resistant or susceptible to Plasmodium parasites. These forces may favour the introduction of species genotypes that adapt to new breeding habitats, potentially having an impact on malaria transmission. Thioester-containing protein 1 (TEP1) of Anopheles gambiae complex plays an important role in innate immune defenses against parasites. This study aims to characterize the distribution pattern of TEP1 polymorphisms among populations of An. gambiae sensu lato (s.l.) in western Kenya. METHODS: Anopheles gambiae adult and larvae were collected using pyrethrum spray catches (PSC) and plastic dippers respectively from Homa Bay, Kakamega, Bungoma, and Kisumu counties between 2017 and 2020. Collected adults and larvae reared to the adult stage were morphologically identified and then identified to sibling species by PCR. TEP1 alleles were determined in 627 anopheles mosquitoes using restriction fragment length polymorphisms-polymerase chain reaction (RFLP-PCR) and to validate the TEP1 genotyping results, a representative sample of the alleles was sequenced. RESULTS: Two TEP1 alleles (TEP1*S1 and TEP1*R2) and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2) were identified. TEP1*S1 and TEP1*R2 with their corresponding genotypes, homozygous *S1/S1 and heterozygous *R2/S1 were widely distributed across all sites with allele frequencies of approximately 80% and 20%, respectively both in Anopheles gambiae and Anopheles arabiensis. There was no significant difference detected among the populations and between the two mosquito species in TEP1 allele frequency and genotype frequency. The overall low levels in population structure (FST = 0.019) across all sites corresponded to an effective migration index (Nm = 12.571) and low Nei's genetic distance values (< 0.500) among the subpopulation. The comparative fixation index values revealed minimal genetic differentiation between species and high levels of gene flow among populations. CONCLUSION: Genotyping TEP1 has identified two common TEP1 alleles (TEP1*S1 and TEP1*R2) and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2) in An. gambiae s.l. The TEP1 allele genetic diversity and population structure are low in western Kenya.

Insecticide resistant Anopheles gambiae have enhanced longevity but reduced reproductive fitness and a longer first gonotrophic cycle.

Widespread insecticide resistance in African malaria vectors raises concerns over the potential to compromise malaria vector control interventions. Understanding the evolution of resistance mechanisms, and whether the selective disadvantages are large enough to be useful in resistance management or designing suitable control strategies is crucial. This study assessed whether insecticide resistance to pyrethroids has an effect on the gonotrophic cycle and reproductive potential of malaria vector Anopheles gambiae. Comparative tests were performed with pyrethroid-resistant and susceptible colonies of Anopheles gambiae colonized from the same geographical area, and the reference Kisumu strain was used as a control. Adult females aged 3 days old were given a blood meal and kept separately for individual egg-laying. The number of days taken to lay eggs post-blood-feeding was recorded to determine the length of the gonotrophic cycle. To measure adult longevity and reproduction potential, newly emerged males and females of equal numbers were aspirated into a cage and females allowed to blood feed daily. The number of eggs laid and the surviving mosquitoes were recorded daily to determine fecundity, net reproduction rate, intrinsic growth rate and adult longevity. Overall, the resistant females had a significantly longer (1.8 days) gonotrophic cycle than susceptible females (F2, 13 = 9. 836, P < 0.01). The proportion of resistant females that laid eggs was lower 31.30% (94/300) compared to 54% (162/300) in the susceptible colony and 65.7% (197/300) in the Kisumu strain. The mean number of eggs laid per female was significantly lower in the resistant colony (88.02 ± 20) compared to the susceptible colony (104.9 ± .28.8) and the Kisumu strain (97.6 ± 34.8). The adult longevity was significantly higher for resistant (39.7 ± 1.6 days) compared to susceptible (29.9 ± 1.7 days) and the Kisumu strain was (29.6 ± 1.1 days) (F2,8 = 45.05, P < 0.0001). Resistant colony exhibited a lower fecundity (4.3 eggs/females/day) and net reproductive rate (2.6 offsprings/female/generation) compared to the susceptible colony (8.6 eggs/female/day; 4.7 offsprings/female/generation respectively) and Kisumu strain (9.7 eggs/female/day; 4.1 offsprings/female/generation respectively). The study suggests high fitness cost on reproductive parameters of pyrethroid-resistant mosquitoes particularly on the duration of gonotrophic cycle, fecundity and net reproductive rate. These fitness costs are likely associated with maintaining both target site and metabolic mechanisms of resistance to pyrethroids. Despite these costs, resistant mosquitoes had longer longevity. These results give insights to understanding the fitness cost of insecticide resistance and thus are critical when predicting the epidemiological impact of insecticide resistance.

Behavioral responses of pyrethroid resistant and susceptible Anopheles gambiae mosquitoes to insecticide treated bed net.

BACKGROUND: Long-lasting insecticidal nets are an effective tool in reducing malaria transmission. However, with increasing insecticide resistance little is known about how physiologically resistant malaria vectors behave around a human-occupied bed net, despite their importance in malaria transmission. We used the Mbita bednet trap to assess the host-seeking behavior of insecticide-resistant Anopheles gambiae mosquitoes under semi-field conditions. The trap incorporates a mosquito netting panel which acts as a mechanical barrier that prevents host-seeking mosquitoes from reaching the human host baiting the trap. METHODS: Susceptible and pyrethroid-resistant colonies of female Anopheles gambiae mosquitoes aged 3-5 days old were used in this study. The laboratory-bred mosquitoes were color-marked with fluorescent powders and released inside a semi-field environment where a human subject slept inside a bednet trap erected in a traditional African hut. The netting panel inside the trap was either untreated (control) or deltamethrin-impregnated. The mosquitoes were released outside the hut. Only female mosquitoes were used. A window exit trap was installed on the hut to catch mosquitoes exiting the hut. A prokopack aspirator was used to collect indoor and outdoor resting mosquitoes. In addition, clay pots were placed outside the hut to collect outdoor resting mosquitoes. The F1 progeny of wild-caught mosquitoes were also used in these experiments. RESULTS: The mean number of resistant mosquitoes trapped in the deltamethrin-impregnated bed net trap was higher (mean = 50.21± 3.7) compared to susceptible counterparts (mean + 22.4 ± 1.31) (OR = 1.445; P<0.001). More susceptible mosquitoes were trapped in an untreated (mean = 51.9 ± 3.6) compared to a deltamethrin-treated bed net trap (mean = 22.4 ± 1.3) (OR = 2.65; P<0.001). Resistant mosquitoes were less likely to exit the house when a treated bed net was present compared to the susceptible mosquitoes. The number of susceptible mosquitoes caught resting outdoors (mean + 28.6 ± 2.22) when a treated bed net was hanged was higher than when untreated bednet was present inside the hut (mean = 4.6 ± 0.74). The susceptible females were 2.3 times more likely to stay outdoors away from the treated bed net (OR = 2.25; 95% CI = [1.7-2.9]; P<0.001). CONCLUSION: The results show that deltamethrin-treatment of netting panels inside the bednet trap did not alter the host-seeking behavior of insecticide-resistant female An. gambiae mosquitoes. On the contrary, susceptible females exited the hut and remained outdoors when a treated net was used. However, further investigations of the behavior of resistant mosquitoes under natural conditions should be undertaken to confirm these observations and improve the current intervention which are threatened by insecticide resistance and altered vector behavior.

Association of TNF-Alpha, MBL2, NOS2, and G6PD with Malaria Outcomes in People in Southern Ghana.

Background: One major issue that has set back the gains of the numerous malaria control interventions that national malaria control programs have implemented is asymptomatic malaria. Certain host genetic factors are known to influence symptomatic malaria; however, not much is known about how host genetics influences the acquisition of asymptomatic malaria. Methods: Genomic DNA was extracted from whole blood collected from 60 symptomatic and 149 nonfebrile (asymptomatic, N = 109, and uninfected, N = 40) volunteers aged between 2 and 69 years from a high (Obom) and a low (Asutsuare) malaria transmission setting in Southern Ghana. Restriction fragment length polymorphism (RFLP) was used to determine polymorphisms at the MBL2 54, TNF-α 308, NOS2 954, and G6PD 202/376 gene loci. Results: Polymorphisms at the MBL2 54 and TNF-α 308 loci were significantly different amongst the three categories of volunteers in both Asutsuare (p = 0.006) and Obom (p=0.05). In Asutsuare, a low malaria transmission area, the allele G has significantly higher odds (3.15) of supporting asymptomatic malaria as against symptomatic malaria. There were significantly higher odds of TNF-α genotype GA being associated with symptomatic malaria as against asymptomatic malaria in both sites, Obom (p=0.027) and Asutsuare (p=0.027). The allele B of the G6PD gene was more prevalent in symptomatic rather than asymptomatic parasite-infected individuals in both Obom (p=0.001) and Asutsuare (p=0.003). Conclusion: Individuals in Southern Ghana carrying the TNF-α 308 GA genotype are more likely to exhibit symptoms of malaria when infected with the malaria parasite as opposed to harboring an asymptomatic infection. Also, the B allele of the G6PD gene is likely to prevent a P. falciparum-infected person from exhibiting symptoms and thereby promote asymptomatic parasite carriage.

Spatiotemporal distribution and insecticide resistance status of Aedes aegypti in Ghana.

BACKGROUND: Vector control is the main intervention used to control arboviral diseases transmitted by Aedes mosquitoes because there are no effective vaccines or treatments for most of them. Control of Aedes mosquitoes relies heavily on the use of insecticides, the effectiveness of which may be impacted by resistance. In addition, rational insecticide application requires detailed knowledge of vector distribution, dynamics, resting, and feeding behaviours, which are poorly understood for Aedes mosquitoes in Africa. This study investigated the spatiotemporal distribution and insecticide resistance status of Aedes aegypti across ecological extremes of Ghana. METHODS: Immature mosquitoes were sampled from containers in and around human dwellings at seven study sites in urban, suburban, and rural areas of Ghana. Adult Aedes mosquitoes were sampled indoors and outdoors using Biogents BG-Sentinel 2 mosquito traps, human landing catches, and Prokopack aspiration. Distributions of immature and adult Aedes mosquitoes were determined indoors and outdoors during dry and rainy seasons at all sites. The phenotypic resistance status of Aedes mosquitoes to insecticides was determined using World Health Organization susceptibility bioassays. The host blood meal source was determined by polymerase chain reaction. RESULTS: A total of 16,711 immature Aedes were sampled, with over 70% found in car tyres. Significantly more breeding containers had Aedes immatures during the rainy season (11,856; 70.95%) compared to the dry season (4855; 29.05%). A total of 1895 adult Aedes mosquitos were collected, including Aedes aegypti (97.8%), Aedes africanus (2.1%) and Aedes luteocephalus (0.1%). Indoor sampling of adult Aedes yielded a total of 381 (20.1%) and outdoor sampling a total of 1514 (79.9%) mosquitoes (z = - 5.427, P = 0.0000) over the entire sampling period. Aedes aegypti populations were resistant to dichlorodiphenyltrichloroethane at all study sites. Vectors showed suspected resistance to bendiocarb (96-97%), permethrin (90-96%) and deltamethrin (91-96%), and were susceptible to the organophosphate for all study sites. Blood meal analysis showed that the Aedes mosquitoes were mostly anthropophilic, with a human blood index of 0.9 (i.e. humans, 90%; human and dog, 5%; dog and cow, 5%). CONCLUSIONS: Aedes mosquitoes were found at high densities in all ecological zones of Ghana. Resistance of Aedes spp. to pyrethroids and carbamates may limit the efficacy of vector control programmes and thus requires careful monitoring.

Genetic diversity and population structure of the human malaria parasite Plasmodium falciparum surface protein Pfs47 in isolates from the lowlands in Western Kenya.

Plasmodium falciparum parasites have evolved genetic adaptations to overcome immune responses mounted by diverse Anopheles vectors hindering malaria control efforts. Plasmodium falciparum surface protein Pfs47 is critical in the parasite's survival by manipulating the vector's immune system hence a promising target for blocking transmission in the mosquito. This study aimed to examine the genetic diversity, haplotype distribution, and population structure of Pfs47 and its implications on malaria infections in endemic lowlands in Western Kenya. Cross-sectional mass blood screening was conducted in malaria endemic regions in the lowlands of Western Kenya: Homa Bay, Kombewa, and Chulaimbo. Dried blood spots and slide smears were simultaneously collected in 2018 and 2019. DNA was extracted using Chelex method from microscopic Plasmodium falciparum positive samples and used to genotype Pfs47 using polymerase chain reaction (PCR) and DNA sequencing. Thirteen observed haplotypes of the Pfs47 gene were circulating in Western Kenya. Population-wise, haplotype diversity ranged from 0.69 to 0.77 and the nucleotide diversity 0.10 to 0.12 across all sites. All the study sites displayed negative Tajima's D values although not significant. However, the negative and significant Fu's Fs statistical values were observed across all the study sites, suggesting population expansion or positive selection. Overall genetic differentiation index was not significant (FST = -0.00891, P > 0.05) among parasite populations. All Nm values revealed a considerable gene flow in these populations. These results could have important implications for the persistence of high levels of malaria transmission and should be considered when designing potential targeted control interventions.

Larval ecology and bionomics of Anopheles funestus in highland and lowland sites in western Kenya.

BACKGROUND: An. funestus is a major Afrotropical vector of human malaria. This study sought to investigate the larval ecology, sporozoite infection rates and blood meal sources of An. funestus in western Kenya. METHODS: Larval surveys were carried out in Bungoma (Highland) and Kombewa (lowland) of western Kenya. Aquatic habitats were identified, characterized, georeferenced and carefully examined for mosquito larvae and predators. Indoor resting mosquitoes were sampled using pyrethrum spray catches. Adults and larvae were morphologically and molecularly identified to species. Sporozoite infections and blood meal sources were detected using real-time PCR and ELISA respectively. RESULTS: Of the 151 aquatic habitats assessed, 62/80 (78%) in Bungoma and 58/71(82%) in Kombewa were positive for mosquito larvae. Of the 3,193 larvae sampled, An. funestus larvae constitute 38% (1224/3193). Bungoma recorded a higher number of An. funestus larvae (85%, 95%, CI, 8.722-17.15) than Kombewa (15%, 95%, CI, 1.33-3.91). Molecular identification of larvae showed that 89% (n = 80) were An. funestus. Approximately 59%, 35% and 5% of An. funestus larvae co-existed with An. gambiae s.l, Culex spp and An. coustani in the same habitats respectively. Of 1,221 An. funestus s.l adults sampled, molecular identifications revealed that An. funestus constituted 87% (n = 201) and 88% (n = 179) in Bungoma and Kombewa, respectively. The Plasmodium falciparum sporozoite rate of An. funestus in Bungoma and Kombewa was 2% (3/174) and 1% (2/157), respectively, and the human blood index of An. funestus was 84% (48/57) and 89% (39/44) and for Bungoma and Kombewa, respectively. CONCLUSION: Man-made ponds had the highest abundance of An. funestus larvae. Multiple regression and principal component analyses identified the distance to the nearest house as the key environmental factor associated with the abundance of An. funestus larvae in aquatic habitats. This study serves as a guide for the control of An. funestus and other mosquito species to complement existing vector control strategies.

Influence of insecticide resistance on the biting and resting preferences of malaria vectors in the Gambia.

BACKGROUND: The scale-up of indoor residual spraying and long-lasting insecticidal nets, together with other interventions have considerably reduced the malaria burden in The Gambia. This study examined the biting and resting preferences of the local insecticide-resistant vector populations few years following scale-up of anti-vector interventions. METHOD: Indoor and outdoor-resting Anopheles gambiae mosquitoes were collected between July and October 2019 from ten villages in five regions in The Gambia using pyrethrum spray collection (indoor) and prokopack aspirator from pit traps (outdoor). Polymerase chain reaction assays were performed to identify molecular species, insecticide resistance mutations, Plasmodium infection rate and host blood meal. RESULTS: A total of 844 mosquitoes were collected both indoors (421, 49.9%) and outdoors (423, 50.1%). Four main vector species were identified, including An. arabiensis (indoor: 15%, outdoor: 26%); An. coluzzii (indoor: 19%, outdoor: 6%), An. gambiae s.s. (indoor: 11%, outdoor: 16%), An. melas (indoor: 2%, outdoor: 0.1%) and hybrids of An. coluzzii-An. gambiae s.s (indoors: 3%, outdoors: 2%). A significant preference for outdoor resting was observed in An. arabiensis (Pearson X2 = 22.7, df = 4, P<0.001) and for indoor resting in An. coluzzii (Pearson X2 = 55.0, df = 4, P<0.001). Prevalence of the voltage-gated sodium channel (Vgsc)-1014S was significantly higher in the indoor-resting (allele freq. = 0.96, 95%CI: 0.78-1, P = 0.03) than outdoor-resting (allele freq. = 0.82, 95%CI: 0.76-0.87) An. arabiensis population. For An. coluzzii, the prevalence of most mutation markers was higher in the outdoor (allele freq. = 0.92, 95%CI: 0.81-0.98) than indoor-resting (allele freq. = 0.78, 95%CI: 0.56-0.86) mosquitoes. However, in An. gambiae s.s., the prevalence of Vgsc-1014F, Vgsc-1575Y and GSTe2-114T was high (allele freq. = 0.96-1), but did not vary by resting location. The overall sporozoite positivity rate was 1.3% (95% CI: 0.5-2%) in mosquito populations. Indoor-resting An. coluzzii had mainly fed on human blood while indoor-resting An. arabiensis fed on animal blood. CONCLUSION: In this study, high levels of resistance mutations were observed that could be influencing the mosquito populations to rest indoors or outdoors. The prevalent animal-biting behaviour demonstrated in the mosquito populations suggest that larval source management could be an intervention to complement vector control in this setting.