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Aedes simpsoni complex has a wide distribution in Africa and comprises at least three described sub-species including the yellow fever virus (YFV) vector Ae . bromeliae . To date, the distribution and relative contributions of the sub-species and/or subpopulations including bionomic characteristics in relation to YF transmission dynamics remain poorly studied. In this study conducted in two areas with divergent ecosystems: peri-urban (coastal Rabai) and rural (Rift Valley Kerio Valley) in Kenya, survival rate was estimated by parity in Ae . simpsoni s . l . mosquitoes sampled using CO 2 -baited BG Sentinel traps. We then applied PCR targeting the nuclear internal transcribed spacer 2 (ITS2), region followed by sequencing and phylogenetic analytics to identify the sibling species in the Ae . simpsoni complex among parous and blood fed cohorts. Our results show that Ae . bromeliae was the most dominant sub-species in both areas, exhibiting high survival rates, human blood-feeding, and potentially, high vectorial capacity for pathogen transmission. We document for the first time the presence of Ae . lilii in Kenya and potentially yet-to-be described species in the complex displaying human feeding tendencies. We also infer a wide host feeding range on rodents, reptile, and domestic livestock besides humans especially for Ae . bromeliae . This feeding trend could likely expose humans to various zoonotic pathogens. Taken together, we highlight the utility of genotype-based analyses to generate precision surveillance data of vector populations for enhanced disease risk prediction and to guide cost-effective interventions (e.g. YF vaccinations).

The global spread of vector-borne diseases remains a worrying public health threat, raising the need for development of new combat strategies for vector control. Knowledge of vector ecology can be exploited in this regard, including plant feeding; a critical resource that mosquitoes of both sexes rely on for survival and other metabolic processes. However, the identity of plant species mosquitoes feed on in nature remains largely unknown. By testing the hypothesis about selectivity in plant feeding, we employed a DNA-based approach targeting trnH-psbA and matK genes and identified host plants of field-collected Afro-tropical mosquito vectors of dengue, Rift Valley fever and malaria being among the most important mosquito- borne diseases in East Africa. These included three plant species for Aedes aegypti (dengue), two for both Aedes mcintoshi and Aedes ochraceus (Rift Valley fever) and five for Anopheles gambiae (malaria). Since plant feeding is mediated by olfactory cues, we further sought to identify specific odor signatures that may modulate host plant location. Using coupled gas chromatography (GC)-electroantennographic detection, GC/mass spectrometry and electroantennogram analyses, we identified a total of 21 antennally-active components variably detected by Ae. aegypti, Ae. mcintoshi and An. gambiae from their respective host plants. Whereas Ae. aegypti predominantly detected benzenoids, Ae. mcintoshi detected mainly aldehydes while An. gambiae detected sesquiterpenes and alkenes. Interestingly, the monoterpenes β-myrcene and (E)-β-ocimene were consistently detected by all the mosquito species and present in all the identified host plants, suggesting that they may serve as signature cues in plant location. This study highlights the utility of molecular approaches in identifying specific vector-plant associations, which can be exploited in maximizing control strategies such as such as attractive toxic sugar bait and odor-bait technology.

Dengue (DEN) and yellow fever (YF) are re-emerging in East Africa, with contributing drivers to this trend being unplanned urbanization and increasingly adaptable anthropophilic Aedes (Stegomyia) vectors. Entomological risk assessment of these diseases remains scarce for much of East Africa and Kenya even in the dengue fever-prone urban coastal areas. Focusing on major cities of Kenya, we compared DEN and YF risk in Kilifi County (DEN-outbreak-prone), and Kisumu and Nairobi Counties (no documented DEN outbreaks). We surveyed water-holding containers for mosquito immature (larvae/pupae) indoors and outdoors from selected houses during the long rains, short rains and dry seasons (100 houses/season) in each County from October 2014-June 2016. House index (HI), Breteau index (BI) and Container index (CI) estimates based on Aedes (Stegomyia) immature infestations were compared by city and season. Aedes aegypti and Aedes bromeliae were the main Stegomyia species with significantly more positive houses outdoors (212) than indoors (88) (n = 900) (χ2 = 60.52, P < 0.0001). Overall, Ae. aegypti estimates of HI (17.3 vs 11.3) and BI (81.6 vs 87.7) were higher in Kilifi and Kisumu, respectively, than in Nairobi (HI, 0.3; BI,13). However, CI was highest in Kisumu (33.1), followed by Kilifi (15.1) then Nairobi (5.1). Aedes bromeliae indices were highest in Kilifi, followed by Kisumu, then Nairobi with HI (4.3, 0.3, 0); BI (21.3, 7, 0.7) and CI (3.3, 3.3, 0.3), at the respective sites. HI and BI for both species were highest in the long rains, compared to the short rains and dry seasons. We found strong positive correlations between the BI and CI, and BI and HI for Ae. aegypti, with the most productive container types being jerricans, drums, used/discarded containers and tyres. On the basis of established vector index thresholds, our findings suggest low-tomedium risk levels for urban YF and high DEN risk for Kilifi and Kisumu, whereas for Nairobi YF risk was low while DEN risk levels were low-to-medium. The study provides a baseline for future vector studies needed to further characterise the observed differential risk patterns by vector potential evaluation. Identified productive containers should be made the focus of community-based targeted vector control programs.

Outdoor biting by anopheline mosquitoes is one of the contributors to residual malaria transmission, but the profle of vectors driving this phenomenon is not well understood. Here, we studied the bionomics and genetically characterized populations of An. gambiae and An. funestus complexes trapped outdoors in three selected dryland areas including Kerio Valley, Nguruman and Rabai in Kenya. We observed a higher abundance of Anopheles funestus group members (n= 639, 90.6%) compared to those of the An. gambiae complex (n= 66, 9.4%) with An. longipalpis C as the dominant vector species with a Plasmodium falciparum sporozoite rate (Pfsp) of 5.2% (19/362). The known malaria vectors including An. funestus s.s. (8.7%, 2/23), An. gambiae (14.3%, 2/14), An. rivulorum (14.1%, 9/64), An. arabiensis (1.9%, 1/52) occurred in low densities and displayed high Pfsp rates, which varied with the site. Additionally, six cryptic species found associated with the An. funestus group harbored Pf sporozoites (cumulative Pfsp rate = 7.2%, 13/181). We detected low frequency of resistant 119F-GSTe2 alleles in An. funestus s.s. (15.6%) and An. longipalpis C (3.1%) in Kerio Valley only. Evidence of outdoor activity, emergence of novel and divergent vectors and detection of mutations conferring metabolic resistance to pyrethroid/DDT could contribute to residual malaria transmission posing a threat to efective malaria control.

The coastal region of Kenya has emerged as a focal point for urban dengue virus transmission driven by Aedes aegypti as the primary vector. To gain a deeper understanding of the epidemiological situation, we carried out a year-long longitudinal study (December 2021- November 2022) of the population dynamics of A. aegypti through weekly mosquito surveys using ovitrap and CO 2 -baited Biogents (BG) mosquito traps in Ukunda, an urban township in the coastal region. Aedes eggs laid in ovitraps were exclusively A. aegypti with 80.8% mean hatch rate. A total of 35,109 adult A. aegypti were captured, with twice as many females than males. The density of adult A. aegypti trap captures varied monthly, but there was no discernible delineation by season. Aedes aegypti fed more on humans (human blood index = 0.72). Two dengue-2 virus RNA was detected in two blood-fed specimens that had fed on humans. Multiple linear regression model indicated 59% variation in adult female abundance explained by weather variables including daily range in wind speed (82.9%) and temperature (17.1%). In contrast, random forest model revealed 83% variation in egg abundance attributed to weather variables, being positively influenced by mean daily temperature, wind speed, relative humidity and negatively by total precipitation. Our results confirm year-round vector presence and active circulation of dengue virus indicative of endemicity in urban Kenya. The findings highlight the importance of short-term climatic factors as predictors of A. aegypti dynamics of value in surveillance and control of arboviral diseases such as dengue.

Insects are increasingly being recognized not only as a source of food to feed the ever growing world population but also as potential sources of new products and therapeutic agents, among which are sterols. In this study, we sought to profile sterols and their derivatives present in the desert locust, Schistocerca gregaria, focusing on those with potential importance as dietary and therapeutic components for humans. Using coupled gas chromatography-mass spectrometry (GC-MS), we analyzed and compared the quantities of sterols in the different sections of the gut and tissues of the locust. In the gut, we identified 34 sterols which showed a patchy distribution, but with the highest composition in the foregut (55%) followed by midgut (31%) and hindgut (14%). Fed ad libitum on wheat seedlings, five sterols unique to the insect were detected. These sterols were identified as 7-dehydrocholesterol, desmosterol, fucosterol, (3β, 5α) cholesta-8, 14, 24-trien-3-ol, 4, 4-dimethyl, and (3β, 20R) cholesta-5, 24-dien-3, 20-diol with the first three having known health benefits in humans. Incubation of the fore-, mid- and hindgut with cholesterol-[4-13C] yielded eight derivatives, three of these were detected in the gut of the desert locust after it had consumed the vegetative diet but were not detected in the diet. Our study shows that the desert locust ingests phytosterols from a vegetative diet and, amplifies and metabolizes them into derivatives with potential salutary benefits and we discuss our findings in this context.

Among the factors affecting the effectiveness of malaria control is poor knowledge of the entomologic drivers of the disease. We investigated anopheline populations as part of a baseline study to implement house screening of windows and doors as a supplementary malaria control tool towards elimination in Jabi Tehnan district, Amhara Regional State of Ethiopia. The samples were surveyed monthly using CDC light traps between June 2020 and May 2021. Mosquito trap density (< 3 mosquitoes/trap) was low, however, with a high overall Plasmodium sporozoite rate (9%; indoor = 4.3%, outdoor = 13.1%) comprising P. falciparum (88.9%) and P. vivax (11.1%). Anopheles gambiae s.l., mostly An. arabiensis, comprised > 80% of total anopheline captures and contributed ~ 42% of Plasmodium-infected mosquitoes. On the other hand, morphologically scored Anopheles funestus s.l., constituting about 6% of anopheline collections, accounted for 50% of sporozoite-infected mosquitoes. Most of the infected An. funestus s.l. specimens (86.7%) were grouped with previously unknown or undescribed Anopheles species previously implicated as a cryptic malaria vector in the western Kenyan highlands, confirming its wider geographic distribution in eastern Africa. Other species with Plasmodium infection included An. longipalpis C, An. theileri, An. demillioni, and An. nili. Cumulatively, 77.8% of the infected mosquitoes occurred outdoors. These results suggest efficient malaria parasite transmission despite the low vector densities, which has implications for effective endpoint indicators to monitor malaria control progress. Additionally, the largely outdoor infection and discovery of previously unknown and cryptic vectors suggest an increased risk of residual malaria transmission and, thus, a constraint on effective malaria prevention and control.

House screening (HS) of doors, eaves, and windows using wire-mesh has demonstrated potential in the integrated vector management of malaria. However, limited epidemiological data are available to guide its implementation across different ecological settings. In a 16-month randomized controlled trial (follow-up period) conducted across three agroecological areas (dry mountain, plateau highland, and semi-arid) in Jabi Tehnan district, northwestern Ethiopia, treatment houses were equipped with HS combined with insecticide-treated nets (ITNs), while control houses received ITNs only. The intervention led to a significant 2.3-fold reduction in indoor malaria vector density, the primary entomologic outcome, largely influenced by An. gambiae s.l. mosquitoes. Fewer blood-fed mosquitoes were found in screened houses, indicating reduced human bites, which translated to six-fold decline in malaria prevalence (0.7%), the primary epidemiologic outcome, compared to control houses (4.3%). In contrast, Plasmodium sporozoite infection rates showed no differences between screened and control houses or agroecological zones, with An. arabiensis and An. funestus s.l. identified as the primary vectors. A modest protective effectiveness (22.6%) was observed, based on the estimated entomological inoculation rate of 0.24 and 0.31 infectious bites/person/night in screened and control houses, respectively, with no variation by agroecology. Despite the synergistic impact of HS with existing ITNs in reducing vector densities, human bite rates, and household malaria prevalence, sustained transmission persisted, partly due to the presence of highly competent vectors such as An. funestus s.l. which had an overall sporozoite rate of 68%. Future research should explore the interactions between vector behavioral adaptations, ecological and social factors contributing to residual transmission, even with seemingly effective control measures.

The bioecology of phlebotomine sand flies is intimately linked to the utilization of environmental resources including plant feeding. However, plant feeding behavior of sand flies remains largely understudied for Afrotropical species. Here, using a combination of biochemical, molecular, and chemical approaches, we decipher specific plant-feeding associations in field-collected sand flies from a dry ecology endemic for leishmaniasis in Kenya. Cold-anthrone test indicative of recent plant feeding showed that fructose positivity rates were similar in both sand fly sexes and between those sampled indoors and outdoors. Analysis of derived sequences of the ribulose-1,5-bisphosphate carboxylase large subunit gene (rbcL) from fructose-positive specimens implicated mainly Acacia plants in the family Fabaceae (73%) as those readily foraged on by both sexes of Phlebotomus and Sergentomyia. Chemical analysis by high performance liquid chromatography detected fructose as the most common sugar in sand flies and leaves of selected plant species in the Fabaceae family. Analysis of similarities (ANOSIM) of the headspace volatile profiles of selected Fabaceae plants identified benzyl alcohol, (Z)-linalool oxide, (E)-β-ocimene, p-cymene, p-cresol, and m-cresol, as discriminating compounds between the plant volatiles. These results indicate selective sand fly plant feeding and suggest that the discriminating volatile organic compounds could be exploited in attractive toxic sugar- and odor- bait technologies control strategies.

Phlebotomine sand flies transmit many viral protozoan and bacterial pathogens of public health importance. Knowledge of the ecologic factors influencing their distribution at local scale can provide insights into disease epidemiology and avenues for targeted control. Animal sheds, termite mounds and houses are important peri-domestic and domestic habitats utilized by different sand flies as resting or breeding habitats. However, our knowledge for selection of these habitats by sand flies remains poor. Here, we tested the hypothesis that these habitat types harbor different composition of sand fly species and differ in their volatile chemistry that could influence sand fly selection. To achieve this, we employed CDC light traps following a cross-sectional survey to investigate the distribution of sand flies in the three habitats in an endemic site for leishmaniasis in Kenya. The study was carried out during the dry season, when sand flies are optimally abundant in 2018 and 2020. Sand fly abundance did not vary between the habitats, but species-specific differences in abundance was evident. Measures of sand fly community structure (Shannon diversity and richness) were highest in animal shed, followed by termite mound and lowest inside human dwelling (house). This finding indicates broader attraction of both sexes of sand flies and females of varying physiological states to animal sheds potentially used as breeding or resting sites, but also as a signal for host presence for a blood meal. Furthermore, gas chromatographymass spectrometric analysis of volatiles collected from represented substrates associated with these habitats viz: human foot odor on worn socks (houses indoors), cow dung (animal sheds) and termite mounds (enclosed vent), revealed a total of 47 volatile organic compounds. Of these, 26, 35 and 16 were detected in human socks, cow dung and enclosed termite vent, respectively. Of these volatiles, 1-octen-3-ol, 6-methyl-5-hepten-2-one, α-pinene, benzyl alcohol, m-cresol, p-cresol and decanal, previously known as attractants for sandflies and other blood-feeding insects, were common to the habitats. Our results suggest that habitat volatiles may contribute to the composition of sand flies and highlight their potential for use in monitoring sand fly populations.