Nzioki et al. Parasites & Vectors (2023) 16:376 Parasites & Vectors https://doi.org/10.1186/s13071-023-05944-5 RESEARCH Open Access Differences in malaria vector biting behavior and changing vulnerability to malaria transmission in contrasting ecosystems of western Kenya Irene Nzioki1,2, Maxwell G. Machani1*, Shirley A. Onyango2, Kevin K. Kabui2, Andrew K. Githeko1, Eric Ochomo1, Guiyun Yan3 and Yaw A. Afrane4* Abstract 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 consti- tuted 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 *Correspondence: Maxwell G. Machani machani.maxwe2011@gmail.com Yaw A. Afrane yaw_afrane@yahoo.com Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://c reat iveco mmons. org/ licen ses/b y/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ public doma in/z ero/1.0 /) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Nzioki et al. Parasites & Vectors (2023) 16:376 Page 2 of 12 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. Keywords Malaria vectors, Biting behavior, Human behavior, Western Kenya Background The use of LLINs represents a powerful barrier against The wide-scale implementation of long-lasting insecti- indoor biting and resting malaria vectors, but their effi- cidal nets (LLINs) and indoor residual spraying (IRS) cacy is limited when people are not in bed, such as early as vector control tools has led to a substantial decline morning or outdoors in the evening [25, 26]. Outdoor in the malaria burden in sub-Saharan Africa in recent activities like farming and security jobs, as well as cul- years [1]. However, progress has stalled since 2015, tural practices, also increase the risk of malaria transmis- with the majority of African countries including Kenya sion, as they involve unprotected individuals overlapping experiencing persisting malaria transmission even with vector biting activity [25]. To achieve elimination, with universal LLIN use and limited IRS deployment understanding local changes in vector biting behavior [1, 2]. It appears that a variety of factors are imped- and identifying when and where people are exposed to ing the expected decrease in the incidence of malaria, vectors is crucial. This knowledge is key when evaluating for instance, widespread and increasing resistance to the likely success of the current indoor mosquito control insecticides and drugs [3, 4], weak health systems, soci- strategies and in designing effective interventions consid- oeconomic challenges, ecological changes, and climate ering the local eco-epidemiological context. This study, change [5, 6]. Additionally, malaria vectors have shifted therefore, assessed indoor and outdoor vector biting their behaviors to reduce exposure to insecticides [7, behavior and human habits and sleeping patterns poten- 8]. Such changes in vector populations threaten pro- tially contributing to persistent malaria transmission in gress toward malaria elimination targets [9, 10]. Exten- western Kenya. sive investigations have been conducted on vector responses to control tools [3, 11, 12]; however, under the current vector control conditions, detailed stud- Methods ies are needed to understand the prevailing nocturnal Study sites human activities and vector biting behavior dynamics. The study was carried out in Ahero (0° 0.11′ S, 34° 0.55′ Indoor interventions rely on vector nocturnal human E, altitude 1162  m above sea level), Kisian (00.0749° biting behavior. Historically, the primary malaria vec- S, 034.6663° E, altitude 1137–1330  m above sea level), tors Anopheles gambiae and Anopheles funestus have and Kimaeti (00.54057° N, 034.56410° E, altitude 1386– fed entirely indoors, with late-night peak biting activity 1545 m above sea level) (Fig. 1). The sites were selected [13]. This behavior coincides with the time most people based on past entomological studies [11, 21, 24, 27–29] are indoors and asleep. However, following the upscal- and different environmental settings. The Ahero and ing of control tools in sub-Saharan Africa, there is Kisian sites are malaria-endemic areas in the lowland growing evidence of malaria vectors shifting their bit- plains located adjacent to Lake Victoria in Kisumu ing behaviors toward times and places where people are County. The three malaria vector species, namely Anoph- not protected [14–19]. Host choice and resting patterns eles arabiensis, An. gambiae, and An. funestus, are pre- have also been observed to change to evade insecticide- sent, with An. arabiensis being the dominant species in treated nets (ITNs) [15]. In Kenya, the National Malaria the two lowland sites [21, 24]. Ahero is characterized by Control Program rolled out the universal bednet pro- large irrigation (rice) fields and cattle farming, with the gram in 2011, which led to an increase in the propor- irrigation fields and frequent flooding providing favora- tion of households owning at least one ITN, resulting in ble larval sites for malaria vector proliferation. Kisian an increase in coverage from 56 to 80% in 2015 [20, 21]. is known for cattle farming, which provides vectors Studies following the ITN universal coverage and IRS with alternative blood meal sources and brings them initiative have reported a shift in biting times [12, 18, into increased contact with humans [28, 29]. Kimaeti 21–23] and biting locations [24] of the primary malaria is located in a malaria epidemic-prone highland area in vectors (An. gambiae and An. funestus) from the classi- Bungoma County, western Kenya. The area practices cal known behaviors. The majority of these studies have mixed farming, with the main cash crop being tobacco focused on vector behavior, with less or no attention to and cattle farming. The three malaria vectors are pre- human habits and sleeping patterns in different eco- sent in the highlands, with An. gambiae and An. funes- epidemiological settings. tus being the dominant species depending on the season Nzioki et al. Parasites & Vectors (2023) 16:376 Page 3 of 12 Fig. 1 Map showing mosquito collection site in western Kenya [27, 30]. The highland and lowland sites of western Kenya coordinate the collection activity and carry out random have different levels of insecticide resistance [28]. spot checks during the collection nights to address any The western Kenya region experiences a bimodal rain- challenges and to keep the collectors awake. Partici- fall pattern, with the long rainy season from April to July, pants were screened for malaria parasites and given anti- which is followed by increased malaria incidence and malaria prophylaxis drugs 1 week before the start of the peak transmission. The short rainy season occurs from study to avoid the risk of contracting malaria during October to November. The hot and dry season is from the collection period. Anopheles mosquitoes collected January to March, which marks the lowest transmission each hour were identified morphologically the follow- period [2]. ing morning using a dissecting microscope according to standard taxonomic keys described by Coetzee [31]. Mosquito collection Three weeks following the long rainy season in June–July Anopheline species molecular identification 2021, mosquitoes were collected using the human land- The legs and wings of morphologically identified An. ing catch (HLC) method in four fixed houses that were gambiae sensu lato and An. funestus s.l. specimens were at least 300 m apart. Collections were conducted for four used for DNA extraction using the ethanol precipitation nights in each of the houses in all the study sites. Male method [32]. The sibling species of An. gambiae s.l. and adult volunteers, who acted as both bait and collector, An. funestus s.l. were distinguished using conventional were trained to minimize the variation between collec- polymerase chain reaction (PCR) [33, 34]. tors and to avoid mosquito bites. A total of 16 volun- teers were grouped into four teams. Each team consisted of four people, with two collecting mosquitoes indoors Detection of sporozoites and the other two outside each house. The mosquitoes Heads and thoraces of individual mosquitoes were were captured when they attempted to bite a collector used to test for the presence of Plasmodium falciparum sitting on a chair exposing their lower legs. Collections sporozoites using enzyme-linked immunosorbent assay were performed on four consecutive nights. The volun- (ELISA) [35]. teers collected mosquitoes for 45  min, with a 15-min break, and changed their sitting position to avoid bias Human behavior survey due to their attractiveness and skills. There were two col- A cross-sectional study design was employed to under- lection shifts: one collector worked from 18:00 to 00:00 stand human activity and sleeping patterns in three vil- during each collection night, followed by the second col- lages. These behaviors were assessed during the same lector from 00:00 to 07:00. A supervisor was assigned to period that vector collections were carried out. Fifty Nzioki et al. Parasites & Vectors (2023) 16:376 Page 4 of 12 households were randomly chosen and visited in each Results of the three villages studied. A household was defined Anopheline mosquito species composition and abundance as a house or a compound occupied by a group of indi- Overall, 2037 Anopheles females were collected from viduals during the study. The inhabitants were inter- the three sites during the study period. Of these, 76.7% viewed using a questionnaire containing questions on (n = 1565) were members of the An. funestus group, where they slept at night, what time they slept at night 22.8% (n = 465) belonged to An. gambiae s.l., and the and woke up in the morning, and the activities and remaining 0.5% (n = 7) belonged to the An. coustani cultural practices that kept them out at night. In cases group (Table 1). The An. funestus group was most abun- where a household had more than one adult, individu- dant in Ahero, at 96.7% (n = 1570), followed by An. gam- als were interviewed separately to prevent them from biae s.l. at 3% (n = 45) and An. coustani at 0.5% (n = 7). influencing each other in their responses. In addition, Out of 351 Anopheles females collected in Kisian, 86.6% data on bednet ownership (the proportion of house- (n = 304) were An. gambiae s.l. and 13.4% (n = 47) were holds that owned at least one LLIN) and utilization An. funestus group species. In Kimaeti village, all the (proportion of the population that had used LLINs mosquitoes collected were An. gambiae s.l. (n = 116). the previous night) by the households and other inter- Overall, 58.8% (95% CI 57–61%) of the mosquitoes were vention tools used for protection from mosquito bites captured indoors and 41.2% (95% CI 39–43%) outdoors. were recorded. The start and end times of sleep periods The variation between the indoor and outdoor num- and the time spent indoors and outdoors by inhabit- bers of biting mosquitoes was statistically significant ants were determined from the data collected from the (χ2 = 121.7, df = 1, P < 0.0001). households. Molecular identification confirmed all An. funestus s.l. assayed from the Ahero and Kisian sites to be An. funestus. Anopheles arabiensis was most abundant in the Data analysis lowland sites [Ahero 80.5% (95% CI 68.4–92.6%), Kisian The human biting rates (HBRs), which indicate the 79% (95% CI 75.2–85.1%)], followed by An. gambiae density of Anopheles, were calculated by dividing the [19.3% (95% CI 7.4–31.6%) and 21% (95% CI 14.8–24.8%), number of mosquitoes collected by the number of per- respectively]. In Kimaeti, An. gambiae [98.1% (95% CI sons per night during the sampling period separately 95.5–100%)] was the dominant species, followed by An. for indoor and outdoor venues [36]. Thus, throughout arabiensis [2% (95% CI 0.7–4.5%)]. The relative propor- the study, total Anopheles density during the night and tion of Anopheles species in mosquito samples varied sig- morning was evaluated as well as the hourly biting rate. nificantly among the sites (χ2 = 22.9, df = 2, P < 0.001). The degree of indoor biting was calculated as indoor HBR  18:00 → 06:00/(indoor HBR  18:00 → 06:00 + out- Hourly biting patterns of anophelines door HBR  18:00 → 06:00), while outdoor biting was The human biting activity of An. funestus in Ahero was calculated as outdoor HBR 18:00 → 06:00/(outdoor observed from dawn to dusk both indoors and outdoors, HBR  18:00 → 06:00 + indoor HBR  18:00 → 06:00) [37, with gradual peaks from midnight (00:00 to 01:00) (mean 38]. The nocturnal biting activity of each Anopheles 7.9 bites/person/hour) and a maximum peak at dawn species was expressed as mean number of each Anoph- (03:00–04:00) (mean 11.0 bites/person/hour) indoors. eles species landing per person per hour indoors or Anopheles funestus showed a steady increase in the late outdoors. The number of mosquitoes caught each morning, with peak biting activity at 05:30–06:30 (8.2 hour is assumed to represent the number of mosqui- mean bites/person/hour) outdoors (Fig.  2A) when peo- toes attempting to feed on humans for the same period. ple were out of bed. The biting activity of An. arabiensis The sporozoite rate was estimated as the proportion of was generally higher outdoors than indoors, with two mosquitoes positive for P. falciparum circumsporozo- peaks indoors at midnight and another one late morn- ite protein (CSP) over the total number of mosquitoes ing, 05:00–06:30 (mean 0.2 bites/person/hour). Increased tested. Descriptive statistics were used to summarize outdoor biting activity was observed in the early even- both household survey data and vector behavior data. ing between 19:00 and 20:00, and was pronounced in the The Chi-square test was also used to compare the late morning between 05:30 and 06:30 (0.3 bites/person/ indoor and outdoor biting rhythm of mosquitoes. The hour; Fig. 2B). non-parametric Kruskal–Wallis rank-sum analysis was On the other hand, An. funestus in Kisian showed a used to test for variation in biting rates among villages. steady increase in late morning activity, with peak bit- In all analyses, P < 0.05 was considered significant. Data ing activity at 04:30–05:30 (mean, 0.8 bites/person/hour) analysis was performed using the open-source R pro- indoors. The outdoor peak biting activity began at 04:30– gramming language software platform [39]. 06:30 (mean, 0.4 bites/person/hour; Fig. 2C). The biting N zioki et al. Parasites & Vectors (2023) 16:376 Page 5 of 12 Table 1 Summary of Anopheles species collected indoors and outdoors at different times of the night in Ahero, Kisian and Kimaeti villages Site Anopheles species Indoor Outdoor Total overall Early night Night (00–03 h) Early morning Total indoor Early night Night (00–03 h) Early morning Total outdoor (18–22 h) (03–07 h) (18–22 h) (03–7 h) Ahero An. funestus s.l. 104 394 443 941 41 204 332 577 1518 An. gambiae s.l. 7 8 7 22 9 2 12 23 45 An. coustani 1 0 1 2 1 0 4 5 7 Overall 112 402 451 965 51 206 348 605 1570 Kisian An. funestus s.l. 0 14 23 37 0 2 8 10 47 An. gambiae s.l. 35 37 59 131 39 53 81 173 304 Overall 35 51 82 168 39 55 89 183 351 Kimaeti An. gambiae s.l. 19 29 19 67 12 21 16 49 116 Total 166 482 552 1200 102 282 453 837 2037 Nzioki et al. Parasites & Vectors (2023) 16:376 Page 6 of 12 Fig. 2 Mean hourly human biting patterns of the Anopheles species in A, B Ahero (An. funestus and An. gambiae s.l.), C, D Kisian (An. funestus and An. gambiae s.l.), and E Kimaeti (An. gambiae s.l.) activity of An. gambiae s.l. was pronounced at the end of coustani were collected in Ahero (n = 7), and the major- midnight indoors (mean, 1.6 bites/person/hour, Fig. 2D). ity were collected outdoors (5/7). The indoor HBR of An. The outdoor biting activity was bimodal, with an early funestus was higher than outdoor HBR in Ahero [52.3 and smaller peak at 21:00–22:00 and a major peak late vs. 32.1 mosquitoes/person/night (m/p/n)] and Kisian morning at 04:30–05:30 (mean, 2.6 bites/person/hour; (2.1 vs. 0.5 m/p/n, respectively). The HBR for An. arabi- Fig. 2D), with activity then declining progressively during ensis was slightly higher outdoors than indoors in Kisian the morning. (10 vs. 7 m/p/n), while in Ahero it was similar between In the highlands (Kimaeti), the biting activity of An. indoors and outdoors (1.2 m/p/n). gambiae s.l. (mostly An. gambiae sensu stricto) indoors In the highland site (Kimaeti), 57.8% of An. gambiae s.l. was bimodal, with a major peak at midnight, 01:00–02:00 (mostly An. gambiae s.s.) collected were indoors, clearly (mean, 0.8 bites/person/hour; Fig. 2E), when people were indicating the preference of this species for feeding asleep and another one early in the morning, 03:00–04:00 indoors (endophagy). The indoor HBR for An. gambiae (mean, 0.6 bites/person/hour; Fig.  2E), when people s.l. was 3.7 m/p/n and outdoors was 2.7 m/p/n. were likely to be awake. The outdoor activity peaked late at midnight from 02:00 to 03:00. Additional informa- Sporozoite infectivity rates tion regarding biting based on the proportion of people In total, 489 An. funestus, 337 An. gambiae, 51 An. ara- indoors/outdoors and asleep/awake is given in Fig. 3. biensis, and seven An. coustani samples were tested for the presence of P. falciparum CSP. Overall, four samples Anopheline indoor and outdoor biting activity (two Ahero and two Kimaeti) tested positive, yielding an Overall, the majority of An. funestus collected from infection rate of 0.4% (2/474) in Ahero and 1.9% (2/105) Ahero and Kisian exhibited endophagic behavior (prefer- in Kimaeti. In Ahero, only An. funestus mosquitoes col- ence for feeding indoors) (Ahero, 62% and Kisian, 78.7%; lected indoors (0.3%) and outdoors (0.5%) were positive Table  2), while An. gambiae s.l. (mostly An. arabiensis) for P. falciparum CSP (Table  3). In Kimaeti, CSP was preferred feeding outdoors (exophagy) (Ahero, 51.1% and detected in the indoor and outdoor An. gambiae collec- Kisian, 56.7%, respectively; Table 2). Low numbers of An. tions, with infectivity rates of 1.5% and 2.6%, respectively. N zioki et al. Parasites & Vectors (2023) 16:376 Page 7 of 12 Fig. 3 Indoor and outdoor mean hourly biting rates of Anopheles mosquitoes with the proportion of people outdoors, indoors and awake, and indoors and asleep throughout the night in A Ahero, B Kisian, and C Kimaeti Table 2 Feeding behaviors of Anopheles species collected in Ahero, Kisian, and Kimaeti villages in western Kenya Site Anopheles species Biting activity Indoor (%) Outdoor (%) Protected hours (%) Unprotected hours (%) Ahero An. gambiae s.l. 48.9 51.1 33.3 66.7 An. funestus s.l. 62.0 38.0 57.0 43.0 An. gambiae s.s. 66.7 33.3 51.5 48.5 An. arabiensis 31.4 68.6 30.0 62.5 An. funestus s.s. 60.4 39.6 58.9 41.8 Kisian An. gambiae s.l. 43.1 56.9 47.0 53.0 An. funestus s.l. 78.7 21.3 38.3 61.7 An. gambiae s.s. 50.3 29.6 59.3 40.4 An. Arabiensis 86.1 23.8 22.2 69.4 An. funestus s.s. 80.0 13.0 66.7 28.6 Kimaeti An. gambiae s.l. 57.8 42.2 33.6 66.4 An. gambiae s.s. 62.5 37.5 41.5 58.7 No CSP positivity was detected in An. arabiensis or An. at least one LLIN. LLINs were the primary prevention coustani samples assayed or for mosquitos collected from method against mosquito bites and malaria infection. Kisian (n = 244). Overall, over 50% of the study participants reported hav- ing stayed outdoors or outdoors and indoors until 21:00 Human exposure to mosquito bites and protection (Fig. 3). About 77% of the respondents reported going to by LLINs bed by 22:00. In Ahero, 54% of preschool-age children The survey showed that LLIN use was high across the had gone to sleep, and 35% of school-going children, three study sites, with 91%, 99%, and 96.6% of house- 86% of adult women in Ahero, 46% in Kimaeti, and none holds in Kisian, Ahero, and Kimaeti, respectively, having in Kisian had gone to sleep by 22:00, while 14% of men Nzioki et al. Parasites & Vectors (2023) 16:376 Page 8 of 12 Table 3 Indoor and outdoor sporozoite rates of Anopheles mosquitoes collected from Ahero, Kisian, and Kimaeti villages in western Kenya Study site Sibling species No. tested Indoor (%) No. tested Outdoor (%) Overall (Pf+) Sporozoite infection rate Ahero An. gambiae s.s. 21 0 12 0 0 An. arabiensis 6 0 13 0 0 An. funestus s.s. 291 1 (0.3) 183 1 (0.5) 2 (0.4) An. Coustani 2 0 5 0 0 Kisian An. gambiae s.s. 100 0 99 0 0 An. arabiensis 25 0 5 0 0 An. funestus s.s. 12 0 3 0 0 Kimaeti An. gambiae s.s. 66 1 (1.5) 39 1 (2.6) 2 (1.9) An. arabiensis 2 0 0 0 0 Pf+ = P. falciparum positivity were asleep by 22:00. Overall, at 23:00, the majority (93%) and outdoors across the three sites, and nearly all expo- of the respondents were asleep while 7% were awake sure to malaria vectors peaked at this time (Fig. 3). indoors. The main activities that kept people outdoors between Across the study sites, it was observed that waking time 18:00 and 20:00 included household (domestic) chores, was between 04:00 and 07:00. About 10% of respondents playing, social–economic activities (i.e., selling at gro- were awake but indoors in the early morning (04:00) in cery stores), and social gatherings (Fig.  4). Night vigils Kisian and Ahero, coinciding with the time of high mos- and watching television after dinner were reported to quito biting (Fig. 3A, B). At 05:00, about 60% were awake keep the majority of men awake longer than their female Fig. 4 Indoor- and outdoor-specific human activities overlaid with the proportion of mosquitoes caught throughout the night in A Ahero, B Kisian, and C Kimaeti N zioki et al. Parasites & Vectors (2023) 16:376 Page 9 of 12 counterparts. Respondents woke early in the morning— greater likelihood of host-seeking indoors than outdoors for instance, women to prepare breakfast and children for the two primary vectors [18, 21, 24]. In Ahero (low- to go to school, and milking and other domestic chores land site), as expected, An. arabiensis preferred to bite including cleaning their houses and livestock areas. Agri- outdoors. In contrast, a higher proportion of An. arabi- cultural activities were also a major reason that people ensis were caught biting indoors in Kisian (lowland site), woke up early in the morning, in particular in Ahero, demonstrating that mosquito foraging behavior can vary where rice plantation is the main activity. noticeably within relatively small areas. The outdoor biting activity of this species in Ahero was found to be Discussion largely associated with cattle availability in the region, Understanding the biting behavior of malaria vectors, although this was not quantified in this study. Recent including the period, location. and frequency at which studies in Kisian have noted increased levels of insecti- humans are exposed to infectious mosquito bites in the cide resistance in An. arabiensis caught resting indoors field, plays a crucial role in the fight against malaria. This versus outdoors [28], and this could also explain the study outlines the variety of Anopheles nocturnal biting observed variations in biting activity. Of concern is the activity in different eco-epidemiological settings (high- fraction of An. gambiae and An. funestus observed bit- land and lowland areas of western Kenya), with data on ing outside the classical time (midnight) and whether human behavior that influence when and where disease these behaviors represent resilience or resistance, as transmission may occur. Overall, An. funestus, An. gam- this appears to reduce their chance of encountering biae s.l., and An. coustani were found to be the three indoor interventions (IRS and LLINs) [15, 48]. The sec- human-biting Anopheles species occurring both indoors ondary vector An. coustani was found to prefer foraging and outdoors. Anopheles funestus and An. gambiae were outdoors in Ahero (albeit in very low numbers, n = 7). the dominant vectors biting humans indoors, while An. Although this vector is not given much attention due arabiensis and An. coustani were more likely to bite out- to its exophagic and zoophilic feeding preferences [40], doors. The study further revealed early evening and late it has been reported to be susceptible to P. falciparum morning biting behavior both indoors (when people are infections [24, 49, 50]. The outdoor human biting activ- still active and unprotected by LLINs) and outdoors. ity observed in the current study also implies that it has These behaviors have implications for the risk of malaria a potential role in malaria transmission, pointing to the transmission and the effectiveness of interventions, need for integrated vector management (IVM) control particularly those that target human-feeding vectors strategies with a combination of non-chemical and chem- indoors. ical methods for more effective vector management—for The study revealed An. funestus as the predominant instance, biological larval source management, attractive vector biting humans in Ahero, while An. gambiae s.l. toxic sugar baits (ATSBs), and spatial repellents. prevailed in Kisian and Kimaeti. This difference in spe- The biting behaviors of An. arabiensis in the lowland cies abundance was attributed to the type of breeding sites revealed an early peak in the evening (19:00–20:00) habitats available in the study sites, season, degree of pre- outdoors and intense biting activity late in the morning disposition to biting humans, scaling up of insecticide- (04:30–06:30) both indoors and outdoors, a time when based interventions [40–43], and mosquito sampling local people are awake and not protected by LLINs. Our method employed [40, 44]. For instance, An. funestus is findings are in agreement with previous studies from the known to breed in permanent habitats with aquatic veg- same regions [18, 40] and elsewhere in Africa [26, 51] that etation cover [41], habitats typically found in Ahero rice observed an increase in morning outdoor biting of this irrigation schemes. Anopheles gambiae and An. arabien- species between 03:00 and 05:00. The increased biting sis prefer breeding in small, sunlit temporary water pools activity outdoors despite the equal chances of females bit- [45], the type of habitats found in the Kisian and Kimaeti ing the human bait in either of the two locations (indoors areas [46]. Studies on the distribution of anopheline spe- vs. outdoors) may have arisen from its preference for cies in rice fields have documented a succession between host-seeking outdoors. Anopheles arabiensis is known An. arabiensis and An. funestus [40, 47]. The increased to be flexible in behaviors, and in the presence of LLINs abundance of An. funestus indicates a significant contri- indoors and livestock outdoors, human–vector contact bution of this species in the transmission of malaria in is likely to be minimal as the vector seeks an alternative this region despite the widespread use of LLINs. host [24, 40, 52]. Anopheles funestus was responsible for Anopheles funestus and An. gambiae exhibited most vectors biting indoors in the lowlands, and this endophagic behavior, with higher proportions seeking observation accords with previous findings in the same a host indoors than outdoors. These findings corrobo- region [21]. In contrast to early studies on biting behav- rate earlier reports from western Kenya documenting a ior, which reported this vector maintaining its classical Nzioki et al. Parasites & Vectors (2023) 16:376 Page 10 of 12 biting habits (late night) in the same regions [11, 12], this high infection rates indoors. It is worth mentioning that study revealed a shift from classical to late-morning bit- high bednet ownership and usage of > 90% was con- ing activity (05:30–06:30) indoors and outdoors in both firmed in all three sites. The reaction of malaria vectors lowland sites. A plausible explanation for the extended to indoor-based interventions such as the excito-repel- periods of foraging to late in the morning could be a lence effects of pyrethroids used in LLINs [22] may force failure to access the preferred host (human) during the mosquitoes to shift their biting times, thus explaining the feeding hours (late night), forcing the mosquito to wait increase in outdoor transmission. This phenomenon can for the times the host is unprotected. Previous studies in be exacerbated by human behavior in areas where peo- western Kenya have shown pre-biting resting behavior in ple remain outdoors for long periods without protection An. funestus, where the vectors were seen resting on the [57]. In this study, over 50% of the population inter- walls before attacking the host [40, 47]. Recent studies viewed stayed outdoors or between outdoors and indoors have reported shifts in the biting behavior of An. funestus until 21:00. The majority of the respondents were asleep after universal LLIN coverage and IRS, from its historical by 23:00 (93%), and the waking time across the sites was biting times (late night) to late morning or daytime biting between 04:00 and 07:00, with about 10% waking up and [18, 22, 23]; however, it is not clear whether this behavior staying indoors at 04:00 and about 60% observed out- is due to plasticity or has a genetic basis. The observed doors in the morning at 05:00. Human behavior coincides behavior is worrisome, as this species (An. funestus) is with the vector biting patterns observed in this study. efficient in malaria transmission [24, 53, 54], and biting Previous reports indicated that people spend more time during times that people are not protected (indoors and outdoors before retiring to bed [21], with a high risk of outdoors) presents a gap in protection. infectious bites from An. funestus outdoors. Agricultural In the highlands (Kimaeti), only An. gambiae were col- practices (rice farming, milking), domestic chores, and lected, with previous studies confirming the species to social–economic activities (selling at grocery stalls) were be dominant in the region [27, 28]. This vector showed the main activities that kept people outdoors. Elsewhere, no change in biting activity, as the results indicate that electricity has been shown to influence community out- the species preferred feeding indoors, with pronounced door activity and sleeping times, as people stay up or out activity late at night between 01:00 and 02:00. Historical of bed for longer in the evening hours [57, 58], although studies have reported humans as the principal host for in this study we did not record the number of houses this species, unlike its sibling species An. arabiensis [13]. with electricity. However, this can be confirmed in this The persistence in feeding late at night indoors when study, as men were observed watching television indoors people are likely to be protected by LLINs can be par- and going to social gatherings (to watch football games) tially explained by increased resistance levels observed for longer hours in the evening. Therefore, the study find- in this species [28, 55]. Machani et  al. [56] investigated ings support previous claims that current control strat- the host-seeking activity of highly pyrethroid-resistant egies focusing on indoor-based interventions may not An. gambiae when a human bait was protected with a be sufficient to eliminate malaria transmission in most treated LLIN, and observed that, unlike susceptible mos- endemic regions [59]. quitoes, resistant mosquitoes attempted to bite a host sleeping under a treated bednet. The late-night biting behavior indoors by An. gambiae implies that compliance Conclusion with LLIN usage could offer protection from infective Anopheles funestus and An. gambiae were found to be bites during this period, as the peaks correspond to the responsible for malaria transmission in the region. The times of sleeping. Of concern is the small peak observed shifting in time of biting from classical biting to late in the early morning indoors (03:00–04:00) when people morning biting (indoor and outdoor) of An. funestus are waking up and remain unprotected by LLINs, as this and the early evening outdoor biting of An. arabiensis, could have implications for persistence of malaria trans- together with the high outdoor malaria transmission, mission indoors. could be due to pressure from the LLINs or humans Anopheles funestus and An. gambiae were responsi- spending more time unprotected outdoors. These find- ble for malaria transmission both indoors and outdoors ings have important implications for the epidemiology in the lowland and highland sites, respectively, with and strategies for the control of malaria in the study area. the majority of malaria infections occurring outdoors. Additional control strategies are needed for ongoing These findings are in agreement with previous studies interventions to better address the issue of residual trans- that observed the two vectors to be the main drivers of mission and reduce indoor and outdoor biting vectors malaria transmission in the region [21, 24, 27]; however, using a more diverse toolbox with IVM strategies. contrary to the present study, the earlier studies reported Nzioki et al. Parasites & Vectors (2023) 16:376 Page 11 of 12 Abbreviations 3. Ranson H, Lissenden N. Insecticide resistance in African Anopheles HLC Human landing catch mosquitoes: a worsening situation that needs urgent action to main- HBR Human biting rate tain malaria control. Trends Parasitol. 2016;32:187–96. CSP Circumsporozoite protein 4. WHO, 2022. World malaria report 2022. ITN I nsecticide-treated net 5. Okumu F, Gyapong M, Casamitjana N, Castro MC, Itoe MA, Okonofua LLIN Long-lasting insecticidal net F, et al. What Africa can do to accelerate and sustain progress against IRS Indoor residual spraying malaria. PLoS Glob Public Health. 2022;2:e0000262. 6. Yewhalaw D, Kweka EJ, Yewhalaw D, Kweka EJ. Insecticide resistance Acknowledgements in East Africa—history, distribution and drawbacks on malaria vectors The authors wish to thank the volunteers for their participation in this study. and disease control. Insecticides resistance; 2016. We acknowledge the Entomology Laboratory at Kenya Medical Research Insti- 7. Killeen GF, Chitnis N. Potential causes and consequences of behav- tute, Kisumu, for providing technical support. Permission to publish this study ioural resilience and resistance in malaria vector populations: a math- was granted by the director of Kenya Medical Research Institute. ematical modelling analysis. Malar J. 2014;13:97. 8. Carrasco D, Lefèvre T, Moiroux N, Pennetier C, Chandre F, Cohuet A. Author contributions Behavioural adaptations of mosquito vectors to insecticide control. MM, EO, and YAA conceived and designed the study. IN, MGM, and SAO Curr Opin Insect Sci. 2019;34:48–54. performed data collection. IN, MGM, and SAO performed data analysis and 9. Antonio-Nkondjio C, Kerah CH, Simard F, Awono-Ambene P, Chouaibou drafted the manuscript. EO, GY, and YAA supervised data collection and manu- M, Tchuinkam T, et al. Complexity of the malaria vectorial system in script writing. All authors have read and approved the final manuscript. Cameroon: contribution of secondary vectors to malaria transmission. J Med Entomol. 2006;43:1215–21. Funding 10. Killeen GF, Seyoum A, Sikaala C, Zomboko AS, Gimnig JE, Govella NJ, This study was supported by grants from the National Institutes of Health et al. Eliminating malaria vectors. Parasit Vectors. 2013;6:172. (R01 AI123074, U19 AI129326, R01 AI050243, D43 TW001505). There was no 11. Githeko AK, Mbogo CM, Atieli FK. Resting behaviour, ecology and additional external funding received for this study. genetics of malaria vectors in large-scale agricultural areas of Western Kenya. Parassitologia. 1996;38:481–9. Availability of data and materials 12. Bayoh MN, Walker ED, Kosgei J, Ombok M, Olang GB, Githeko AK, et al. The dataset supporting the conclusions of this article is included within the Persistently high estimates of late night, indoor exposure to malaria article. vectors despite high coverage of insecticide treated nets. Parasit Vec- tors. 2014;7:380. 13. Gillies M, Coetzee M. A supplement to the Anophelinae of Africa Declarations south of the Sahara (Afrotropical Region). Publ Sth Afr Inst Med Res. 1987;55:1–143. Ethics approval and consent to participate 14. Briët OJ, Chitnis N. Effects of changing mosquito host searching behav- Ethical approval was obtained from the Ethical Review Board (Ref.: KEMRI/ iour on the cost effectiveness of a mass distribution of long-lasting, SERU/085/3434) at the Kenya Medical Research Institute. The area chief, insecticidal nets: a modelling study. Malar J. 2013;12:1–11. sub-chief, village elders, and relevant county authorities were sensitized on 15. Gatton ML, Chitnis N, Churcher T, Donnelly MJ, Ghani AC, Godfray HCJ, the study activities planned and permission obtained. Household heads and et al. The importance of mosquito behavioural adaptations to malaria participants provided written consent to be interviewed and their houses control in Africa. Evolution. 2013;67:1218–30. used for mosquito collections. Informed consent was obtained from the 16. Reddy MR, Overgaard HJ, Abaga S, Reddy VP, Caccone A, Kiszewski volunteers who were trained to collect landing mosquitoes to minimize the AE, et al. Outdoor host seeking behaviour of Anopheles gambiae mos- risk of malaria transmission. All experiments and methods were carried out in quitoes following initiation of malaria vector control on Bioko Island. accordance with the relevant guidelines and regulations of SERU. Equator Guinea Malar J. 2011;10:1–10. 17. Cooke MK, Kahindi SC, Oriango RM, Owaga C, Ayoma E, Mabuka D, Competing interests et al. ‘A bite before bed’: exposure to malaria vectors outside the times The authors have declared that they have no competing interests. of net use in the highlands of western Kenya. Malar J. 2015;14:1–15. 18. Wamae PM, Githeko AK, Otieno GO, Kabiru EW, Duombia SO. Early bit- Consent for Publication ing of the Anopheles gambiae s.s. and its challenges to vector control Not applicable. using insecticide treated nets in western Kenya highlands. Acta Trop. 2015;150:136–42. Author details 19. Meyers JI, Pathikonda S, Popkin-Hall ZR, Medeiros MC, Fuseini G, Matias 1 Centre for Global Health Research, Kenya Medical Research Institute, Kisumu, A, et al. Increasing outdoor host-seeking in Anopheles gambiae over 6 Kenya. 2 School of Zoological Sciences, Kenyatta University, Nairobi, Kenya. years of vector control on Bioko Island. Malar J. 2016;15:1–13. 3 Program in Public Health, College of Health Sciences, University of Califor- 20. Zhou G, Li JS, Ototo EN, Atieli HE, Githeko AK, Yan G. Evaluation of nia, Irvine, CA 92697, USA. 4 Department of Medical Microbiology, University universal coverage of insecticide-treated nets in western Kenya: field of Ghana Medical School, College of Health Sciences, University of Ghana, surveys. Malar J. 2014;13:351. 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Am J Trop Med Hyg. 2014;90:597. • support for research data, including large and complex data types 44. Mathenge EM, Misiani GO, Oulo DO, Irungu LW, Ndegwa PN, Smith TA, et al. Comparative performance of the Mbita trap, CDC light trap and the • gold Open Access which fosters wider collaboration and increased citations human landing catch in the sampling of Anopheles arabiensis, An. funestus • maximum visibility for your research: over 100M website views per year and culicine species in a rice irrigation in western Kenya. Malar J. 2005;4:7. 45. Gimnig JE, Ombok M, Kamau L, Hawley WA. Characteristics of larval At BMC, research is always in progress. anopheline (Diptera: Culicidae) habitats in Western Kenya. J Med Ento- mol. 2001;38:282–8. Learn more biomedcentral.com/submissions