Resting behaviour of malaria vectors in highland and lowland sites of western Kenya: Implication on malaria vector control measures

Maxwell G Machani; Eric Ochomo; Fred Amimo; Jackline Kosgei; Stephen Munga; Guofa Zhou; Andrew K Githeko; Guiyun Yan; Yaw A Afrane

doi:10.1371/journal.pone.0224718

. 2020 Feb 25;15(2):e0224718. doi: 10.1371/journal.pone.0224718

Resting behaviour of malaria vectors in highland and lowland sites of western Kenya: Implication on malaria vector control measures

Maxwell G Machani ^1,², Eric Ochomo ¹, Fred Amimo ², Jackline Kosgei ¹, Stephen Munga ³, Guofa Zhou ⁴, Andrew K Githeko ³, Guiyun Yan ⁴, Yaw A Afrane ^5,^*

Editor: Friedrich Frischknecht⁶

PMCID: PMC7041793 PMID: 32097407

Abstract

Background

Understanding the interactions between increased insecticide resistance and resting behaviour patterns of malaria mosquitoes is important for planning of adequate vector control. This study was designed to investigate the resting behavior, host preference and rates of Plasmodium falciparum infection in relation to insecticide resistance of malaria vectors in different ecologies of western Kenya.

Methods

Anopheles mosquito collections were carried out during the dry and rainy seasons in Kisian (lowland site) and Bungoma (highland site), both in western Kenya using pyrethrum spray catches (PSC), mechanical aspiration (Prokopack) for indoor collections, clay pots, pit shelter and Prokopack for outdoor collections. WHO tube bioassay was used to determine levels of phenotypic resistance of indoor and outdoor collected mosquitoes to deltamethrin. PCR-based molecular diagnostics were used for mosquito speciation, genotype for knockdown resistance mutations (1014S and 1014F) and to determine specific host blood meal origins. Enzyme-linked Immunosorbent Assay (ELISA) was used to determine mosquito sporozoite infections.

Results

Anopheles gambiae s.l. was the most predominant species (75%, n = 2706) followed by An. funestus s.l. (25%, n = 860). An. gambiae s.s hereafter (An. gambiae) accounted for 91% (95% CI: 89–93) and An. arabiensis 8% (95% CI: 6–9) in Bungoma, while in Kisian, An. arabiensis composition was 60% (95% CI: 55–66) and An. gambiae 39% (95% CI: 34–44). The resting densities of An. gambiae s.l and An. funestus were higher indoors than outdoor in both sites (An. gambiae s.l; F₁, ₆₅₅ = 41.928, p < 0.0001, An. funestus; F₁, ₆₅₅ = 36.555, p < 0.0001). The mortality rate for indoor and outdoor resting An. gambiae s.l F1 progeny was 37% (95% CI: 34–39) vs 67% (95% CI: 62–69) respectively in Bungoma. In Kisian, the mortality rate was 67% (95% CI: 61–73) vs 76% (95% CI: 71–80) respectively. The mortality rate for F1 progeny of An. funestus resting indoors in Bungoma was 32% (95% CI: 28–35). The 1014S mutation was only detected in indoor resitng An. arabiensis. Similarly, the 1014F mutation was present only in indoor resting An. gambiae. The sporozoite rates were highest in An. funestus followed by An. gambiae, and An. arabiensis resting indoors at 11% (34/311), 8% (47/618) and 4% (1/27) respectively in Bungoma. Overall, in Bungoma, the sporozoite rate for indoor resting mosquitoes was 9% (82/956) and 4% (8/190) for outdoors. In Kisian, the sporozoite rate was 1% (1/112) for indoor resting An. gambiae. None of the outdoor collected mosquitoes in Kisian tested positive for sporozoite infections (n = 73).

Conclusion

The study reports high indoor resting densities of An. gambiae and An. funestus, insecticide resistance, and persistence of malaria transmission indoors regardless of the use of long-lasting insecticidal nets (LLINs). These findings underline the difficulties of controlling malaria vectors resting and biting indoors using the current interventions. Supplemental vector control tools and implementation of sustainable insecticide resistance management strategies are needed in western Kenya.

Background

Malaria still remains a major public health concern in sub-Saharan Africa, responsible for an estimated 228 million cases and 405,000 deaths despite the massive investments in scaling-up indoor anti-vector interventions [1]. Remarkable advances in the fight against malaria have been achieved within the past decade mainly through the massive scale-up of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) in many localities [2,3]. Despite the increased efforts, it is worrying that no significant progress has been made in reducing global malaria cases in the year 2015–2017 period [4], with some regions in sub-Saharan Africa, previously reported to experience a resurgence of malaria including western Kenya [5]. This transmission recurrence is partly attributed to the emergence of insecticide resistance and behavioural modification that have arisen as an adaptation by mosquitoes in response to high use of insecticides for vector control [6,7]. All these factors have the potential to weaken malaria control programs thus posing a serious threat in the fight against malaria [8].

The current vector control interventions take advantage of susceptible mosquito behaviors. These interventions are based on the observation that malaria vectors prefer to bite humans indoors late at night and often rest inside houses after blood feeding hence, they will be exposed to sufficient levels of insecticides which will either kill them or reduce their longevity thus affecting their vectorial capacity [4]. In sub-Saharan Africa insecticide-treated net (ITN) ownership is estimated to have increased from 3% in 2000 to 83% in the period of 2015–2017[4]. In Kenya, the government rolled out the universal bed net programme where every two persons in a household were provided with a free ITN. The ITN ownership rose from 12.8% in 2004 to over 80% in 2015[9,10] The increased use of indoor interventions may pose stress on the indoor feeding and resting of malaria vectors leading to either behavioural defense [11] or physiological defense [6]. Malaria vectors have been shown to adapt to changing environment due to either behavioural avoidance or selection of mutations and recombination that favour their survival in the presence of insecticides threatening the efficacy of the current indoor-based vector control tools [6,12] and the resulting increase in residual transmission [13]. Insecticide resistance is common in sub-Saharan Africa with some regions reporting resistance to all classes of insecticides [14,15]. In Kenya, the target site and metabolic resistance mechanisms play a major role in pyrethroid resistance [16,17]. The primary malaria vectors in Kenya belong to An. gambiae complex and An. funestus group due to their anthropophilic and endophilic behaviours that makes them be more efficient in malaria transmission [18–20]. With the scale-up of indoor-based vector control tools mosquitoes have changed behaviours; some are biting and resting indoors whilst others have changed to prefer biting and resting outdoors. Behavioral modifications including changes in biting time and location [21–23], changes in host choice and shift from endophilic (i.e. resting in houses) to exophilic (i.e. resting outdoors) behavior have been associated with long-term use of insecticide-based interventions [24,25]. Knowledge of the resting habits of resistant vectors and their feeding preference may predict the intensity of malaria transmission. It is hypothesized that insecticide-resistant malaria vectors could bite and rest indoors in the presence of interventions whilst susceptible ones bite and rest outdoors. Additionally, behaviors of malaria vectors have been shown to differ on small geographical scales, further complicating malaria elimination efforts[26]. Understanding how the resting habits of malaria vectors change in response to current indoor-based vector control interventions is important for sustaining vector control. These behavioral modifications and physiological resistance in most of the malaria vectors have been shown to contribute to maintaining malaria transmission [12,27].

In order to improve vector control intervention strategies, it is crucial to characterize the behavioral patterns of each species of a particular vectorial system in their specific settings over time and in a range of environmental changes, especially with increasing pyrethroid resistance. The objective of this study was to investigate the species diversity of malaria vectors, their resting behavior, and the distribution of infections in two ecological settings of western Kenya with different levels of insecticide resistance. This information could provide a better understanding of the interactions between increased insecticide resistance phenotypes in field malaria vector population and the subsequent resting behaviour patterns in the presence of the current indoor intervention.

Methods

Study site

The study was carried out in Kisian (00.02464°S, 033.60187°E, altitude 1,280–1,330 m above sea level), Kisumu county and Bungoma (00.54057°N, 034.56410°E, altitude 1386–1,545 m above sea level) in Bungoma County, all in western Kenya. Kisian is located in the lowland area around Lake Victoria in western Kenya. An. gambiae sensu stricto (s.s.), An. arabiensis and An. funestus are the main vectors of malaria in this region [17,28]. Bungoma County is located in malaria epidemic-prone highland area. The sites experience a bimodal pattern of rainfall, with the long rainy season (April—July) which triggers peak malaria transmission period and the short rainy season (October-November) and year-to-year variation. The hot and dry season is from January to March which marks the lowest transmission period[5] Both sites are endemic for Plasmodium falciparum malaria. The malaria vector population in both sites include An. gambiae and An. arabiensis and Anopheles funestus [16,17]

Mosquito sampling

Mosquitoes were sampled during the middle of the long dry season (February-March) and four weeks after the start of the long rainy season (May-July) in 2018. Indoor resting malaria vectors were sampled using pyrethrum spray catches (PSCs) in sixty (60) randomly selected houses from 06:00 to 09:00 h [23]. The prokopack aspirator (John W Hock, Gainesville, FL, USA) was used to collect mosquitoes resting indoors and outdoors from the selected houses every morning. For indoor collections, mosquitoes resting on the walls and under the roof of the house or ceiling, under the beds were systematically aspirated. Outdoor sampling points included granaries, kitchens and evening outdoor human resting points. Outdoor resting mosquitoes were additionally collected from pit shelters constructed according to Muirhead-Thomson's method [29] within 20 m of each selected house, resting mosquitoes in the cavities created in the pit shelter were collected from 06:00 to 09:00 h by using Prokopack. Clay-pots were used to collect outdoor resting mosquitoes. The pots were placed then left outdoors from 18:00 to 06:00h at about 5m from the window of selected houses [30]. Resting mosquitoes in the pots were collected in the morning from 06:00 to 09:00h by placing a white mesh from a mosquito cage over the mouth and agitating the mosquitoes inside the pot, causing them to fly and move to the cage [30,31]. The pot was checked for the remaining mosquitoes and were collected using an aspirator to a well-labeled paper cup. Anopheline mosquitoes were sorted morphologically according to the identification keys described by [32]. Female mosquitoes were further classified according to their gonotrophic status. Mosquitoes from each collection method were stored in separately labeled vials and preserved by desiccation.

Some of the collected indoor and outdoor resting mosquitoes that were either blood-fed or gravid from the two sites were kept in paper cups covered with moistened cotton towels and transported to the insectary at Kenya medical research institute in Kisumu. Gravid An. gambiae s.l and An. funestus s.l females were provided with oviposition cups. Eggs laid were allowed to hatch in spring water in small trays and larvae reared on a mixture of tetramin (fish food) and brewer’s yeast provided daily under controlled standard insectary conditions with a temperature range of 26± 2°C and 70% to 80% relative humidity. Emerging adults were provided with a 10% sugar solution until ready to be used for bioassay tests.

WHO resistance bioassays

To assess susceptibility or resistance of F1 progeny of mosquitoes caught from different locations(indoor and outdoor) and study sites, emerging female adults aged 2–5 days were exposed to 0.05% deltamethrin following the standard WHO tube test protocol[33] for 1 h. The knockdown time (KDT) of females was reported every 10 min during the 60 min exposure period. After the 1 h exposure, surviving mosquitoes were transferred to recovery tubes and provided with 10% sucrose for 24 h holding period. Mosquitoes alive 24 h after the 60-min insecticide-exposure time were classified as resistant. Mortality was scored after the 24 h recovery period.

Anopheline species discrimination

Sibling species of the An. gambiae and An. funestus complexes were distinguished using conventional PCR [34,35]. DNA was extracted from mosquito legs and wings using ethanol precipitation method [36].

Detection of sporozoite infectivity

The head and thorax of individual mosquitoes samples collected were used to detect the presence of P. falciparum sporozoites using enzyme-linked immunosorbent assays (ELISA) method as described by Wirtz et al. [37].

Detection of blood meal sources using polymerase chain reaction (PCR)

The abdominal section of blood-fed Anopheles mosquitoes were cut transversely between the thorax and the abdomen. Genomic DNA was extracted from mosquito abdomens using ethanol precipitation method as described by Collins et al. [36]. One universal reverse primer and five animal-specific forward primers (human, cow, goat, pig, and dog) were used for amplification of cytochrome b gene, encoded in the mitochondrial genome to test for specific host blood meal origins using conventional PCR [38].

Genotyping for kdr mutations

DNA was extracted from adult An. gambiae and An. arabiensis mosquitoes as earlier described [34]. Real-time (RT) PCR was used to detect mutations at amino acid position 1014 of the voltage-gated sodium channel (Vgsc) using a modification of the protocol by Bass et al. [39]. Samples were genotyped for both Vgsc-1014S and 1014F kdr alleles.

Scientific and ethical clearance

The institutional review board of Kenya Medical Research Institute granted ethical review and approval (Ref: KEMRI/SERU/CGHR/085/3434). Prior to the commencement of data collection, a detailed explanation of the aims, study procedures, risks and benefits were provided to community leaders and participants of each study site. Informed consent was obtained from the household heads. Participation was voluntary and household heads were free to withdraw from the study in case of any inconvenience.

Data analysis

Resting density of Anopheline mosquitoes was calculated as the number of female mosquitoes per trap/night for each trapping method. Analysis of variance (ANOVA) was used to compare malaria vector density between indoor and outdoor locations. Chi-square was used to test the difference in seasonal abundance and malaria vector species composition between resting locations (indoor and outdoor). Human blood index (HBI) was calculated as the proportion of blood-fed mosquito samples that had fed on humans to the total tested for blood meal origins [40]. Bovine, goat, dog, and pig blood indices were also calculated in a similar way. Mixed blood meals were included in the calculation of blood meal indices [41].

The sporozoite infection rate (IR) expressed as the proportion of mosquitoes positive for Plasmodium sporozoite was calculated by dividing the number of sporozoite positive mosquitoes by the total number of mosquitoes assayed. The frequency of the resistance allele was calculated using the Hardy-Weinberg equilibrium test for kdr genotypes. Data were analyzed using R software packages.

Results

Indoor and outdoor Anopheline mosquito composition

A total of 2,706 and 860 female Anopheline mosquitoes were collected from Bungoma (highland site) and Kisian (lowland site) respectively during the study period. Anopheles gambiae s.l was the most abundant species accounting for 70% (95% CI: 68–71) in Bungoma and 91% (95% CI: 89–93) in Kisian followed by An. funestus 31% (95% CI: 29–32) and 10% (95% CI: 7–11) respectively. In Bungoma out of 1880 An. gambiae s.l collected, 85% (1606/1880) was caught resting indoors and 15% (274/1880) were caught resting outdoors. For An. funestus, 97% (798/826) were caught resting indoors and 3% (28/826) were caught outdoors. In Kisian, 58% (453/781) An. gambiae s.l were resting indoors and 42% (328/781) were caught resting outdoors. For An. funestus, 91% (72/79) were collected indoors and 9% (7/79) was caught resting outdoors (S1 Table). The mean indoor resting density of An. gambiae s.l from both sites was significantly higher than outdoor resting density (F₁, ₆₅₅ = 41.928, p < 0.0001). The mean indoor resting density for An. funestus was also higher than outdoor resting density (F₁, ₆₅₅ = 36.555, p < 0.0001) (Fig 1).

Anopheles gambiae and An. funestus sibling species composition

For species identification, a sub-sample of 1,566 from both sites (1,172 An. gambiae s.l and 394 An. funestus s.l) were used to discriminate the sibling species. In Bungoma, 843 samples of An. gambiae s.l were analysed. Of these 91% (95% CI: 89–93) were An. gambiae and 8% (95% CI: 6–9) were An. arabiensis. Overall, of the three vector species, An. gambiae (66%, 95% CI: 64–70) was the predominant malaria vector in Bungoma followed by An. funestus (28%, 95% CI: 25–30) and An. arabiensis (5%, 95% CI: 4–7). There was a significant difference between indoor and outdoor locations in terms of mosquito species composition in Bungoma (X² = 122.96, df = 2, p < 0.0001). In Kisian, out of the 329 samples analysed 60% (95% CI: 55–66) were An. arabiensis and 39% (95% CI: 34–44) were An. gambiae. Overall An. arabiensis (49%, 95% CI: 44–54) was the most abundant vector species followed by An. gambiae (32%, 95% CI: 27–36) and An. funestus (19%, 95% CI: 15–23, Fig 2). All the An. funestus s.l assayed from the two study sites were all An. funestus s.s (hereafter An. funestus).

In Bungoma, the seasonal composition of An. gambiae and An. funestus species was higher during the rainy season (57%, 95% CI: 53–61) and (67%, 95% CI: 63–73) compared to the dry season (43%, 95% CI: 39–47) and vs (32%, 95% CI: 25–37)(X² = 16.28, df = 2, p < 0.0003) respectively. In contrast, in Kisian the overall seasonal prevalence of the three vector species composition was higher during the dry season than rainy season (An. arabiensis, 68% (95% CI: 61–76) and 32% (95% CI: 24–40), An. funestus, 63 (95% CI: 52–74) and 37% (95% CI: 21–42); X² = 30.42, df = 2, p < 0.0001, Fig 2).

Phenotypic resistance

All the F1 mosquito populations tested from Bungoma and Kisian showed remarkable resistance to deltamethrin, with mortality rates ranging from 32–76% (Fig 3). Reduced mortality was observed for F1 progeny of Anopheles gambiae s.l resting indoors (37%, 95% CI: 34–39) than outdoors (67%, 95% CI: 62–69) in Bungoma. In Kisian the F1 progeny of Anopheles gambiae s.l resting indoors had lower mortality rates (67%, 95% CI: 61–73) than outdoors (76%, 95% CI: 71–80). Though the levels of deltamethrin resistance observed were higher for mosquitoes resting indoors compared to outdoors across the sites, there was no significant difference between the means (F₃, ₂₈ = 1.391, p < 0.266). The mortality rate for F1 progeny of Anopheles funestus resting indoors from Bungoma was 32% (95% CI: 28–35). Due to technical difficulties in raising An. funestus and the small numbers collected resting outdoors, susceptibility test was not done in Kisian and for the outdoor population from Bungoma. The An. gambiae s.s Kisumu susceptible laboratory strain showed 100% mortality.

Fig 3 — Dotted lines represent upper (98%) and lower (90%) cut-offs for WHO classifications; values above the upper line indicate susceptibility and values below the lower red line indicate resistance (WHO, 2016). Error bars for the mean are shown.

Target site genotyping

In total 693 Anopheles, gambiae s.l samples were genotyped for the presence of Vgsc-1014S and 1014F mutations. In Bungoma, overall the frequency of Vgsc-1014S was 88% (290/328) and 6% (20/328) for Vgsc-1014F in An. gambiae, whereas only Vgsc-1014S was observed in An. arabiensis with a low frequency of 4% (2/52) (Table 1). The frequency of Vgsc-1014S and 1014F was 90 (177/195) vs 9% (20/195) respectively for indoor resting An. gambiae. Only Vgsc-1014S was observed for outdoor collections with a frequency of 85% (113/133). The Vgsc-1014S was the only kdr mutation observed in An. arabiensis resting indoors (10%, 2/20) and was not detected in the outdoor resting collections (S2 Table). In Kisian, the frequency of Vgsc-1014S in An. gambiae was 70% (89/125) and that of 1014F was 1% (2/125), whereas only Vgsc-1014S was observed in An. arabiensis with a frequency of 18% (36/188). The frequency of Vgsc-1014S mutation for An. gambiae resting indoors was 72% (78/107) and 61% (11/18) for outdoor collections. The same mutation was present in An. arabiensis collected from indoors 18% (20/110) and outdoors 18% (16/78). The Vgsc-1014F was only observed in An. gambiae caught resting indoors at a low frequency of 1%.

Table 1. Frequency of Kdr mutations in An. gambiae s.s and An. arabiensis populations of western Kenya.

Site	Species	Location	An. gambiae s.s			An. arabiensis
Site	Species	Location	N tested	1014S	1014F	N tested	1014S	1014F
Bungoma		Indoor	195	90	9	20	10	0
		Outdoor	133	85	0	32	0	0
	An. gambiae		328	86	6
	An. arabiensis					52	3.8	0
Kisian		Indoor	107	72	1	110	18	0
		Outdoor	18	61	0	78	18	0
	An. gambiae		125	70	1
	An. arabiensis					188	18	0

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Blood meal sources

A total of 857 blood-fed (719 An. gambiae s.l and 138 An. funestus) mosquito specimens were analysed for blood meal origins (Table 2). An. funestus was the most anthropophagic species and An. arabiensis the least from both sites. In Bungoma, the human blood index (HBI) for An. gambiae, An. arabiensis and An. funestus caught resting indoors were 65% (273/422), 25% (6/24) and 75% (83/111) respectively. For outdoor resting An. gambiae, An. arabiensis and An. funestus the HBI was 46% (18/39), 25% (2/8) and 75% (3/4) respectively. In Kisian, the overall HBI of An. gambiae, An. arabiensis and An. funestus was 60, 7 and 83% respectively. The HBI for An. gambiae resting outdoors was 100% (3/3 and 60% (46/77) for indoor collections. The HBI for indoor and outdoor resting An. arabiensis was 7% (8/114) vs 6% (2/32). The HBI of An. funestus resting indoors was 83% (19/23).

Table 2. Blood meal origins of Anopheline mosquitoes collected from indoor and outdoor in Bungoma and Kisian, western Kenya.

Site	Blood-meal origins	An.gambiae s.s		An. arabiensis		An. funestus
Site	Blood-meal origins	Indoor	Outdoor	Indoor	Outdoor	Indoor	Outdoor
Bungoma	Number tested	422	39	24	8	111	4
	Human	229 (54)	13 (33)	6 (25)	1 (13)	72 (65)	3 (75)
	Bovine	60 (14)	9 (23)	16 (67)	3 (38)	14 (13)	1 (25)
	Human+Bovine	31 (7)	4 (10)	0	1 (13)	9 (8)	0
	Other	24 (6)	4 (1)	2 (8)	1 (13)	3 (3)	0
	Un-identified	78 (19)	9 (23)	0	2 (25)	13 (12)	0
	HBI	65	46	25	25	75	75
	BBI	22	36	67	63	21	25
Kisian	Number tested	77	3	114	32	23	0
	Human	42 (56)	2 (67)	6 (5)	0	18 (78)	0
	Bovine	26 (34)	0	104 (91)	29 (91)	4 (17)	0
	Human+Bovine	3 (4)	1 (33)	2 (2)	1 (3)	1 (4)	0
	Other	4 (5)	0	1 (1)	2 (6)	1 (4)
	Un-identified	2 (3)	0	1 (1)	0	0	0
	HBI	60	100	7	6	83	0
	BBI	40	33	93	97	22	0

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Others: dog, goat, pig, or multi-source excluding human+bovine

HBI, human blood Index; BBI, bovine blood index

Sporozoites infection rates

A total of 1,517 samples comprising of 1,156 An. gambiae s.l and 361 An. funestus specimens were tested for Plasmodium falciparum Circumsporozoite (CSP) (Table 3). Ninety-one samples (90 Bungoma and 1 Kisian) tested positive giving an overall infection rate of 8% in Bungoma and 0.3% in Kisian. Overall, the sporozoite rate was higher indoors (9%, 82/956) than outdoors (4%, 8/190) in Bungoma, whereas in Kisian the sporozoite rate was 0.3%, 1/332) indoors. None of the samples collected outdoors in Kisian tested positive (n = 73). The sporozoite for An. funestus resting indoors was 11% (34/311). None of An. funestus collected outdoors was positive. The sporozoite rate for indoor resting An. gambiae was 8% (47/618) and outdoors at 5% (7/148). For An. arabiensis caught resting indoors, the sporozoite rate was 4% (1/27) and 3% (1/35) for outdoors. In Kisian, only 1/112 (1%) An. gambiae collected from indoor was CSP positive. No CSP positives were detected in An. arabiensis and An. funestus resting indoors and outdoors in Kisian. The overall entomological inoculation rates (EIRs) of the three vector species from indoor resting collections and outdoor in Bungoma was 66 and 10 infective bites/person/year respectively. In Kisian the overall EIR from indoor collections was 1.2 infective bites/person/year.

Table 3. Sporozoite rates of Anopheles mosquitoes from indoor and outdoor collections in Bungoma and Kisian, western Kenya.

Site	Season	Location	An. gambiae s.s		An. arabiensis		An. funestus
Site	Season	Location	N tested	Pf +ve	N tested	Pf +ve	N tested	Pf +ve
Bungoma		Indoor	618	47(8)	27	1(4)	311	34(11)
		Outdoor	148	7(5)	35	1(3)	7	0
	Dry		290	21(7)	26	0	100	18(18)
	Rainy		476	33(7)	36	2(6)	218	17(8)
Kisian		Indoor	112	1(1)	147	0	73	0
		Outdoor	16	0	54	0	3	0
	Dry		41	1(2)	127	0	47	0
	Rainy		87	0	74	0	29	0

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Pf, Plasmodium falciparum

+ve, Positive

Discussion

Given the widespread occurrence of pyrethroid insecticide resistance in malaria vectors in western Kenya [17,19,42], little is known about the behavioural response of these mosquito populations to the wide use of LLINs. Evidence has shown that successful malaria elimination strategies require vector control interventions that target the changing vector behaviour [43]. Overall, this study investigated the behavioral heterogeneity of malaria vectors for resting behavior, feeding choices and infection rates in the context of increased use of LLINs. The study revealed high resting densities, infection rates and insecticide resistance indoors compared to outdoors.

Of the three major malaria vectors in western Kenya, An. gambiae was the major vector in Bungoma followed by An. funestus. In Kisian An. arabiensis was predominant species followed by An. gambiae. The predominance of the two malaria vectors in the two different ecological settings is consistent with previous studies [16,44,45] observing increased frequency of An. gambiae s.s at sites further away from the lake Victoria basin and increase in the frequency of An. arabiensis at sites around the lake basin. There was a rise in the abundance of An. funestus s.s in Bungoma, a phenomena that has been observed previously in some regions in western Kenya [20]. Despite the high coverage and usage of long-lasting insecticidal nets (LLIN) in the region, increased indoor resting tendency of An. gambiae and An. funestus was observed, while An. arabiensis was found mostly resting outdoors. The variation in the relative frequency and behaviour of the three vectors has been observed earlier from the region [23,46]. It is worth noting that, the proportion of An. arabiensis was higher during the dry season in Kisian (lowland). This vector has been shown to survives best in areas with lower relative humidy and high temperatures and therefore prefers the lowland areas with such climates compared to the highland areas. The dry season also has low relative humidity and high temperatures [47].

This study observed reduced susceptibility of the F1 progeny of An. funestus and An. gambiae s.l captured resting indoors compared to those resting outdoors to deltamethrin insecticide. Earlier studies from western Kenya have reported increasing phenotypic resistance of An. gambiae s.l to pyrethroids [16,45,48,49]. Few studies have reported on An. funestus insecticide resistance[50] probably because of difficulties associated with rearing the larvae. The frequency of Vgsc-1014S and Vgsc-1014F from indoors resting An. gambiae was higher than outdoors, from all sites. The Vgsc-1014F mutation was only detected in An. gambiae resting indoors, this mutation was absent in the mosquitoes collected outdoors. This might be that mosquitoes with some mutations are able to rest indoors in the presence of insecticides for vector control. Previous studies have observed the occurrence of Vgsc-1014F at low frequencies in East Africa including Kenya [42,51,52]. The presence of Vgsc-1014F in indoor resting mosquitoes may be of particular concern given that the mutation has been found to be strongly associated with pyrethroid resistance in West Africa [53]. The presence of kdr mutations at Bungoma where it was first detected in Kenya in An. gambiae [16] and now at the lowland site of Kisian indicate the widespread occurrence of the mutations among the An. gambiae population. The increased resistance level could be a result of selection pressure from insecticides used in vector control such as the widespread use of LLINs leading to the selection of resistant strains [6]. Some studies have shown a relationship between the spread of kdr alleles with the use of LLINs [54–56]. The extensive use of agriculture insecticides may also contribute to the occurrence of new mutations to existing insecticides [57–61].

Even though we observed high frequencies of Vgsc-1014S in An. gambiae, the allele was at low frequency in An. arabiensis, with a higher frequency of Vgsc-1014S, detected for indoor resting individuals than outdoors. Most recently, the presence of kdr mutations in An. arabiensis from the lowlands of western Kenya has been reported [17,49]. The low kdr frequency observed in An. arabiensis could be due to the reduced insecticide selection pressure as they resort to feed and rest outdoors in the absence of insecticides, unlike An. gambiae that feeds and rests indoors. The high frequency of kdr mutations, behavioural resilience and an increased proportion of An. arabiensis resting outdoors could all raise further concerns on the future utility of the current indoor interventions.

Sporozoite infection rates were high in An. funestus and An. gambiae collected from Bungoma, with An. funestus showing considerably higher sporozoite rates than the other species. These findings are in agreement with previous studies that observed high sporozoite rates in An. funestus and An. gambaie, implying the importance of the two vectors in malaria transmission in the region [20,46]. The higher sporozoite rates in An. funestus in this study further indicates its reemergence and as one of the primary important vector in malaria transmission in the region. The blood meal analysis showed a large proportion of the two species preferred feeding on humans than animals, with An. funestus observed to be highly anthropophagic in the region. This consistency in host choice has been frequently observed in An. funestus in Kenya and other parts of Africa [46,62,63]. This human-host choice and higher indoor resting proportions of An. funestus together with increased resistance poses a great concern in malaria elimination efforts due to its efficiency in transmitting malaria. These findings confirm earlier reports from other regions in western Kenya that have documented the re-emergence of An. funestus and its role in malaria transmission [20,64]. The infection rates were higher for vectors collected indoors than outdoors, suggesting ongoing malaria transmission regardless of the use of LLINs. Earlier reports from western Kenya have shown increased EIR for indoor collected mosquitoes [46]. Some studies have linked the rebound of malaria in western Kenya with increasing insecticide resistance after high coverage of LLINs [54,56,65]. The observed sporozoite infection rates outdoors might be attributed to changing in the biting behaviour of malaria vectors as some vectors could be feeding on humans when they are active and unprotected outdoors [66]. Complementary malaria control intervenions are thus, needed to control outdoor resting and biting mosquitoes, as the current tools only target indoor resting and biting mosquitoes.

Despite the low sporozoite rates of An. arabiensis reported in this study, its importance in outdoor malaria transmission should not be taken for granted due to its opportunistic behavior, as the vector could continue with transmission outdoors in the region, which is a major threat to effective malaria vector control.

Conclusion

The study shows high densities of An. gambiae and An. funestus resting indoors despite the use of indoor interventions with the increasing importance of An. funestus in sustaining malaria transmission in western Kenya highlands. An. arabiensis were more outdoors than indoors. This behavioural plasticity increases its survival and potential in continuing residual transmission after the main endophilic and endophagic vectors have been reduced by the interventions. The Vgsc-1014S and Vgsc-1014F mutations were observed at high frequencies in An. gambiae resting indoors. This calls for further screening of other resistance mutations in this population for better resistance management. Sporozoite rates were higher indoors than outdoors, showing that transmission occurs more indoors than outdoors in these sites. Insecticide resistance management strategies and/or new vector control interventions that may not be insecticide based are needed in western Kenya to reduce malaria transmission.

Supporting information

S1 Table. Summary of female Anopheles mosquitoes collected from indoor and outdoor in highland (Bungoma) and lowland (Kisian) settings of western Kenya.

(DOCX)

Click here for additional data file.^{(17.3KB, docx)}

S2 Table. Summary results of KDR alleles for indoor and outdoor resting mosquitoes in Bungoma and Kisian sites in western Kenya.

(DOCX)

Click here for additional data file.^{(14.7KB, docx)}

Acknowledgments

The authors wish to thank the villagers and community leaders in Bungoma and Kisian for their permission to collect mosquitoes in their houses. We acknowledge the Entomology Laboratory at Kenya Medical Research Institute, Kisumu for providing technical and laboratory space for the study. The permission to publish this study was granted by the director of Kenya Medical Research Institute.

Abbreviations

BBI: bovine blood index
CSP: circumsporozoite protein
EIR: entomological inoculation rate
ELISA: enzyme-linked immunosorbent assay
F1: first generation offspring
HBI: human blood index
IRS: indoor residual spray
KDR: Knockdown resistant gene
LLINs: long-lasting insecticidal nets
PCR: polymerase chain reaction
PSC: pyrethrum spray catch
Vgsc: voltage-gated sodium channel

Data Availability

All relevant data are within the paper and its two Supporting Information files.

Funding Statement

This study was supported by grants from the National Institute of Health (R01 A1123074, U19 AI129326, R01 AI050243, D43 TW001505). There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Resting behaviour of malaria vectors in a highland and a lowland site of western Kenya: Implication on malaria vector control measures

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The study by Afrane and colleagues investigates resting behavior, host preference and Pf infection of malaria vectors in two settings of western Kenya. The paper is in parts fairly well written; in other parts very repetitive and unstructured. The methodologies are routine in field entomology, the findings are robust and valuable. They support a large body of evidence of high levels of resistance in the area.

The paper would benefit from some serious re-writing but the paper The intro is gives detailed background information without being overly long. It is also well written and would not need much work. The Results, on the other hand, can be greatly shortened. A lot of details are provided in the long first paragraph. All percentages that are reported should come with some indicator of precision. Either a confidence interval around the proportion or mentioning numerator and denominator (n/N). The discussion section is not very focused and would benefit from re-writing. It could, without problems, be shortened with 25-33% to make the paper more attractive, focusing on time trends in resistance or feeding behavior in Western Kenya and the implications for control. The structure of the discussion can also be greatly improved: dealing with each of the important elements in a single clearly structured paragraph (instead of mixing all messages throughout the discussion) would really improve this part of an, otherwise valuable, paper.

SPECIFIC COMMENTS

The abstract lists many findings without a very clear selection or any statistical tests. It could be improved by presenting only the key results: sporozoite rate, tube assay (?), resistance genetic findings, and present them in a bit more detail and with stats.

Abstract. Trapping methods may be listed in the abstract.

Abstract: confidence intervals or details of the underlying number of mosquitoes (n/N) should be mentioned in the abstract. Also a statement ‘None of the outdoor collected mosquitoes in Kisian tested positive for sporozoite infections’ can only be interpreted if the number of examined mosquitoes is given.

Abstract: .’Vgsc-1014S was observed at a slightly higher frequency in An. gambiae s.s hereafter(An. gambiae) resting indoor than outdoor (89.7 vs 84.6% and 71.5 vs 61.1%) in Bungoma and Kisian respectively’ is unclear. Vgsc is not defined in the abstract. And the estimates are possibly not statistically significant. If so, the finding should receive less prominence in the results section.

Abstract: ‘high densities of insecticide-resistant… [mosquitoes]’ in the conclusion can only be presented if also mosquito burden is presented in the abstract. What was the average number of mosquitoes per trapping night? It is really high? Or did the authors (simply) want to say that among mosquitoes there was a large proportion of resistant mosquitoes?

Intro, line 112. ‘Insecticide resistance could make mosquitoes respond numerically…’ is unclear to me. What is intended? This sentence could be simplified, probably by splitting the sentence in two.

Methods, line 152. Clay pots are notoriously difficult to standardize. A useful reference is Van den Bijllaardt, Acta Tropica 111 (2009) 197–199 where very detailed procedures are explained. For the current method, more details should be given on where traps were placed and how sampling was done. This applies to all trapping methods, not just clay pots.

Methods, line 195/196. The authors may want to comment on reports from SE Asia (Coosemans and team) that there is cross-reactivity in ELISA results in mosquitoes that fed on cattle blood. This might have contributed to the fairly high sporozoite rates observed in this study.

Methods: the dry versus wet season sampling is useful but needs some indication to where in the season samples were taken. How many weeks after start of the rains was ‘wet season’? How dry was the dry season?

Results, line 218-220. ‘Overall, the proportion of Anopheles species resting indoors was significantly higher by 82.4% than outdoor location 17.6% across the study sites (z = -8.47, p < 0.0001) ‘. This is very imprecise. I am not sure what is compared here. Is it the proportion of anophelines among caught mosquitoes? Or is it the absolute number of anophelines caught indoors vs outdoors? If so: is this appropriate? The sampling surface was probably very different. Only sporozoite rates and blood meal sources can be compared indoors vs outdoors since trapping methods differed profoundly.

Similarly, line 232 is unclear ‘There were more An. gambiae and An. funestus resting indoors than outdoors (80.7 vs 19.3% and 97.8 vs 2.2% respectively; X2 =122.96, df = 2, p < 234 0.0001). ‘ How can this be a percentage? Is it the percentage of all gambiae that was caught indoors? I find these presentations very difficult and dangerous since the sampling intensity was so different. I would advise to simply present for indoor and outdoor sampled mosquitoes separately: numbers per trapping night, species composition, sporozoite rates, resistance phenotype, genotype, blood source. Compare the % sporozoite positive between indoor vs outdoor but do not compare absolute numbers! Reconsidering what the authors really want to present would also allow this very long and difficult first section of the results section to be shortened considerably. That would really improve the paper. At the moment, it is the weaker part of the paper. Make use of the display figures (tables and figures) and do not add too much detail on proportions etc in the main text.

Throughout the results, give n/N or confidence intervals. Percentages without any indication of precision (either numbers of confidence intervals) are not very informative.

For blood sources, the authors make statements on higher vs lower between species and indoor vs outdoor. Present statistics and indicate precision of estimates. If a finding is not statistically significant, do not claim differences.

I am puzzled by Figure 2. There seems to be much less variation in mosquito exposure than is typically reported. Are these only successful trapping efforts or are zeroes (no mosquitoes caught) also included in the figure? These findings typically follow a Poisson or Negative Binomial distribution. The current presentation suggests a normal distribution with very few negative trapping events. That would be unusual. It is important to explain this and present the % of traps with at least one mosquito and the distribution of mosquitoes in positive traps.

Figure 3: adjust title. This figure doesn’t present abundance. Only composition.

Discussion

Line 331 ‘…leading to increased vector-human contact and ongoing malaria transmission in the region despite the high coverage and usage of LLINs’ is not an appropriate sentence. This was in no way proven by the data. It is pure speculation and should be recognizable as such (and probably placed somewhere later in the discussion and not in the crucial first paragraph).

As indicated above, the discussion is slightly unstructured. Findings and interpretations are mixed. It would be recommended to really work in clearly structured paragraphs that could explain in detail and with good references:

Paragraph 1. Conclusion. Pretty much as it is now.

Paragraph 2. Species composition in the areas. How does this relate to other findings?

Paragraph 3. How does resistance differ between species?

Paragraph 4. The resistance phenotype and genotype indoors and outdoors. Did this change compared to earlier studies in the area? Is this an indicator that biting increases indoors as a consequence of increased resistance (which again could be because of increased pressure due to widescale use of interventions)

Paragraph 5. Sporozoite rate per species among outdoor mosquitoes and for wet versus dry season. How does this compare to other studies? What does it mean for what vector is most important for transmission in the region? Include the blood meal preference in this section. Its relevance lies in this (explaining human biting and thus vector importance)

Paragraph 6. Final conclusion.

Reviewer #2: This manuscript describes the composition, bionomics and epidemiology of Anopheles malaria vectors in Western Kenya. The work is technically well done. The results are valuable and interesting, because malaria transmission continues and has even rebounded in parts of Africa despite decades of vector control.

The manuscript is well written and contains thoughtful analysis. It can be published after only several very minor points that should be corrected, as follows.

line 199 (and other locations): "anopheline" is an adjective, not a Latin name, and should not be italicized or capitalized.

line 252: unclear wording where it says "High resistance levels" of 36.6% or 65.5%, but the numbers refer to the 24 hour mortality rate, not actually resistance. Please reword this.

line 319, 320 (and other locations): be consistent about Latin names. Names should be spelled out at first use, and thereafter genus is abbreviated.

line 352 (and other locations): be consistent about "kdr", which is not capitalized, but here is shown as Kdr

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Feb 25;15(2):e0224718. doi: 10.1371/journal.pone.0224718.r002

Author response to Decision Letter 0

22 Jan 2020

``Response to reviewer comments

The following is our response to the comments by the reviewers. We really thank reviewers for their constructive criticism and insights that has helped to improve this paper.

Reviewer #1:

The study by Afrane and colleagues investigates resting behavior, host preference and Pf infection of malaria vectors in two settings of western Kenya. The paper is in parts fairly well written; in other parts very repetitive and unstructured. The methodologies are routine in field entomology, the findings are robust and valuable. They support a large body of evidence of high levels of resistance in the area.

Specific comments

Abstract

Reviewer: The abstract lists many findings without a very clear selection or any statistical tests. It could be improved by presenting only the key results: sporozoite rate, tube assay (?), resistance genetic findings, and present them in a bit more detail and with stats.

Response: The result section has been revised and only key findings i.e. [Species composition per study site, indoor and outdoor resting densities with statistical evidence, phenotypic resistance (WHO tube assays) with confidence intervals, genotypic resistance per site per species and sporozoite infections] have been presented in details

Reviewer: Trapping methods may be listed in the abstract

Response: The trapping methods have been included in the methods section in Line 41-43

Reviewer: Confidence intervals or details of the underlying number of mosquitoes (n/N) should be mentioned in the abstract. Also a statement ‘None of the outdoor collected mosquitoes in Kisian tested positive for sporozoite infections’ can only be interpreted if the number of examined mosquitoes is given.

Response: The confidence intervals around the percentages, numerator and denominator have been included in the result section. Lines 51-67. Number of outdoor resting mosquitoes analysed for sporozoite infection in Kisian has been included in Line 69

Reviewer: Vgsc-1014S was observed at a slightly higher frequency in An. gambiae s.s hereafter(An. gambiae) resting indoor than outdoor (89.7 vs 84.6% and 71.5 vs 61.1%) in Bungoma and Kisian respectively’ is unclear. Vgsc is not defined in the abstract. And the estimates are possibly not statistically significant. If so, the finding should receive less prominence in the results section.

Response: This finding has been deleted from the abstract.

Reviewer: high densities of insecticide-resistant… [mosquitoes]’ in the conclusion can only be presented if also mosquito burden is presented in the abstract. What was the average number of mosquitoes per trapping night? It is really high? Or did the authors (simply) want to say that among mosquitoes there was a large proportion of resistant mosquitoes?

Response: This statement has been revised and now it reads “….high indoor resting densities of An. gambiae and An. funestus, insecticide resistance, and persistence of malaria transmission indoors with high entomological inoculation rates (EIR) regardless of the use of Long-lasting insecticidal nets (LLINs)”.

Introduction

Reviewer: line 112. ‘Insecticide resistance could make mosquitoes respond numerically…’ is unclear to me. What is intended? This sentence could be simplified, probably by splitting the sentence in two.

Response: The sentence has been deleted from the introduction

Methods

Reviewer: line 152. Clay pots are notoriously difficult to standardize. A useful reference is Van den Bijllaardt, Acta Tropica 111 (2009) 197–199 where very detailed procedures are explained. For the current method, more details should be given on where traps were placed and how sampling was done. This applies to all trapping methods, not just clay pots

Response: This has been revised with more details given in the methodology section for all trapping methods. Van den Bijllaardt et al 2009 and Odiere et al 2007 have been added as the most relevant reference for clay pot collections. Line 152-166

Reviewer: line 195/196. The authors may want to comment on reports from SE Asia (Coosemans and team) that there is cross-reactivity in ELISA results in mosquitoes that fed on cattle blood. This might have contributed to the fairly high sporozoite rates observed in this study.

Response: It is true our study observed high sporozoite rates, this may not be as a result of cross reactivity in ELISA assays with cattle blood as the majority of the samples were fed on human blood in Bungoma. If there was cross-reactivity we could have experienced it in Kisian where a large number of collected mosquitoes were zoophilic and have taken cattle blood with low sporozoite rates observed.

Reviewer: The dry versus wet season sampling is useful but needs some indication to where in the season samples were taken. How many weeks after start of the rains was ‘wet season’? How dry was the dry season?

Response: The details on the dry and wet season has been included in the methodology section. “…

“Mosquitoes were sampled during the middle of long dry season (February-March) and four weeks after the start of long rainy season (May-July)” Meaning mosquitoes were collected after 4 weeks the start of the hot dry and long rain season. Line 150-151

Reviewer: line 218-220. ‘Overall, the proportion of Anopheles species resting indoors was significantly higher by 82.4% than outdoor location 17.6% across the study sites (z = -8.47, p < 0.0001) ‘. This is very imprecise. I am not sure what is compared here. Is it the proportion of anophelines among caught mosquitoes? Or is it the absolute number of anophelines caught indoors vs outdoors? If so: is this appropriate? The sampling surface was probably very different. Only sporozoite rates and blood meal sources can be compared indoors vs outdoors since trapping methods differed profoundly.

Response: That sentence on the comparison has been deleted from the result section.

Reviewer: Similarly, line 232 is unclear ‘There were more An. gambiae and An. funestus resting indoors than outdoors (80.7 vs 19.3% and 97.8 vs 2.2% respectively; X2=122.96, df = 2, p < 234 0.0001). ‘ How can this be a percentage? Is it the percentage of all gambiae that was caught indoors? I find these presentations very difficult and dangerous since the sampling intensity was so different. I would advise to simply present for indoor and outdoor sampled mosquitoes separately: numbers per trapping night, species composition, sporozoite rates, resistance phenotype, genotype, blood source.

Response: The section now presents the resting densities of malaria vectors indoors and outdoors, species composition per study site, overall malaria vector composition per location and seasonal species composition.

Reviewer: Compare the % sporozoite positive between indoor vs outdoor but do not compare absolute numbers! Reconsidering what the authors really want to present would also allow this very long and difficult first section of the results section to be shortened considerably. That would really improve the paper. At the moment, it is the weaker part of the paper. Make use of the display figures (tables and figures) and do not add too much detail on proportions etc in the main text.

Response: This result section has been revised according to reviewers suggestions. The n/N for sporozoite percentages is included in the table. Table 3

Reviewer: Throughout the results, give n/N or confidence intervals. Percentages without any indication of precision (either numbers of confidence intervals) are not very informative.

Response: The confidence intervals around the percentages, n/N have been included in the text and also in the tables

Reviewer: For blood sources, the authors make statements on higher vs lower between species and indoor vs outdoor.present statistics and indicate precision of estimates. If a finding is not statistically significant, do not claim differences

Response: The statements indicating higher and lower in the blood meal findings has been revised throughout the text and the precision of estimates (n/N) included in the table. Table 2

Reviewer: I am puzzled by Figure 2. There seems to be much less variation in mosquito exposure than is typically reported. Are these only successful trapping efforts or are zeroes (no mosquitoes caught) also included in the figure? These findings typically follow a Poisson or Negative Binomial distribution. The current presentation suggests a normal distribution with very few negative trapping events. That would be unusual. It is important to explain this and present the % of traps with at least one mosquito and the distribution of mosquitoes in positive traps.

Response: The figure 2 has been changed and it now presents successful trapping efforts.

Reviewer: Figure 3: adjust title. This figure doesn’t present abundance. Only composition.

Response: This has been revised and now reads “ Seasonal and Anopheles sibling species composition resting indoors and outdoors in A: Highland site (Bungoma) and B: lowland site (Kisian), western Kenya”

Discussion

Reviewer: Line 331 ‘…leading to increased vector-human contact and ongoing malaria transmission in the region despite the high coverage and usage of LLINs’ is not an appropriate sentence. This was in no way proven by the data. It is pure speculation and should be recognizable as such (and probably placed somewhere later in the discussion and not in the crucial first paragraph).

Response: The sentence has been revised to read “The study revealed high resting densities, infection rates and insecticide resistance indoors compared to outdoors”

Reviewer: As indicated above, the discussion is slightly unstructured. Findings and interpretations are mixed. It would be recommended to really work in clearly structured paragraphs that could explain in detail and with good references:

Paragraph 1. Conclusion. Pretty much as it is now.

Paragraph 2. Species composition in the areas. How does this relate to other findings?

Paragraph 3. How does resistance differ between species?

human biting and thus vector importance)

Paragraph 6. Final conclusion.

Response: This has been addressed and discussed further according to the reviewer’s suggestion . Paragraph 1 Presents the significance of the study and the main findings of the study.

Paragragh 2: discussion on the species composition in the region and how it relates with previous work from the same region

Paragraph 3&4 discussion on the phenotypic and genotypic resistance per species per location(indoor and outdoor location) per study sites comparing with earlier findings

Paragragh 5 discussion on sporozoite rates per species among indoor and outdoor collections blood meal sources compared with earlier reports. The primary vector in the region.

Paragraph 6 final conclusion

Response to Reviewer # 2

This manuscript describes the composition, bionomics and epidemiology of Anopheles malaria vectors in Western Kenya. The work is technically well done. The results are valuable and interesting, because malaria transmission continues and has even rebounded in parts of Africa despite decades of vector control.

The manuscript is well written and contains thoughtful analysis. It can be published after only several very minor points that should be corrected, as follows.

Reviewer: Line 199 (and other locations): "anopheline" is an adjective, not a Latin name, and should not be italicized or capitalized.

Response: This has been revised throughout the text

Reviewer: line 252: unclear wording where it says "High resistance levels" of 36.6% or 65.5%, but the numbers refer to the 24 hour mortality rate, not actually resistance. Please reword this.

Response: Revision has been done and the sentence now reads, “Reduced mortality was observed for F1 progeny of Anopheles gambiae s.l resting indoors (37%, 95%......”

Reviewer: line 319, 320 (and other locations): be consistent about Latin names. Names should be spelled out at first use, and thereafter genus is abbreviated.

Response: Corrected, consistency checked throughout the text

Reviewer: Line 352 (and other locations): be consistent about "kdr", which is not capitalized, but here is shown as Kdr

Response: Corrected, consistency checked throughout the text “kdr” with lower case used

Technical Comments from the Editorial Office

Editor: Please do not report values like '57.3' - only two digits are meaningful, so instead such a value should simply read 57.

Response: This has been revised and the two digits value maintained throughout the result section.

Editor: Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now

Response: There was no additional external funding received for this study.

Editor: We note that [Figure 1] in your submission contains [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

Response: Figure 1 has been removed

Editor: We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/dataavailability# loc-unacceptable-data-access-restrictions.

Response: The statement on data availability has been included and now it reads…… “The datasets used for the current study are available at the repository of the Kenya Medical Research Institute”

Editor: Your ethics statement must appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also ensure that your ethics statement is included in your manuscript, as the ethics section of your online submission will not be published alongside your manuscript.

Response: The ethical statement has been moved to the methods section.

Editor: Please ensure that you refer to Table 2 in your text as, if accepted, production will need this reference to link the reader to the Table.

Response: Table 2 has been referred in the text

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(26KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0224718.r003

Decision Letter 1

Friedrich Frischknecht

5 Feb 2020

Resting behaviour of malaria vectors in highland and lowland sites of western Kenya: Implication on malaria vector control measures

PONE-D-19-29030R1

Dear Dr. Afrane,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Friedrich Frischknecht

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thanks for returning a well done revision, in my view you addressed all the important issues raised by the reviewers as well as their minor comments. After careful reading your revised manuscript, I thus decided not to send it to the reviewers again. There are still a few minor improvements possible in terms of wording (e.g. first sentence of abstract needs an 'and'; first sentence of discussion needs attention) and the reference list needs attention as the cited literature is not all in the journal style. I trust you can make this changes without a need for further external review. Congratulation to a nice study.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0224718.r004

Acceptance letter

Friedrich Frischknecht

11 Feb 2020

PONE-D-19-29030R1

Resting behaviour of malaria vectors in highland and lowland sites of western Kenya: Implication on malaria vector control measures

Dear Dr. Afrane:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Friedrich Frischknecht

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Summary of female Anopheles mosquitoes collected from indoor and outdoor in highland (Bungoma) and lowland (Kisian) settings of western Kenya.

(DOCX)

Click here for additional data file.^{(17.3KB, docx)}

S2 Table. Summary results of KDR alleles for indoor and outdoor resting mosquitoes in Bungoma and Kisian sites in western Kenya.

(DOCX)

Click here for additional data file.^{(14.7KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(26KB, docx)}

Data Availability Statement

All relevant data are within the paper and its two Supporting Information files.

[pone.0224718.ref001] 1.WHO (2019) World Malaria Report 2018. Geneva: World Health Organisation.

[pone.0224718.ref002] 2.Hamel MJ, Adazu K, Obor D, Sewe M, Vulule J, et al. (2011) A reversal in reductions of child mortality in western Kenya, 2003–2009. Am J Trop Med Hyg 85: 597–605. 10.4269/ajtmh.2011.10-0678 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Resting behaviour of malaria vectors in highland and lowland sites of western Kenya: Implication on malaria vector control measures

Maxwell G Machani

Eric Ochomo

Fred Amimo

Jackline Kosgei

Stephen Munga

Guofa Zhou

Andrew K Githeko

Guiyun Yan

Yaw A Afrane

Roles

Abstract

Background

Methods

Results

Conclusion

Background

Methods

Study site

Mosquito sampling

WHO resistance bioassays

Anopheline species discrimination

Detection of sporozoite infectivity

Detection of blood meal sources using polymerase chain reaction (PCR)

Genotyping for kdr mutations

Scientific and ethical clearance

Data analysis

Results

Indoor and outdoor Anopheline mosquito composition

Fig 1. Indoor and outdoor resting density of female Anopheles mosquitoes collected per trapping method A: Highland site (Bungoma) and B: Lowland site (Kisian), western Kenya.

Anopheles gambiae and An. funestus sibling species composition

Fig 2. Seasonal composition and Anopheles sibling species composition resting indoors and outdoors in A: Highland site (Bungoma) and B: Lowland site (Kisian), western Kenya.

Phenotypic resistance

Fig 3. Mortality rates of indoor and outdoor resting Anopheles gambiae s.l and An. funestus F1 progeny in standard WHO tube bioassays after exposure to 0.05% deltamethrin test papers and 24 hr recovery period.

Target site genotyping

Table 1. Frequency of Kdr mutations in An. gambiae s.s and An. arabiensis populations of western Kenya.

Blood meal sources

Table 2. Blood meal origins of Anopheline mosquitoes collected from indoor and outdoor in Bungoma and Kisian, western Kenya.

Sporozoites infection rates

Table 3. Sporozoite rates of Anopheles mosquitoes from indoor and outdoor collections in Bungoma and Kisian, western Kenya.

Discussion

Conclusion

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Friedrich Frischknecht

Roles

Author response to Decision Letter 0

Decision Letter 1

Friedrich Frischknecht

Roles

Acceptance letter

Friedrich Frischknecht

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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