Indoor and outdoor malaria vector surveillance in western Kenya: implications for better understanding of residual transmission

Abstract

Background

The widespread use of indoor-based malaria vector control interventions has been shown to alter the behaviour of vectors in Africa. There is an increasing concern that such changes could sustain residual transmission. This study was conducted to assess vector species composition, feeding behaviour and their contribution to indoor and outdoor malaria transmission in western Kenya.

Methods

Anopheles mosquito collections were carried out from September 2015 to April 2016 in Ahero and Iguhu sites, western Kenya using CDC light traps (indoor and outdoor), pyrethrum spray catches (PSCs) (indoor) and pit shelters (outdoor). Species within Anopheles gambiae s.l. and Anopheles funestus s.l. were identified using polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was used to determine mosquito blood meal sources and sporozoite infections.

Results

A total of 10,864 female Anopheles mosquitoes comprising An. gambiae s.l. (71.4%), An. funestus s.l. (12.3%), Anopheles coustani (9.2%) and Anopheles pharoensis (7.1%) were collected. The majority (61.8%) of the anopheline mosquitoes were collected outdoors. PCR result (n = 581) revealed that 98.9% An. arabiensis and 1.1% An. gambiae s.s. constituted An. gambiae s.l. in Ahero while this was 87% An. gambiae s.s. and 13% An. arabiensis in Iguhu. Of the 108 An. funestus s.l. analysed by PCR, 98.1% belonged to An. funestus s.s. and 1.9% to Anopheles leesoni. The human blood index (HBI) and bovine blood index (BBI) of An. arabiensis was 2.5 and 73.1%, respectively. Anopheles gambiae s.s. had HBI and BBI of 50 and 28%, respectively. The HBI and BBI of An. funestus was 60 and 22.3%, respectively. Forage ratio estimate revealed that An. arabiensis preferred to feed on cattle, An. gambiae s.s. showed preference for both human and cattle, while An. funestus preferred human over other hosts. In Ahero, the sporozoite rates for An. arabiensis and An. funestus were 0.16 and 1.8%, respectively, whereas in Iguhu, the sporozoite rates for An. gambiae s.s. and An. funestus were 2.3 and 2.4%, respectively. In Ahero, the estimated indoor and outdoor entomological inoculation rate (EIR) was 108.6 infective bites/person/year (79.0 from An. funestus and 29.6 from An. arabiensis) and 43.5 infective bites/person/year (27.9 from An. arabiensis and 15.6 from An. funestus), respectively. In Iguhu, the estimated indoor and outdoor EIR was 24.5 infective bites/person/year (18.8 from An. gambiae s.s. and 5.7 from An. funestus) and 5.5 infective bites/person/year (all from An. gambiae s.s.), respectively.

Conclusion

Anopheles gambiae s.s. showed an increasing tendency to feed on cattle. Anopheles arabiensis was highly zoophagic, whereas An. funestus showed anthropophagic behaviour. While the majority of malaria transmission occurred indoor, the magnitude of outdoor transmission was considerably high. Additional control tools that complement the existing interventions are required to control residual transmission.

Background

Malaria is a serious vector-borne disease affecting hundreds of millions of people in Africa. In the past decade, a substantial reduction in malaria incidence has been observed in Africa, including Kenya, due to the scale-up of interventions. Vector control is one of the key elements in achieving the remarkable decline of malaria, with the scale-up of insecticide-treated nets (ITNs) and expansion of indoor residual spray (IRS) contributing significantly [1–4]. The proportion of households owning at least one ITN in sub-Saharan Africa is estimated to have risen from 3% in 2000 to 67% in 2015 [1]. In western Kenya, the ITN ownership rose from 12.8% in 2004 to over 80% in 2015 [5–7].

Despite the progress made in scaling-up of the interventions, malaria transmission continues to occur. Several factors are responsible for this transmission, including the spread of insecticide resistance [5, 8], shift in vector species composition [9–12] and increasing vector behavioural change towards more zoophagic, exophagic and/or exophilic tendencies following the widespread use of ITNs and IRS [13, 14].

Recent reports from East Africa showed strong evidence for shifts in Anopheles gambiae sensu lato (s.l.) sibling species composition from predominantly endophagic An. gambiae sensu stricto (s.s.) to predominantly exophagic Anopheles arabiensis following the scale-up of ITNs [9, 11–13, 15]. In the lowlands of western Kenya, the proportion of An. gambiae s.s. declined from about 85% in 1998 to 1% in 2009 following massive distribution of ITNs, whereas An. arabiensis population showed proportionate increment [9]. While malaria transmission by An. gambiae s.s. declined significantly, residual transmission continued to occur by An. arabiensis. Similarly, the proportion of An. arabiensis in the highlands of western Kenya has been increasing gradually [5].

Vector behavioural modifications including changes in host-preference, biting locations (indoor or outdoor) and resting behaviours have been reported following the long-term use of ITNs. For instance, ITN use was associated with shift in host preference of An. gambiae s.s. from human to cattle in Burkina Faso [16]. The long-term use of ITN increased the outdoor feeding proportion of An. gambiae s.s. in Bioko Island [17, 18] and Anopheles funestus in Tanzania [13]. However, these changes are not universal. A recent study in Asembo district of western Kenya showed that the majority of biting by An. arabiensis, An. gambiae s.s. and An. funestus occurred indoors despite high ITN coverage in the area [19].

Malaria is mesoendemic and holoendemic in the highland and lowland areas of western Kenya, respectively [20]. The transmission is maintained by An. gambiae s.s., An. funestus and An. arabiensis. Anopheles gambiae s.s and An. funestus are considered as highly endophagic and anthropophagic, while An. arabiensis is considered as zoophagic and endophilic. However, most of the studies on their feeding and resting behaviour were conducted before the scale-up of vector control interventions [21–23]. It is possible that the anthropophagic and endophilic individuals could shift to zoophagic and exophilic tendencies or be reduced to leave zoophagic and exophilic sibling species following the scale-up of ITNs as has been observed elsewhere.

In view of the increasing concern about residual malaria transmission in Africa, there is a pressing need to enhance our understanding about vector behaviours to evaluate the likely success of the current vector control tools. The main aim of this study was to assess vector species composition, feeding behaviour and their contribution to indoor and outdoor malaria transmission in western Kenya.

Methods

Study sites

The study was conducted in lowland and highland settings of western Kenya. Two sites were selected (Fig. 1): Ahero (0°.11′S, 34°.55′E, altitude 1162 m) in Kisumu county and Iguhu (0°.17′N; 34°.74′E, altitude 1430–1580 m a.s.l) in Kakamega county. Iguhu site is highland characterized by valleys and depressions surrounded by densely populated hills whereas Ahero is lowland plain area. The sites have bimodal pattern of rainfall, with long rainy season from April to June, which triggers peak malaria transmission period and short rainy season from October to November with minimal transmission [24]. The hot and dry season is from January to March and this marks the lowest transmission [5]. Plasmodium falciparum is the predominant malaria species in the area and is transmitted by An. gambiae s.s., An. arabiensis and An. funestus [5, 25].

Fig. 1.

Map of the study sites

Mosquito collections

Adult mosquito collections were carried out monthly during the short rainy season (September to November) in 2015 and dry season (February to April) in 2016. Indoor and outdoor host-seeking mosquitoes were collected using Centers for Disease Control and Prevention (CDC) light traps (John W. Hock Ltd, Gainesville, FL., USA). For indoor host-seeking mosquito collection, CDC light traps were set inside houses near the bed at a height of 1.5 m from 18:00 to 06:00 h in twenty randomly selected houses per month in each study site. For the outdoor host-seeking mosquito sampling, CDC light traps were set outdoor in the vicinity (within 2 m) of sentinel houses. The same houses were used for mosquito collections each month.

Indoor resting mosquitoes were sampled using pyrethrum spray catches (PSCs) from another twenty randomly selected houses from 06:00 to 09:00 h following standard protocol [26]. Outdoor resting mosquitoes were collected monthly in the mornings (06:00 to 09:00 h.) from twenty artificial outdoor pit shelters constructed according to the method of Muirhead-Thomson [27], in the compound of 20 selected houses in each study site. The collections were repeated using the same pit shelters each month.

Along with mosquito collection, data on the numbers of potential hosts in the study area including human, bovine, goat, dog and chicken were collected using questionnaire surveys. All collected mosquitoes were identified morphologically to species using keys [28]. Female Anopheles mosquitoes were further classified as unfed, blood fed, half-gravid and gravid. Each mosquito was kept in a labelled 1.5 ml Eppendorf tube containing silica gel desiccant and cotton wool. Samples were stored at − 20 °C refrigerator at Climate and Human Health Research Laboratory of Kenya Medical Research Institute until used for further processing.

Identification of vector species complexes

Members of An. gambiae s.l. and An. funestus s.l. groups were identified to species by polymerase chain reaction (PCR), following the protocols developed by Scott et al. for An. gambiae s.l. [29] and Koekemoer et al. for An. funestus s.l. [30].

Detection of blood meal sources

The blood meal sources of freshly fed Anopheles mosquitoes were analyzed by a direct enzyme-linked immunosorbent assay (ELISA) [31] using human, bovine, goat, chicken and dog antibodies. Positive controls were included for each host during the assay. Laboratory reared unfed An. gambiae was used as negative control.

Sporozoite ELISA

Dried head and thorax of the preserved Anopheles mosquito specimens were carefully separated from the abdomen and tested for P. falciparum circumsporozoite proteins (CSPs) using sand-witch ELISA method [32, 33].

Data analysis

The density of adult anopheline mosquitoes was calculated as the number of female mosquitoes per trap/night for each collection method. Analysis of variance (ANOVA) was used to compare malaria vector density between indoor and outdoor locations. χ²-test was employed to test the difference in vector species composition between indoor and outdoor.

Human blood index (HBI) was calculated as the proportion of Anopheles mosquitoes that fed on human over the total Anopheles tested for blood meal origins [34]. Bovine, goat, dog and chicken blood indices were also calculated in similar way. Mixed blood meals were included in the calculation of blood meal indices [35]. The forage ratio (FR), a measure of host preference by mosquitoes, was determined as the percent of engorged Anopheles mosquitoes which have fed on a given host (human, bovine, goat, dog or chicken) divided by the percent which it comprises in the total population of hosts available in the study area [36]. The FR w _i for species i was calculated as:

$$w_i=\frac{o_i}{p_i}$$

where w _i is the FR for mosquito species i, o _i is the proportion of host species i in the blood meals, and p _i is the proportion of host species i available in the environment.

Statistical significance of the FR estimate for each host was based on overlap of the 95% confidence interval (CI) of the estimate with the value one [37]. A host was considered to have been preferred if the lower 95% confidence limit for the FR estimate was greater than one. A host was inferred to have been avoided if the upper 95% confidence limit for the FR estimate was less than one. A host for which the 95% CI for its FR included one was considered to have been feed on opportunistically [37].

The sporozoite rate was estimated as the proportion of mosquitoes positive for P. falciparum CSPs over the total number tested. Annual entomological inoculation rate (EIR) was calculated from mosquito collections by CDC light traps using the formula, 1.605 × (no. CSP-positive ELISA results from CDC light traps/no. mosquitoes tested) × (no. mosquitoes collected from CDC light traps/no. trap-nights) × 365 [38, 39]. The multiplication factor 1.605 is a conversion factor for CDC light trap catches vs. man biting catches [38]. The annual EIR of Anopheles mosquitoes collected by PSCs was determined as: (no. fed mosquitoes caught by PSC/no. human occupants who spent the night in the sprayed house) × (no. mosquitoes fed on human/no. mosquitoes tested for human blood meal) × (PSC based sporozoite rate) × 365 [40].

The annual EIR for Anopheles mosquitoes collected from pit shelters was also estimated as (no. fed mosquitoes caught in the pit shelters/no. human occupants who spent the night in a house nearest to the pit shelter) × (no. human fed mosquitoes/no. mosquitoes tested for human blood meal) × (sporozoite rate from pit shelters) × 365. This formula was employed based on the assumption that all Anopheles mosquitoes collected from pit shelters have got their human blood meals from occupants of the nearest house, either indoor or outdoor.

Data were analyzed using STATISTICA 8.0 (StatSoft, Tulsa, USA) and SPSS version 20.0 (SPSS, Chicago, IL, USA) software packages. p < 0.05 was considered statistically significant during the analysis.

Results

Anopheline mosquito species composition and abundance

A total of 10,864 female Anopheles mosquitoes belonging to four species were collected during the study period (Table 1). Anopheles gambiae s.l. was the predominant species accounting for 71.4% of the total captures, followed by An. funestus s.l. (12.3%), Anopheles coustani complex (9.2%) and Anopheles pharoensis (7.1%). In addition, 3263 male anopheline mosquitoes and 5206 Culex species (males and females together) were collected over the study period. There was a significant difference in anopheline mosquito species co-occurrence between the study sites (F _{1, 952} = 423.02, p < 0.0001). There was also significant difference in anopheline mosquito species co-occurrence between indoor and outdoor locations (F _{1, 956} = 29.44, p < 0.0001). The majority (61.8%) of the anopheline mosquitoes were collected outdoors.

Table 1.

Summary of female Anopheles mosquitoes collected from indoor and outdoor in lowland (Ahero) and highland (Iguhu) settings of western Kenya (n = 120 trap-nights for each trap)

Study sites and Anopheles spp.	Indoor		Outdoor		Total
	Light trap	PSC	Light trap	Pit shelter
Ahero
An. gambiae s.l.	1592	1009	1636	3262	7499
An. funestus s.l.	628	204	270	142	1244
An. coustani	321	2	652	15	990
An. pharoensis	78	0	688	0	766
Iguhu
An. gambiae s.l.	108	51	56	41	256
An. funestus s.l.	49	30	13	4	96
An. coustani	3	0	10	0	13
Total	2779	1296	3325	3464	10,864

PSC, pyrethrum spray catch

Indoor and outdoor Anopheles mosquito density

Figure 2 shows the mean indoor and outdoor density of host-seeking and resting female Anopheles mosquitoes. In Ahero, the mean outdoor resting density of An. gambiae s.l. was significantly higher than indoor resting density (t ₂₃₈ = 8.45, p < 0.0001), whereas the difference in mean indoor and outdoor resting density of An. funestus s.l. was not significant (p > 0.05). The mean outdoor host-seeking density of An. gambiae s.l. was also higher than indoor, although the difference was not statistically significant (t ₂₃₈ = 0.14, p = 0.889). The mean indoor host-seeking density of An. funestus s.l. was significantly higher than outdoor (t ₂₃₈ = 2.37, p = 0.019). Significantly higher outdoor host-seeking density than indoor was observed for An. coustani (t ₂₃₈ = 2.589, p = 0.01) and An. pharoensis (t ₂₃₈ = 4.923, p < 0.0001).

Fig. 2.

Indoor and outdoor host-seeking and resting density of female Anopheles mosquitoes collected from Ahero and Iguhu, western Kenya

In Iguhu, the indoor host-seeking densities of An. gambiae s.l. and An. funestus s.l. were significantly higher than outdoor (An. gambiae s.l., t ₂₃₈ = 2.12, p = 0.034; An. funestus s.l., t ₂₃₈ = 3.09, p = 0.002). The difference in mean indoor and outdoor resting density of An. gambiae s.l. was not significant (t ₂₃₈ = 0.97, p = 0.335), while the mean indoor resting density of An. funestus s.l. was significantly higher (t ₂₃₈ = 3.23, p = 0.001) than outdoor.

Composition of Anopheles gambiae and Anopheles funestus sibling species

A total of 750 specimens (628 An. gambiae s.l. and 122 An. funestus s.l.) were analysed for identification of their respective sibling species. Of theses, 581 An. gambiae s.l. and 108 An. funestus s.l. specimens were successfully amplified and identified to species by PCR. Figure 3 shows member species of An. gambiae s.l. In Ahero, of the An. gambiae s.l. assayed, An. arabiensis and An. gambiae s.s accounted for 98.9 and 1.1%, respectively. In contrast in Iguhu, An. gambiae s.s. and An. arabiensis constituted 87 and 13%, respectively of the assayed An. gambiae s.l. specimens. Overall, there was significant difference between indoor and outdoor locations in terms of An. gambiae s.l. species composition (χ² = 26.443, df = 1, p < 0.0001). The proportion of An. arabiensis was higher outdoors than indoors. Of the 108 An. funestus s.l. confirmed by PCR, An. funestus s.s. (hereafter An. funestus) and Anopheles leesoni, accounted for 98.1 and 1.9%, respectively. All the An. leesoni were from outdoor CDC light traps. The member species of An. funestus s.l. did not vary between the study sites.

Fig. 3.

Composition of Anopheles gambiae sibling species in Ahero and Iguhu, western Kenya

Physiological status

In both indoor and outdoor collections, the majority (> 70%) of the host-seeking anophelines were unfed. About 55% of the indoor resting and 39% of the outdoor resting An. arabiensis were blood fed. One-third of the indoor resting and 31.7% of the outdoor resting An. gambiae s.s. were blood fed. About half of the indoor resting An. funestus were blood fed, while this was 11.6% for the outdoor resting An. funestus.

Blood meal indices

Table 2 shows the host blood indices of An. arabiensis and An. funestus in Ahero. The HBI of An. arabiensis from indoor CDC light traps and PSCs was 8.2 and 1.2%, respectively, whereas the HBI of An. arabiensis from outdoor CDC traps and pit shelters was 3.4 and 0.7%, respectively. The overall HBI of An. arabiensis was 2.5%. The HBI of An. funestus from indoor CDC light traps and PSCs was 72.7 and 63.6%, respectively, while the HBI of An. funestus from both outdoor CDC light traps and pit shelters was 50%. In Ahero, the overall HBI for An. funestus was 62%.

Table 2.

Blood meal origins of An. arabiensis and An. funestus from indoor and outdoor collections in Ahero, western Kenya

Blood-meal origins	An. arabiensis				An. funestus
	Indoor		Outdoor		Indoor		Outdoor
	Light trap	PSC	Light trap	Pit shelter	Light trap	PSC	Light trap	Pit shelter
Number tested	122	165	59	298	11	44	4	12
Human	7 (5.7)	1 (0.6)	2 (3.4)	2 (0.7)	8 (72.7)	23 (52.3)	2 (50.0)	6 (50.0)
Bovine	74 (60.7)	108 (65.5)	30 (50.8)	251 (84.2)	0	10 (22.7)	1 (25.0)	5 (41.7)
Goat	5 (4.1)	5 (3.0)	1 (1.7)	4 (1.3)	0	0	0	0
Dog	1 (0.8)	5 (3.0)	1 (1.7)	5 (1.7)	0	0	0	0
Chicken	2 (1.6)	0	0	1 (0.3)	0	0	0	0
Human + bovine	1 (0.8)	1 (0.6)	0	0	0	2 (4.6)	0	0
Human + dog	2 (1.6)	0	0	0	0	3 (6.8)	0	0
Bovine + dog	1 (0.8)	1 (0.6)	0	4 (1.3)	0	0	0	0
Goat + dog	0	1 (0.6)	0	0	0	0	0	0
Dog + chicken	0	0	0	1 (0.3)	0	0	0	0
Unknown	29 (23.8)	43 (26.1)	25 (42.4)	30 (10.1)	3 (27.3)	6 (13.6)	1 (25.0)	1 (8.3)
HBI	8.2	1.2	3.4	0.7	72.7	63.6	50.0	50.0

HBI was calculated as the number of mosquito positive for human (including mixed blood meal) divided by the total number tested

HBI, human blood index; PSC, pyrethrum spray catches

In contrast, the bovine blood index (BBI) of An. arabiensis from indoor CDC light traps, PSCs, outdoor CDC light traps and pit shelters was 62.3, 66.7, 50.8, and 85.6%, respectively. Overall, the BBI of An. arabiensis was 73.1%. The BBI of An. funestus from PSCs, outdoor CDC light traps and pit shelters was 27.3, 22.7 and 41.7%, respectively. None of the An. funestus from indoor CDC light traps was positive for bovine blood meal. In Ahero, the overall BBI of An. funestus was 25.4%. Blood meal indices for other vertebrate hosts (goat, dog and chicken) were low (< 4%).

Table 3 shows the host blood indices of An. gambiae s.s. and An. funestus in Iguhu. The HBI of An. gambiae s.s. from indoor CDC light traps and PSCs was 70.0 and 76.5%, respectively, whereas the HBI of An. gambiae s.s. from outdoor CDC light traps and pit shelters was 20.0 and 23.1% respectively. The overall HBI of An. gambiae s.s. was 50.0%. The HBI of An. funestus from indoor CDC light traps and PSCs was 53.8 and 61.1%, respectively. In outdoor CDC light traps, very small number of fed An. funestus was caught, which yielded a HBI of 50%. Hence, in Iguhu, the overall HBI of An. funestus was 55.9%.

Table 3.

Blood meal origins of An. gambiae s.s. and An. funestus from indoor and outdoor collections in Iguhu, western Kenya

Blood-meal origins	An. gambiae s.s.				An. funestus
	Indoor		Outdoor		Indoor		Outdoor
	Light trap	PSC	Light trap	Pit shelter	Light trap	PSC	Light trap	Pit shelter
Number tested	10	17	10	13	13	18	2	1
Human	7 (70)	11 (64.7)	2 (20)	3 (23.1)	7 (53.8)	11 (61.1)	1 (50.0)	0
Bovine	0	3 (17.6)	4 (40)	6 (46.1)	2 (15.4)	3 (16.7)	1 (50.0)	0
Goat	0	0	0	0	1 (7.7)	0	0	0
Dog	0	0	0	1 (7.7)	1 (7.7)	0	0	1 (100)
Human + bovine	0	1 (5.9)	0	0	0	0	0	0
Human + dog	0	1 (5.9)	0	0	0	0	0	0
Unknown	3 (30)	1 (5.9)	4 (40)	3 (23.1)	2 (15.4)	4 (22.2)	0	0
HBI	70.0	76.5	20	23.1	53.8	61.1	50.0	0

HBI was calculated as the number of mosquito positive for human (including mixed blood meal) divided by the total number tested

HBI, human blood index; PSC, pyrethrum spray catches

The BBI of An. gambiae s.s. from PSCs, outdoor CDC light traps and pit shelters was 23.5, 40.0, and 46.1%, respectively. None of the tested An. gambiae s.s. from indoor CDC light traps was positive for bovine blood meal. The overall BBI of An. gambiae s.s. was 28%. The BBI of An. funestus from indoor CDC light traps, PSCs and outdoor CDC light traps was 15.4, 16.7, and 50%, respectively. In Iguhu, the overall BBI of An. funestus was 17.6%.

Feeding preference of malaria vectors

The overall blood meal indices and host preferences of Anopheles mosquitoes are shown in Table 4. Regardless of higher proportion of humans compared to domestic animals in Ahero, An. arabiensis showed a strong preference to feed on bovine (Forage ratio, FR = 3.9, 95% CI 3.7–4.9). Anopheles gambiae s.s. showed preference to both human (FR = 1.8, 95% CI 1.3–2.3) and bovine (FR = 2.3, 95% CI 1.3–3.3). Anopheles funestus showed a preference to human in both Ahero (FR = 2.2, 95% CI 1.8–2.6) and Iguhu (FR = 2.0, 95% CI 1.6–2.4).

Table 4.

Overall blood meal indices and host-preferences of malaria vectors from indoor and outdoor collections in Ahero and Iguhu, western Kenya

Site and species	Parameters	Human	Bovine	Goat	Dog	Chicken
Ahero
Host abundance in the area (%)		27.8	18.8	4.0	6.0	43.4
An. arabensis	Blood index	2.5	73.1	2.5	3.4	0.6
	FR (95% CI)	0.09 (0.05 to 0.13)	3.9 (3.7 to 4.1)*	0.6 (0.3 to 0.9)	0.5 (0.3 to 0.7)	0.01 (− 0.03 to 0.05)
An. funestus	Blood index	62.0	25.4	0	4.2	0
	FR (95% CI)	2.2 (1.8 to 2.6)*	1.4 (0.9 to 1.9)	0	0.7 (− 0.1 to 1.5)	0
Iguhu
Host abundance in the area (%)		27.5	12.4	2.4	2.5	55.2
An. gambiae s.s.	Blood index	50	28	0	2.0	0
	FR (95% CI)	1.8 (1.3 to 2.3)*	2.3 (1.3 to 2.3)*	0	0.8 (− 0.7 to 2.3)	0
An. funestus	Blood index	55.9	17.6	2.9	2.9	0
	FR (95% CI)	2.0 (1.6 to 2.4)*	1.4 (0.4 to 2.4)	1.2 (− 1.2 to 3.6)	1.2 (− 1.1 to 3.5)	0

FR, forage ratio

* Indicates the preferred host

Sporozoite rates

Overall, 2608 Anopheles mosquitoes comprising An. arabiensis (n = 1280), An. gambiae s.s. (n = 214), An. funestus (n = 629), An. coustani (n = 255) and An. pharoensis (n = 230) were tested for P. falciparum CSPs. Of these, 20 specimens (2 An. arabiensis, 5 An. gambiae s.s., 12 An. funestus and 1 An. coustani) were positive for CSPs.

Table 5 shows the sporozoite rates of Anopheles mosquitoes collected from indoors and outdoors. In Ahero, the sporozoite rate of An. arabiensis from indoor and outdoor CDC light traps was 0.38 and 0.35%, respectively. However, none of the An. arabiensis tested from PSCs and pit shelters were positive. The overall sporozoite rate of An. arabiensis was 0.16%. The sporozoite rate of An. funestus from indoor CDC light traps and PSCs was 2.6 and 2.0%, respectively, while this was 1.2% from both outdoor CDC light traps and pit shelters. Hence, in Ahero, the overall spozoite rate of An. funestus was 1.8%. Moreover, one An. coustani specimen from outdoor CDC light trap was positive for CSP.

Table 5.

Sporozoite rates of Anopheles mosquitoes from indoor and outdoor collections in Ahero and Iguhu, western Kenya

Study site and Anopheles sp.	Parameters	Indoor		Outdoor		Total
		Light trap	PSC	Light trap	Pit shelter
Ahero
An. arabiensis	No tested	263	264	286	447	1260
	Pf +ve (%)	1 (0.38)	0	1 (0.35)	0	2 (0.16)
An. funestus	No tested	194	100	169	84	547
	Pf +ve (%)	5 (2.6)	2 (2.0)	2 (1.2)	1 (1.2)	10 (1.8)
An. coustani	No tested	50	0	200	0	250
	Pf +ve (%)	0	0	1 (0.5)	0	1 (0.4)
An. pharoensis	No tested	25	0	205	0	230
	Pf +ve (%)	0	0	0	0	0
Iguhu
An. gambiae s.s.	No tested	84	46	50	34	214
	Pf +ve (%)	3 (3.6)	0	1 (2.0)	1 (2.9)	5 (2.3)
An. funestus	No tested	42	25	13	2	82
	Pf +ve (%)	1 (2.4)	1 (4.0)	0	0	2 (2.4)
An. arabiensis	No tested	8	5	2	5	20
	Pf +ve (%)	0	0	0	0	0
An. coustani	No tested	1	0	4	0	5
	Pf +ve (%)	0	0	0	0	0

Pf, Plasmodium falciparum; Pf +ve, number P. falciparum CSP positive (rate in percent)

In Iguhu, the sporozoite rate of An. gambiae s.s. from indoor CDC light traps was 3.6%, but none of the An. gambiae s.s. tested from PSCs was positive. In contrast, the sporozoite rate of An. gambiae s.s. from outdoor CDC light traps and pit shelters was 2.0 and 2.9%, respectively. Overall, the sporozoite rate of An. gambiae s.s. was 2.3%. The sporozoite rate of An. funestus from indoor CDC light traps and PSCs was 2.4 and 4%, respectively. No CSP was detected in An. funestus collected from outdoor CDC light traps and pit shelters. Thus, in Iguhu, the overall sporozoite rate of An. funestus was 2.4%.

Entomological inoculation rates (EIRs)

The EIRs of Anopheles mosquitoes are shown in Table 6. In Ahero, the estimated P. falciparum EIR of An. arabiensis from indoor and outdoor CDC light traps was 29.6 and 27.9 infective bites/person/year (ib/p/year), respectively, whereas the EIR of An. funestus from indoor and outdoor CDC light traps was 79.0 and 15.6 ib/p/year, respectively. The overall indoor and outdoor EIR was 108.6 and 43.5 ib/p/year, respectively. About 48% of the total infective bites by An. arabiensis and 16.5% by An. funestus occurred outdoor. The EIR of An. arabiensis and An. funestus from PSCs was 0 and 0.92 ib/p/year, respectively.

Table 6.

Entomological inoculation rates (EIR) of malaria vectors from indoor and outdoor collections in Ahero and Iguhu, western Kenya

Site and species	Parameters	Indoor		Outdoor
		Light trap	PSC	Light trap	Pit shelter
Ahero
An. arabiensis	SR	0.38	0	0.35	0
	EIR	29.6	0	27.9	0
An. funestus	SR	2.6	2.0	1.2	1.2
	EIR	79.0	0.92	15.6	0.05
An. coustani	SR	0	0	0.5	0
	EIR	0	0	16.8	0
Iguhu
An. gambiae s.s.	SR	3.6	0	2.0	2.9
	EIR	18.8	0	5.5	0.17
An. funestus	SR	2.4	4.0	0	0
	EIR	5.7	0.82	0	0
An. arabiensis	SR	0	0	0	0
	EIR	0	0	0	0

SR, sporozoite rate in percent; EIR, annual entomological inoculation rate measured as the number of infective bites/person/year; PSC, pyrethrum spray catch

In Iguhu, the estimated P. falciparum EIR of An. gambiae s.s. from indoor and outdoor CDC light traps was 18.8 and 5.5 ib/p/year, respectively, whereas the EIR of An. funestus from indoor and outdoor CDC light traps was 5.7 and 0 ib/p/year, respectively. The overall indoor and outdoor EIR was 24.5 and 5.5 ib/p/year, respectively. About 22.6% of the total infective bites by An. gambiae s.s. occurred outdoor. The EIR of An. gambiae s.s. and An. funestus from PSCs was 0 and 0.82 ib/p/year, respectively.

Discussion

This study showed that An. arabiensis was the most abundant anopheline species in Ahero (lowland), whereas An. gambiae s.s. was the most abundant species in Iguhu (highland) sites of western Kenya. An. funestus was the second most abundant species in both sites, which is consistent with previous studies [5, 6].

Anopheles arabiensis showed increased exophagic tendency in the study area when compared with the findings of studies conducted before the scale up of vector control interventions [22, 41]. For instance, studies by Githeko et al. in 1990s, when ITN coverage was negligeable, showed that An. arabiensis was two times more likely to bite indoors than outdoors [22]. In the present study, the outdoor biting density of An. arabiensis was higher than indoor. The increased outdoor host-seeking tendency of An. arabiensis in this study compared to the previous reports might be due to the scale-up of ITNs. Bayoh et al. also noted that An. arabiensis was more likely to bite outdoors in western Kenya when compared with data collected before the scale-up of ITNs [19]. Moreover, An. arabiensis showed highly exophilic behaviour in this study, with significantly higher outdoor resting density than indoor resting density.

The proportion of An. arabiensis has been increasing in western Kenya highlands. Until 2002, An. gambiae s.s. was the only member of An. gambiae s.l. complex reported in western Kenya highlands > 1400 m a.s.l. [25, 42]. The proportion of An. arabiensis was reported to be 0.8% in 2003 [43] and reached 9.2% in 2010 [5]. In this study, the proportion of adult An. arabiensis increased to 13%. A recent study reported a higher proportion of An. arabiensis (38.2%) in larval population [44]. The continued proportional increase in An. arabiensis population might be due to the increased ITN coverage [9, 10] and/or the zoophilic and exophagic/exophilic behaviour of this species or due to species shift. Other factors such as climatic and environmental change, which resulted in increased temperature or availability of more habitats in the area, might have also contributed as this was found to favor An. arabiensis [45]. Such shift in vector species composition could undermine the efficacy of ITNs as the interventions do not target zoophilic and exophilic vector species which avoids the lethal effect of ITNs and sustain residual malaria transmission [46].

Anopheles gambiae s.s. showed endophagic behaviour, with higher indoor host-seeking density than outdoor. This is in agreement with the earlier reports by Githeko et al. [22]. Recent studies in western Kenya have also showed that An. gambiae s.s. was more likely to seek hosts indoor than outdoor [19, 47]. In contrast, studies in Bioko Island, Equatorial Guinea showed that An. gambiae s.s. seek hosts outdoor than indoor [48]. This difference might be due to the variation in molecular forms of An. gambiae s.s. (S and M/Anopheles coluzzii) from Kenya and Equatorial Guinea [49] although the variability in host-seeking behaviour between the two molecular forms is not yet explicitly described.

It is unusual that An. gambiae s.s. showed similar feeding preference to human and bovine. Two decades ago, the HBI of indoor resting An. gambiae s.s. in western Kenya and other parts of the country was 96–97%, an indication that they had fed exclusively on humans [21, 23, 50]. In this study, the overall HBI of An. gambiae s.s. was only 50.0% although predominantly from indoor collection. Compared to the earlier studies conducted in western Kenya before ITNs were used in large scale [21, 23], the HBI of indoor resting An. gambiae s.s. has significantly dropped by 20% and the drop was entirely replaced by BBI. For outdoor resting An. gambiae s.s., the BBI reached up to 46%. Similar reduction in HBI and increment in BBI has also been reported recently [7, 15]. This suggests an increasing tendency of An. gambiae s.s. to feed on bovine following the increased ITN coverage in the western Kenya highlands.

Anopheles funestus s.s. was the predominant species among Anopheles funestus group in the study area. Similar findings were reported in Tanzania [51]. Kweka et al. [52] also found that An. funestus s.s. was the predominant sibling species in larvae population in western Kenya. However, there was significant difference in terms of the relative proportion of An. funestus s.s. between adult and larvae population. In this study, An. funestus s.s. accounted for 98.1% of the adult An. funestus s.l. population. In contrast, Kweka et al. found only 32.9% An. funestus s.s. in larvae population. This difference could be due to the presence of other zoophilic and exophilic sibling species of An. funestus s.l. in the larvae that do not bite or rest indoor or around human dwellings.

Anopheles funestus showed anthropophagic behaviour in both study sites, feeding predominantly on human. The anthrophagic behaviour of An. funestus was frequently observed in Kenya [21, 50] and elsewhere in Africa [53–56]. Nevertheless, they also fed on bovine, with higher BBI than the previous reports [21, 50, 53–56].

The secondary vectors, An. pharoensis and An. coustani showed exophagic behaviour, with significantly higher outdoor host-seeking density than indoor. Other studies in Kenya [41, 57] and elsewhere in Africa [58–60] reported similar phenomenon for these species. It is worth mentioning that both An. pharoensis and An. coustani were very rare in both indoor resting collections and pit shelters despite their preponderance in CDC light traps. Hence, further studies are required to find out the potential resting places of An. pharoensis and An. coustani.

The EIR data showed that the majority of malaria transmission by An. gambiae s.s. and An. funestus occurred indoors, while An. arabiensis contributed almost equally to both outdoor and indoor transmission. The higher indoor EIRs despite high ITN coverage could be attributed to inconsistent ITN use [61], increasing insecticide resistance among vectors [5, 8], and shifts in malaria vector biting times from mid-night to early evening and morning when people are still indoor but unprotected by ITNs [47, 62]. However, the magnitude of the outdoor EIRs was also considerably high compared to previous reports [19]. The ongoing shifts in vector species composition and changes in vector behaviours might have contributed to the high outdoor EIRs.

In addition to the primary vectors, a single specimen of An. coustani from outdoor CDC light trap was found to be positive for P. falciparum CSP based on ELISA, although not yet confirmed by PCR. Studies are increasingly reporting the importance of the secondary vectors in residual malaria transmission [57, 60, 63–65]. Several studies have demonstrated the susceptibility of An. coustani to P. falciparum infection [57, 59, 60, 66]. Although ELISA technique is not specific enough to incriminate zoophagic mosquitoes as a vector [33], a recent study in Madagascar confirmed the presence of Plasmodium CSP in An. coustani by both ELISA and PCR [60], suggesting that this species could play a role in outdoor malaria transmission.

Conclusion

Anopheles arabiensis was highly exophilic and zoophagic. Anopheles gambiae s.s. showed an increasing tendency to feed on bovine while An. funestus showed anthropophagic behaviour. While most of malaria transmission occurred indoor, the magnitude of outdoor transmission was considerably high. Additional control tools that complement the existing interventions are required to control residual transmission. Further studies are required to comprehend the role of secondary vectors in malaria transmission.

Abbreviations

BBI

bovine blood index

CDC

Centers for Disease Control and Prevention

CSP

circumsporozoite protein

EIR

entomological inoculation rate

ELISA

enzyme-linked immunosorbent assay

FR

forage ratio

HBI

human blood index

IRS

indoor residual spray

ITN

insecticide-treated net

PCR

polymerase chain reaction

PSC

pyrethrum spray catch