Biting behaviour and the spatio-temporal dynamics of malaria vectors in different ecological zones in Ghana

doi:10.21203/rs.3.rs-1975878/v1

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Biting behaviour and the spatio-temporal dynamics of malaria vectors in different ecological zones in Ghana

https://doi.org/10.21203/rs.3.rs-1975878/v1

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Background: Significant decrease in malaria morbidity and mortality have been attained using long-lasting insecticide treated nets and indoor residual spraying. Selective pressure from these control methods influences change in vector bionomics and behavioural pattern. There is a need to understand how insecticide resistance drives behavioural changes within vector species. This study aimed to determine the spatio-temporal dynamics and biting behaviour of malaria vectors in different ecological zones in Ghana in an era of high insecticide use for public health vector control.

Methods: Adult mosquitoes were collected during the dry and the rainy seasons in 2017 and 2018 from five study sites in Ghana in different ecological zones. Indoor and outdoor biting mosquitoes were collected per hour from 18:00 to 06:00 hours employing the human landing catches (HLC) technique. Morphological and molecular species identification of vectors were done using identification keys and PCR respectively. Genotyping of insecticide resistant markers was done using the TaqMan SNP genotyping probe-based assays. Detection of P. falciparum sporozoites was determined using PCR.

Results: A total of 50,322 mosquitoes belonging to four different genera were collected from all the study sites during the sampling seasons in 2017 and 2018. Among the Anophelines were Anopheles gambiae s.l. 93.16%, (31055/33,334), An. funestus 2.07%, (690/33,334), An. pharoensis 4.63%, (1545/33,334), and An. rufipes 0.13% (44/33,334). Overall, 76.40%, (25,468/33,334) of Anopheles were collected in the rainy season and 23.60%, (7,866/33,334) in the dry season. There was a significant difference (z = 2.410; p = 0.0160) between indoor biting (51.09%; 15,866/31,055) and outdoor biting An. gambiae s.l. (48.91%; 15,189/31,055). The frequency of the Vgsc-1014F mutation was slightly higher in indoor biting mosquitoes (54.90%) than outdoors (45.10%). Overall, forty-four pools (44) of samples were positive for P. falciparum CSP giving an overall sporozoite rate of 0.07%.

Conclusion: Anopheles gambiae s.l. were more abundant indoors across all ecological zones of Ghana. The frequency of G119S was higher in indoor than outdoor from all the study sites, but higher sporozoite rates in outdoor mosquitoes in Dodowa and Kpalsogu. There is thus, an urgent need for a supplementary malaria control intervention to control outdoor biting mosquitoes.

Anopheles

indoor and outdoor densities

biting time

sporozoite rate

genotypic resistance

The malaria burden in Africa is generally attributed to the relatively effective vector system that is made up of An. gambiae s.l, and An. funestus [1, 2]. The transmission potential of these vectors varies between climatic seasons, ecological zones, and sometimes among areas in close proximity [3, 4]. In Ghana, studies have shown that An. gambiae s.l. and An. funestus are the predominant malaria vectors and they occur in sympatry over much of their range [5, 6]. Information on the various malaria vector species and their distribution under the diverse ecological conditions is essential in their control strategies [7, 8].

There are three main ecological zones in Ghana – the coastal savannah zone in the south, the forest zone in the middle part, and the sahel savannah zone in the northern part of Ghana. There are also transition areas between these zones. The coastal and forest zones have a bimodal rainfall pattern which allows for two peaks of malaria transmission, while the Sahel savannah zone has unimodal rainfall pattern which makes malaria transmission seasonal. The different climatic conditions experienced in the various ecological areas contribute to differences in malaria vector species composition, hence malaria transmission [9, 10].

Vector control is key in the malaria control strategy [11] and like many other African countries long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS) are used in Ghana [12]. These methods which target only indoor biting mosquitoes have achieved remarkable successes towards malaria elimination but progress of these achievements has plateaued [13] due to the development and fast spread of insecticide resistance. The success of IRS and LLINs are largely based on the anthropophilic, endophagic and endophilic behaviours of Anopheles vectors, however, there is the growing threat of both physiological and behavioural resistance to the insecticides used in these vector control strategies [14, 15]. The complexity of controlling malaria is attributed to changes in species and the behavioural pattern of the malaria vectors [14, 16–19] and these variations in mosquito bionomics is attributed to the possible influence of IRS, LLINs and the development of insecticide resistance [20, 21]. The development of resistance by these vectors may maintain transmission where control interventions have been successful [22]. The wide and prolong use of LLINs and IRS causes certain behavioural changes in Anopheles vectors that helps them circumvent or eschew insecticide-treated areas [14, 23]. The most principal of such adaptations include a change in feeding behaviour.

Anopheles mosquitoes tend to change from their historically late night, indoor biting to early night and outdoor biting times [14]. These mosquitoes avoid IRS and LLINs control by feeding and resting outdoors. They also feed in the early hours of the evening when people are outside and not in bed and/or early in the morning when people are out of their bed nets [14, 15, 24]. The local dynamics of insecticide resistance may be impacted by the spatio-temporal variation in insect vectors [25] albeit selection pressure may have resulted in the variations in mechanism of insecticide resistance in malaria vectors [25]. Studies have shown strong association between observed frequency of knock down resistance (kdr) mutations and acetylcholine esterase (Ace-1), and resistance to pyrethroids and DDT in field mosquito populations [26, 27] thus, the presence of either kdr or Ace-1 gene in a field population of mosquito is a reliable indicator of both resistance prevalence and high individual resistance [28].

The variations in species distribution in different ecological zones could be influenced by some landscape barriers gene flow and exposure of the vector population to different levels of insecticide pressures[29]. Mutations caused by these factors at the neurons might be having a pleiotropic effect on the mosquito behaviour. Therefore, this study was to determine the spatio-temporal dynamics and biting behaviour of malaria vectors in different ecological zones in Ghana in an era of high insecticide use for public health vector control. This information will provide a better understanding of how the ecology in Ghana affect vector seasonal dynamics and explain the interactions between increased insecticide resistance in the malaria vector population and the ensuing biting behaviour patterns.

Study sites

Adult mosquitoes were collected during the dry season (February –March) and the rainy season (May-July) in 2017 and 2018 from five (5) study sites in Ghana. The study sites were selected from four ecological zones. Anyakpor (5° 46'51.96 "N 0° 35'12.84 "E) in the coastal savannah zone; Dodowa (5° 52' 58.3212'' N 0° 5' 52.9548'' W) in the coastal-forest transition zone; Dwease (6° 32'3.05 "N 1° 14'42.22 "W) in the forest zone; Pagazaa (9° 22'33.34 "N 0° 42'29.67 "W), and Kpalsogu (9° 33'45.2 "N 1° 01'54.6 "W) both in the Sahel savannah zone. These sites are shown in Fig. 1.

Anyakpor is a rural coastal community in the Ada East District of Ghana. It has a dry equatorial climate with temperatures between 23°C – 33°C and a bimodal rainfall pattern with a long rainy season from April to June and a short rainy season from October to November. Farming activities occur all year round which is supported by an irrigation scheme. This allows for uninterrupted farming activities throughout the year. There are dug-out wells and other water impoundments which collect water during the dry and rainy season, and these may serve as suitable breeding sites and eventually affect the densities of mosquitoes. A previous study by Hinne et al., [30], reported that, the most dominant species present in this site was An. coluzzii [30].

Dodowa is a town in the Shai Osudoku district with an average temperature of 27℃ and a bimodal rainfall pattern like Anyakpor. It has a secondary forest type vegetation with little original virgin forest left as a result of deforestation. Dwease is also a rural community close to Konongo in the Asante-Akim Central municipality with a wet-semi equatorial climate characterized by a bimodal rainfall just like Anyakpor. Dwease has a semi-deciduous forest vegetation of open and closed forests.

Kpalsogu and Pagazaa are rural communities in the Kumbungu and Tamale municipalities respectively. They have a unimodal rainfall pattern from May to November and a long dry season from December to April. The mean annual temperature is around 28°C but can get to a maximum of 42°C. Kpalsogu is in close proximity to dam linked to an irrigation scheme which allows uninterrupted farming activities throughout the year. There are other water impoundments which collect water during the rainy season for irrigation in the dry season. In the rainy season, these dam overflow, creating many swamps which are suitable breeding habitats for Anopheles mosquitoes. Water from the dam which is diverted through canals to farms also serves as breeding sites for mosquitoes and may affect mosquito densities within the arear. There is also an IRS campaign supported by the President’s Malaria Initiative (PMI) to prevent malaria ongoing within this community. Exposure of mosquitoes to sublethal doses of insecticide may facilitate resistance within the vectors. Moreover, this indoor vector control strategy may facilitate outdoor biting by the malaria vectors present there.

Mosquito collections

Indoor and outdoor biting mosquitoes were collected per hour from 18:00 to 06:00 hours for four different nights in four houses per season by a two-person team of trained catchers in 8 randomly selected houses employing the human landing catches (HLC) technique [31]. Collections were done in the 5 sentinel sites in 4 different ecological zones of Ghana. Each study site was sectioned into 4 to ensure a fair representation of the mosquito population at the study site. Briefly, volunteers sat in the dark with their lower limbs exposed and with the aid of a flashlight, located and collected the blood-seeking mosquitoes with a collection tube when they landed in search of a blood meal. Indoor and outdoor collectors were rotated hourly to avoid differences in individual attractiveness or repulsiveness to mosquitoes and as a precaution against dozing. Outdoor human biting catches were carried out at the same household ten meters away [32].

Hourly mosquitoes caught were kept separately in individual tubes containing silica gel and pre-labeled with date, time and location of capture and taken to the laboratory for identification [33]. Independent staff supervised rotations and regularly walked between different groups for whole-night quality control of collectors placed inside and outside dwellings. Mosquito biting pattern was classified as follows: Early evening (EE) (6:00pm-10:00pm), late evening (LE) (10:00pm – 4:00am), and early morning (EM) (4:00am – 6:00am).

Morphological and molecular identification of vector mosquitoes

Mosquitoes collected were identified morphologically using a simplified key adopted from Gillies and Coetzee [34]. Members of the An. gambiae complex were further identified by PCR. The legs of each mosquito were used for DNA extraction as previously described [35]. Four sets of oligonucleotide primers (An. gambiae, An. arabiensis, An. melas and universal primer) were used in PCR for the identification of members of the Anopheles gambiae s.l species complex [35]. Anopheles gambiae s.s and An. coluzzii were distinguished by PCR-RFLP using the method of Hinne et al. [30].

Genotyping for insecticide resistance mutations

Genomic DNA extracted from the legs of the indoor and outdoor mosquito samples were used to detect the presence of insecticide resistance mutations. Single nucleotide polymorphism (SNP) markers were screened from DNA of each specimen using a TaqMan SNP genotyping probe-based assays [36]. These markers include Vgsc-1014F and Vgsc-1014S. The same set of samples were also genotyped for Ace1-119S mutation [36].

Detection of sporozoite

The head and thoracis of mosquito samples were used to detect the presence of P. falciparum sporozoite using polymerase chain reaction (PCR) as described by Echeverry et al. [37]. Twenty (20) mosquitoes were pooled according to the site, species and collection time for the detection of sporozoite; a total of 643 pools were constituted from a total of 12,860 Anopheles gambiae s.l.

Data analysis

Chi-square was used to test the difference in seasonal abundance and malaria vector species composition between biting locations (indoor and outdoor). The sporozoite infection rate (IR) expressed as the proportion of mosquitoes positive for Plasmodium sporozoite was calculated according to the method previously described by Maia et al. [38]. In brief, Sporozoite rate equals the number of positive pools divided by the multiplication of number of pools and maximal pool size, all multiply by 100%. The kdr L1014F and Ace 1 G119S mutation frequencies were calculated according to the following formula:

F (kdr) = (2RR + RS) / 2n

where RR is the number of homozygotes, RS is the number of heterozygotes, and n is the total number of specimens analysed according to Ahadji-Dabla et al., [39].

Abundance and seasonal distribution of malaria vectors

A total of 50,322 mosquitoes belonging to four different genera were collected from all the study sites during the sampling seasons in 2017 (n = 26415) [Anopheline = 18153/26415, Culicine = 5856/26415, Mansonia = 2406/26415] and 2018 (n = 23907) [Anopheline = 15181/23907, Culicine = 6614/23907, Mansonia = 2112/23907]. Overall, 66.24%, (33,334/50,322) of mosquitoes sampled were Anophelines, 24.78%, (12,470/50,322) were Culicine, 8.98%, (4,736/50,322) were Mansonia mosquitoes. Among the Anophelines were Anopheles gambiae s.l. 93.16%, (31055/33,334), An. pharoensis 4.63%, (1545/33,334), An. funestus 2.07%, (690/33,334), and An. rufipes 0.13% (44/33,334).

Overall, 76.40%, (25,468/33,334) of Anophelines were collected in the rainy season and 23.60%, (7,866/33,334) in the dry season. Across the different ecological zones, the abundance of An. gambiae s.l. were high in the Sahel savannah [2017 (7,818/17,245); 2018 (6,883/13,810)] followed by the coastal savannah [2017 (7,110/17,245); 2018 (5,695/13,810)], and then the forest zone [2017 (2,317/17,245); 2018 (1,232/13,810)]. An. gambiae s.l. were more abundant in the rainy season (75.51%; 23,449/31,055) than in the dry season (24.49%; 7,606/31,055) during both sampling years (z = -36.037; p _< 0.0001). In all the study sites, An. gambiae s.l. were more abundant in the rainy season than in the dry season except in Anyakpor and Dwease where An. gambiae s.l. were more abundant in the dry season (60.50%; 3082/5094), (68.83%; 848/1232) respectively, than in the rainy season during the 2018 sampling period. The most abundant other Anopheline mosquitoes were sampled from Kpalsogu = 692 [An. pharoensis = 641/692, An. funestus = 44/692, An. rufipes = 7/692]; Anyakpor = 151 [An. pharoensis = 149/151, An. funestus = 0/151, An. rufipes = 2/151]; Pagaza = 48 [An. pharoensis = 11/48, An. funestus = 35/48, An. rufipes = 2/48]; Dodowa = 15 [An. pharoensis = 12/15, An. funestus = 1/15, An. rufipes = 2/15]; Dwease = 2 [An. pharoensis = 2/2, An. funestus = 0/2, An. rufipes = 0/2] during the 2017 sampling season, and Pagaza = 563 [An. pharoensis = 25/563, An. funestus = 537/563, An. rufipes = 1/563); Anyakpor = 391 [An. pharoensis = 388/391, An. funestus = 0/391, An. rufipes = 3/391]; Kpalsogu = 351 [An. pharoensis = 310/351, An. funestus = 15/351, An. rufipes = 26/351]; Dwease = 58 [An. pharoensis = 0/58, An. funestus = 58/58, An. rufipes = 0/58]; Dodowa = 8 [An. pharoensis = 7/8, An. funestus = 0/8, An. rufipes = 1/8] during the 2018 sampling season. The most predominant species sampled were An. pharoensis [2017(815/908), 2018 (730/1371)], followed by An. funestus [2017(80/908), 2018 (610/1371)], and then An. rufipes [2017(13/908), 2018 (31/1371)]. High abundance of the other Anopheline species were sampled during rainy season [2017 (pharoensis = 681/908, funestus = 80/908; rufipes = 10/908); 2018 (pharoensis = 635/1371, funestus = 609/1371, rufipes = 4/1371], as compared to the dry season [2017 (pharoensis = 134/908; funestus = 0/908; rufipes = 3/908); 2018 (pharoensis = 95/1371, funestus = 1/1371, rufipes = 27/1371)].

Indoor and outdoor distribution of vectors

Overall, there was a significant difference (z = 2.410; p = 0.0160) between indoor biting (51.09%; 15,866/31,055) and outdoor biting An. gambiae s.l. (48.91%; 15,189/31,055). The abundance of An. funestus biting outdoor (59.28%; 409/690) was higher than those that were caught biting indoor (40.72%; 281/690) but the difference was not significant (z = -1.928; p = 0.0538).

In 2017, 48.19%, (8,311/17,245) An. gambiae s.l. were collected indoors and 54.70% (7555/13,810) in 2018 as shown in Fig. 2, at all the study sites. The abundance of indoor biting An. gambiae s.l. increased in the 2018 sampling period except in Dwease in the forest area where the abundance of indoor An. gambiae s.l. reduced from 53.73% (1245/2317) in 2017 to 50.57% (623/1232) 2018. During the 2017 sampling period, Anopheles gambiae s.l. preferred to bite indoors in all sites, except in Dodowa where there was more outdoor biting (59.64%; 3,139/5,263) than indoor biting (40.36%; 2,124/5,263) (Fig. 2).

Species discrimination in the Anopheles gambiae complex

A subsample of 1,670 of An. gambiae s.l. from all the study sites were randomly selected and used to discriminate the sibling species. An. coluzzii 55.9% (935/1670) was the most abundant species followed by An. gambiae s.s 39.5% (659/1670), An. arabiensis 2.3% (39/1670) and An. melas 2.2% (37/1670). Overall, An. coluzzii was the abundant species sampled in all the ecological zones, except in the Sahel savannah zone, where the species were dominated by the An. gambiae s.s. [Sahel (An. gambiae s.s. = 323/1670; An. coluzzii = 298/1670; An Arabiensis = 39/1670; An. melas = 0/1670); coastal (An. gambiae s.s. = 288/1670; An. coluzzii = 432/1670; An Arabiensis = 0/1670; An. melas = 37/1670); forest (An. gambiae s.s. = 48/1670; An. coluzzii = 205/1670; An Arabiensis = 0/1670; An. melas = 0/1670)].

The composition and distribution of these species differed significantly by study sites and year (Fig. 3; p < 0.001). An. coluzzii was most abundant in Anyakpor (83.65%, 394/471), Dwease (81.03%, 205/253) and Kpalsogu (80.20%, 239/298) while An. gambiae s.s was the most abundant in Dodowa (86.71%, 248/286) and Pagaza (74.03%, 268/362) respectively. All the An. melas collected during the study were only from the coastal savannah site of Anyakpor (7.86%, 37/471). All An. arabiensis were from Sahel savannah sites of Kpalsogu (1.34%, 4/298) and Pagazaa (9.67%, 35/362) (Fig. 3).

Biting times of An. gambiae s.l in the study sites

Anopheles gambiae s.l. were found to bite the most during the late evening (LE) (66.61%; 20685/31055), followed by the early morning (EM) (20.05%; 6228/31055), and less during the early evening (EE) (13.34%; 4142/31055). This was the same pattern for both indoor [LE: (66.12%, 10490/15866); EM: (20.52%, 3255/15866); EE: (11.42%, 2121/18566)] and outdoor biting [LE: (67.12%,10195/15189); EM: (19.57%, 2973/15189), EE: (13.96%, 2021/15189)] (Fig. 4).

With respect to the species of the An. gambiae sl., An. coluzzii and An. melas had a different biting pattern from An. gambiae s.s. and An. arabiensis. An. coluzzii preferred late evening feeding (48.02%; 449/935) followed by early morning (26.52%; 248/935) and early evening (25.45%; 238/935) feeding. Anopheles melas on the other hand preferred to bite in the early evening (43.24%; 16/37) followed by the late evening (40.54%; 15/37) and the early morning (16.22%; 6/37). Anopheles gambiae s.s preferred to bite in the late evening (54.48%; 359/659) followed by the early evening (25.04%; 165/659) and the early morning (20.46%; 135/659). Anopheles arabiensis preferred late evening (41.03%; 16/36) biting followed by early evening (33.33%; 13/36) and early morning (25.64%; 10/36) biting.

Insecticide resistance genotypes in Anopheles gambiae s.l.

Anopheles gambiae s.l samples were genotyped for the presence of Vgsc-1014S and 1014F mutations as well as the G119S mutation. Overall, the frequency of the Vgsc-1014F mutation was slightly higher in indoor biting mosquitoes (54.90%) than those biting outdoors (45.10%). Overall, Anopheles melas had a high Vgsc-1014F mutation (87.1%), whiles An. arabiensis had (50%) (Table 2).

Table 1
	An. gambiae s. s	An. coluzzii	An. melas	An. arabiensis	Total
	No (%)	No (%)	No (%)	No (%)	Total
Anyakpor	40 (8)	394 (84)	37 (8)	0	471
Dodowa	248 (87)	38 (13)	0	0	286
Dwease	48 (19)	205 (81)	0	0	253
Kpalsogu	55 (18)	239 (80)	0	4 (1)	298
Pagazaa	268 (74)	59 (16)	0	35 (10)	362
Total	659 (39)	935 (56)	37 (2)	39 (2)	1670

Table 2

Species of the *Anopheles gambiae* complex and frequency of *kdr L1014F* and *Ace-1 G119S* mutation
	N	Kdr L1014F	n	F (kdr)	N	Ace 1 G119S	n	F (Ace-1)
Indoor	873	RR	328	0.57	870	RR	178	0.54
		RS	338			RS	579
		SS	207			SS	113
Outdoor	752	RR	266	0.55	777	RR	120	0.48
		RS	302			RS	512
		SS	184			SS	145
Species
An. arabiensis	36	RR	13	0.50	39	RR	2	0.44
		RS	10			RS	30
		SS	13			SS	7
An. coluzzii	908	RR	300	0.53	924	RR	187	0.52
		RS	363			RS	591
		SS	245			SS	146
An. gambiae s.s	646	RR	255	0.59	648	RR	96	0.50
		RS	258			RS	451
		SS	133			SS	101
An. melas	35	RR	26	0.87	36	RR	13	0.63
		RS	9			RS	19
		SS	0			SS	4
F(kdr) = 2RR + RS/2n Ahadji-Dabla 2019. F: allelic frequency, N = number of samples tested, n = total number of samples positive for a specific genotype

Similarly, the frequency of the G119S mutation in An. gambiae s.l were slightly higher in indoor host seeking mosquitoes (52.94%) than outdoor biting mosquitoes (47.06%). Anopheles melas had a high frequency of G119S mutation 0.63 (30.14%), whiles An. arabiensis had the lowest frequency of 0.44 (21.01%) (Table 3). The Vgsc-1014S mutation was not detected in any mosquito for this study.

Table 3

Sporozoite rates in the *An. gambiae* s.l
Community	Number per pools	Total Pools	Positive pools	Dry season	Rainy season	Indoor	Outdoor	Sporozoite Rate (SR)	Dry season SR	Rainy season SR	Indoor SR	Outdoor SR
Anyakpor	20	164	8	4	4	7	1	0.24	0.12	0.12	0.21	0.03
Dodowa	20	105	7	5	2	3	4	0.33	0.24	0.10	0.14	0.19
Dwease	20	103	10	5	5	6	4	0.49	0.24	0.24	0.29	0.19
Pagazaa	20	131	11	4	7	6	5	0.42	0.15	0.27	0.23	0.19
Kpalsogu	20	140	8	4	4	3	5	0.29	0.14	0.14	0.11	0.18
		643	44
* Sporozoite Rate SR= ((no of positive pools) / (no of pools × maximal pool size)) × 100 Wei-Dong Gu 1995

Sporozoite infection rates in the sampled vectors

A total of 12,860 Anopheles gambiae s.l were pooled in groups of 20, into 643 pools and tested for Plasmodium falciparum Circumsporozoite (CSP). Overall, forty-four pools (44) were positive for P. falciparum CSP (Anyakpor (n = 8), Dodowa (n = 7), Dwease (n = 10), Kpalsogu (n = 8), and Pagazaa (n = 11)) giving an overall sporozoite rate of 0.07% (Table 3).

Dwease had the highest sporozoite rate (0.49%), followed by Pagazaa (0.42%), Dodowa (0.33%), and Kpalsogu (0.29%), with Anyakpor reporting the lowest sporozoite rate (0.24%). Putting all the sites together, sporozoite rate was quite similar in indoor biting An. gambiae s.l. (0.04%) compared to those that were biting outdoors (0.03%). With respect to the individual study sites, sporozoite rate was higher indoors compared to outdoors except in Dodowa [(indoor (0.14%); outdoor (0.19%)] and Kpalsogu [(indoor (0.11%); outdoor (0.18%)] where outdoor biting An. gambiae s.l. had a higher sporozoite rate.

Furthermore, the sporozoite rates were similar during both the dry and rainy seasons in Anyakpor [dry = 0.12%; rainy = 0.12%], Dwease [dry = 0.24%; rainy = 0.24%], and Kpalsogu [(dry = 0.14%; rainy = 0.14%]. All the study sites had similar sporozoite rates in both seasons as represented in Table 2 except in Dodowa where the sporozoite rate was higher in the dry season (0.24%) compared to the rainy season (0.10%), and in Pagazaa where the rainy season had a higher sporozoite rate (0.27%) compared to the dry season (0.15%).

Evidence has shown that successful malaria elimination strategies require vector control intervention that target the changing vector behaviour [40]. It is therefore, essential to monitor the changing vector behaviour and ecology in the era of increasing malaria intervention to reduce the high disease burden. This study investigated the temporal and spatial dynamics and the effect of resistant genotypes on biting behaviour in malaria vectors.

The majority of vectors were collected during the rainy season, which is likely due to the availability of breeding habitats that facilitates oviposition by gravid females. However, the presence of mosquitoes in high abundance during the dry season in Anyakpor and Kpalsogu was likely due to the irrigated farming, which supports the breeding of vectors during the dry season [5, 30]. During the rainy season, these areas that are low-lying get flooded and therefore disrupts malaria vector breeding, however, during the dry season, the irrigation areas provide breeding habitats for continuous vector breeding. This implies that, malaria transmission may be occurring in these areas all year-round [41, 42].

Malaria vectors in Africa have been efficient in malaria transmission largely due to their anthropophilic and endophilic nature [6, 43]. Therefore, knowledge of the biting behaviour in disease vectors is important to understand the role of the vectors in disease transmission, and hence, the deployment of effective control tools. In this study, the abundance of Anopheles mosquitoes that were biting indoors were relatively similar to the outdoor biting. Compared to a study done in Ghana by Tuno et al., 2010 [44] in which the abundance of outdoor An. gambiae were 15% and 23% during the dry and rainy season respectively, this study has shown a drastic increase in the outdoor biting activity in An. gambiae s.l. According to Sherrard-Smith et al., (2019), mathematical models suggest that even the smallest changes in outdoor host seeking activity of malaria vectors can have a substantial public health impact.

Larger increases in outdoor biting behaviour leads to a reduced effectiveness of LLINs [45]. The shift in biting behaviour may be due to selective pressure mounted by the use of LLINs and IRS. Because LLINs and IRS are indoor based, increase in outdoor biting mosquitoes may indicate possible outdoor malaria transmission which calls for the need of outdoor vector interventions [46, 47]. Historically, the large-scale use of LLINs and IRS leads to an increase in the abundance of outdoor biting vectors [24]. The high densities of outdoor biting An. gambiae contributes to the persistence of malaria in the Sahel savannah area despite LLINs and IRS interventions (Monroe et al., 2015). The presence of outdoor feeding mosquitoes limits the effectiveness of these interventions [24, 48] and may be of major public health concern. It will be important that, vector control strategies which targets both indoor and outdoor malaria vectors should be introduced in these areas.

Overall, An. coluzzii was the most abundant vector, however, An. gambiae s.s. was the predominant species present in the costal savannah and forest zone. This is indicative that the primary malaria vectors are well established across all ecological zones, hence constant surveillance and strengthening of control strategies is of essence. These findings corroborates the findings of Hinne et al., [30] whose study was done in similar study sites which reported high abundance of An. coluzzii as compared to An. gambiae .s.s in these study sites[30].

Findings from this study showed that both indoor and outdoor An. gambiae s.l. preferred to bite late in the night when people were asleep. Peak biting activity in the late night occurs because household members begin to rise as early as 03:00hrs to begin morning chores including fetching water and firewood, feeding animals, cooking, bathing, and preparing for market during market days. Outdoor sleeping is also a major factor contributing to peak outdoor biting in the late evenings. People sleep outside during the early night time due to high temperatures in the rooms and wait till about 02:00hrs [48] when their rooms are cool enough to sleep indoors. During the dry season in the Sahel savannah areas, some people spend the entire night sleeping outdoors, because their rooms become extremely warm. Other outdoor activities such as funerals, church activities and trading are motives for people to stay outside in the late evening [48]. This finding corroborates with a study in Uganda, that reported that the peak biting times for An. gambiae s.l. was between 23:00–5:00 hours [15]. These behavioural activities by An. melas and An. arabiensis in correlation to the vector densities observed for both indoor and outdoor settings could have major public health implications, because with the level of resistance and sporozoites rate observed in the malaria vectors, there could be possible transmission of malaria outdoor (residual malaria). Malaria vector feeding and resting behaviours are likely to change to maximize available feeding opportunities. An. melas and An. arabiensis preferred to bite their host outdoors compared to indoors, whereas An. coluzzii and An. gambiae preferred indoor biting.

The frequency of kdr mutations was very high but similar in both outdoor and indoor biting mosquitoes. This could mean that mosquito population from the study sites are able to resist the presence of insecticides that are employed for vector control and may lead to an increased human-vector contact and malaria transmission in the region despite the high coverage of LLINs. The Vgsc-1014F mutation has been found to be strongly associated with pyrethroids resistance in West Africa, consequently their presence in indoor biting mosquitoes may be of particular concern [49]. The presence of Vgsc-1014F mutation in a mosquito population is a reliable marker of both high individual resistance and resistance prevalence [50, 51]. Studies have shown a relationship between the spread of Vgsc-1014F alleles with the use of LLINs [52, 53]. The lowest frequency of Vgsc-1014F was found in An. arabiensis which could be explained by the biting behaviour of this species; An. arabiensis prefers to bite and rest outdoors and this could have limited their exposure to the insecticides used in vector control.

The results from the study showed that the frequency of G119S from the mosquito population was higher in indoor than outdoor mosquito population from all the study sites. The highest frequency of G119S was observed in An. melas and the lowest was found in An. arabiensis. The high frequency of Ace-1 mutation that was observed in Anyarkpor may be due to the frequent use of pesticide in agricultural activities in the area and exposure to malaria vectors, since most of these pesticides contain the same active ingredients as insecticides used for public health control of malaria vectors [54–56]. This may imply that vector control management tools may fail in such areas and requires careful monitoring. A similar observation was made in Southern Ghana by Essandoh et al. (2013), who reported a high prevalence of resistance in malaria vectors was consistent with agriculture-driven selection [26].

Sporozoite rates determined during the study was similar for both sampling years (2017 and 2018), suggesting that malaria transmission did not change between the years. Higher sporozoite rates was recorded in indoor mosquitoes compared to outdoor biting mosquitoes from all the study sites except in Dodowa and Kpalsogu where the sporozoite rate were higher in mosquitoes collected outdoor than those from indoor, this is suggestive of outdoor malaria transmission (residual malaria) and it is imperative that vector control tools should be implemented to target outdoor biting mosquitoes as well. The infection rates found in the indoor biting mosquitoes could suggest ongoing malaria transmission regardless of vector control tools employed in the study sites.

This study revealed that, Anopheles gambiae s.l. were more abundant indoors across all ecological zones of Ghana. Irrigated farms create breeding habitats for the breeding of malaria vectors during the dry season, thereby supporting malaria transmission year-round. Furthermore, the abundance of Anopheles mosquitoes and frequency of kdr mutations was similar in both indoor and outdoor biting mosquitoes. However, the frequency of G119S from the mosquito population was higher in indoor than outdoor mosquito population from all the study sites. Higher sporozoite rates was recorded in outdoor mosquitoes in Dodowa and Kpalsogu. There is thus, an urgent need for a supplementary malaria control intervention to control outdoor resting and biting mosquitoes, as the current tools only target indoor resting and biting mosquitoes. Continued surveillance of vector behaviours is recommended to help in the control of malaria.

WHO: World Health Organisation; LLINs: Long lasting insecticide treated nets; IRS: Indoor residual sprays; PMI: President Malaria Initiative; Ace-1: Acetylcholinesterase 1; kdr: knockdown resistance; HLC: Human landing catch; EE: Early evening; LE: late evening; EM: early morning; PCR: Polymerase chain reaction; DNA: Deoxyribonucleic acid; PCR-RFLP: Polymerase chain reaction-restriction fragment length polymorphism; Vgsc: voltage-gate sodium channel; SNP: single nucleotide polymorphism; G: glycine; L: leucine; F: phenylalanine; S: serine; RR: homozygote resistant; RS: heterozygote resistant; SS: homozygote susceptible; N: number of samples tested; n: total number of positive samples; NMIMR: Noguchi Memorial Institute for Medical Research; CSP: Circumsporozoite protein

Acknowledgments

We thank the residents at our study sites for their support during the study. Our sincere gratitude goes to Michelle Asigbaase, Andy Asafu-Adjaye, Dr. Joseph Harold Nyarko Osei, Sakyi Kojo Yirenkyi, Dr. Sellase Pi-Bansa and Dr. Kojo Frimpong who are staffs in the Parasitology unit of the Noguchi memorial institute for medical research (NMIMR) for their support during laboratory work ; and Isaac Sraku , Anisa Abdulai and Prince Ashong of the IMAT research team of the Medical Microbiology Department, University of Ghana for their help during the laboratory activities as well. We also thank Thomas Peprah Agyekum for his help during data analysis.

Funding

This study was supported by grants from the National Institute of Health (RO1A1123074 and D43TW011513), and it is also part of the research undertaken towards the award of Doctor of Philosophy (PhD) Degree in Entomology under the sponsorship of the Deutscher Akademischer Austauschdienst (DAAD).

Availability of data and materials

The datasets used for this study are available from the corresponding author on reasonable request.

Authors’ contribution

Conceived and designed the experiments: OKA, SKD, YAB, MDW, YAA. Supervised data collection: OKA, SBD, ARM, CMO-A, SC, YAA. Performed the experiments: OKA, SBD, ARM, CMO-A. Analysed the data: OKA, SBD, IAH, CMO-A, YAA. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Scientific and ethical approval for the study was obtained from the Institutional Review Board of the Noguchi Memorial Institute for Medical Research (NMIMR). Verbal and written informed consent of the village elders and compound/household heads and the mosquito collectors were taken before mosquitoes were collected from the study sites. The mosquito catchers were recruited from the various sampling sites to foster acceptance from residents. The catchers were trained to collect landing mosquitoes prior to blood feeding to minimize the risk of malaria transmission.

Consent for publication

Not applicable

Competing interest

The authors declare that they have no competing interests.

Author details

¹Department of Parasitology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon.²African Regional Post-Graduate Programme in Insect Science, College of Basic and Applied Science, University of Ghana, Legon. ³Department of Medical Microbiology, University of Ghana Medical School, University of Ghana, Korle-Bu, Accra.⁴Department of Animal biology and conservation Science, College of Basic and Applied Sciences, University of Ghana, Legon. ⁵Dentistry (SMD) - College of Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi – Ghana.

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No competing interests reported.

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Biting behaviour and the spatio-temporal dynamics of malaria vectors in different ecological zones in Ghana

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Abstract

Figures

Background

Methods

Study sites

Mosquito collections

Morphological and molecular identification of vector mosquitoes

Genotyping for insecticide resistance mutations

Detection of sporozoite

Data analysis

Results

Abundance and seasonal distribution of malaria vectors

Indoor and outdoor distribution of vectors

Sporozoite infection rates in the sampled vectors

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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