Seasonal distribution and abundance of host-seeking malaria vectors
A total of 39,863 mosquitoes consisting of four different genera were collected using HLC across all study sites during the sampling period: Anopheles (52.54%, 20,945/39,863), Culex (42.10%, 16,742/39,863), Aedes (3.74%, 1,491/39,863) and Mansonia (1.72%, 685/39,863). About 41.75% (16,642/39,863) of mosquitoes were collected during the dry season [Anopheles (30.47%, 5,071/16,642), Culex (64.46%, 10,727/16,642), Aedes (1.44%, 240/16,642), Mansonia (2.55%, 424/16,642)] and 58.25% (23,221/39,863) in the rainy season [Anopheles (68.36%, 15,874/23,221), Culex (25.90%, 6,015/23,221), Aedes (4.61%, 1,071/23,221), Mansonia (1.12%, 261/23,221)]. Overall, Anopheles dominated (52.54%, 20,945/39,863), followed by Culex (42.00%, 16,742/39,863), Aedes (3.74%, 1,491/39,863), and Mansonia (1.72%, 685/39,863). Among the Anopheles, the highest abundance was An. gambiae s.l. (99.93%, 20,931/20,945), with smaller proportions of An. funestus s.l. (0.02%, 4/20,945), An. pharoensis (0.03%, 6/20,945), and An. rufipes (0.02%, 4/20,945) (Table 1).
Among the site categories, irrigated urban farming (IUF) had the most abundant of An. gambiae s.l. (48.85%, 10,224/20931), followed by peri-urban (PU) (17.86%, 3738/20931), and the lowest was from low socioeconomic (LS) (2.84%, 595/20931). The rainy season had a higher abundance of Anopheles gambiae s.l. in all sites categories when compared to the dry season [IUF (dry = 3,067, rainy = 7,157), PU (dry = 805, rainy = 2,933), LS (dry = 127, rainy = 468), MS (dry = 469, rainy = 2,402) and HS (dry = 598, rainy = 2,905)]. Among the individual sites, Tuba in the irrigated urban farming category recorded the highest abundance of An. gambiae s.l. in both dry and rainy season [dry (47.55%, 2,409/5,066), rainy (38.20%, 6,061/15,865)], whereas Chorkor in the LS categories had the lowest abundance of mosquitoes in both seasons [dry (1.11%, 56/5,066), rainy (1.32%, 210/15,865)]. An. funestus species were exclusively collected from Medie in the peri-urban site category (Table 1) during the dry season.
Among the sampled anophelines collected, only 0.07% (14/20,945) comprised of other Anopheles species (Table 1). These species consist of An. rufipes (28.57%, 4/14), An. funestus (28.57, 4/14) and An. pharoensis (42.86%, 6/14). An. pharoensis species were exclusively collected during the rainy season, whereas, An. funestus were sampled during the dry seasons.
Seasonal distribution and abundance of resting malaria vectors
A total of 1,884 resting mosquitoes were collected from all sites. The most abundant genera of mosquitoes sampled were the culicines (57.22%, 1,078/1,884), followed by the anophelines (31.26%, 589/1,884) and then Aedines (11.52%, 217/1,884). More Anopheles gambiae s.l. were sampled during the rainy season (77.08%, 454/589) compared to the dry season (22.92%, 135/589) (t = −0.0405, df = 587, P = 0.48). Tuba, from the irrigated urban farming site category, had the highest abundance of resting An. gambiae s.l., 26.49%, 156/589 (rainy = 116, dry = 40), with the least sampled from East Legon from the HS site category, 1.70%, 10/589 (rainy = 7, dry = 3).
Indoor and outdoor abundance of resting Anopheles gambiae s.l.
Of the total (n = 589) resting Anopheles gambiae s.l. mosquitoes that were sampled during the study period, significantly abundant Anopheles gambiae s.l were collected indoors (60.44%, 356/589) as compared to outdoors (49.56%, 233/589) (t = 1.9103, df = 587, P = 0.03). Among the sites, Tuba (IUF) had the highest abundance of An. gambiae s.l. in both indoor (24.72%, 88/356) and outdoor (29.18%, 68/233), whereas the least indoor (1.40%, 5/356) and outdoor (2.15%, 5/233) abundance were recorded in East Legon (HS) (Table 2).
Species discrimination in host-seeking Anopheles gambiae complex
From the host-seeking An. gambiae s.l. collected, a subsample of 2,232 from all the study sites in both seasons were randomly selected and used to discriminate the sibling species. An. gambiae s.s (68.86%, 1,537/2,232) was the most abundant species followed by An. coluzzii (27.24%, 608/2,232), and hybrids of An. gambiae s.s. and An. coluzzii (3.90%, 90/2,232) (Table 3). With respect to socioeconomic characterization, more An. coluzzii was collected from LS [An. gambiae s.s. = 26.14% (40/153); An. coluzzii = 67.97% (104/153); hybrids = 5.88% (9/153)] and MS [An. gambiae s.s. = 36.65% (103/281); An. coluzzii = 62.28% (175/281); hybrids = 1.07% (3/281)] site categories. However, An. gambiae s.s. was more abundant in the other site categories [PU, HS, IUF] (Tables 3).
Species discrimination in resting Anopheles gambiae complex
With resting mosquitoes, the total An. gambiae s.l. (n = 589) from all the study sites were used to discriminate the sibling species. An. coluzzii (64.52%, 380/589) were the most abundant, followed by Anopheles gambiae s.s, (26.99%, 159/589) and then Hybrids of An. coluzzii and An. gambiae s.s. (8.49%, 50/589). During both seasons, An. coluzzii was the most abundant sampled, Dry [indoor (48); outdoor (41)]; Rainy [indoor (164); outdoor (127)], followed by An. gambiae s.s, Dry [indoor (24); outdoor (9)]; Rainy = [indoor (85); outdoor (41)], and then hybrids; Dry [indoor (8); outdoor (5)] Rainy = [indoor (22); outdoor (15)], (Fig. 2).
Indoor and outdoor seasonal distribution of resting Anopheles gambiae species
Biting times of An. gambiae s.l. in the study sites
An. gambiae s.l. exhibited the highest biting activity during the late evening (LE) at 71.64% (14,995/20,931), followed by the early morning (EM) at 17.56% (3,675/20,931), and the lowest during the early evening (EE) at 10.80% (2,261/20,931). This pattern was consistent across all sites and site categories. Notably, significantly higher biting activity was recorded in the LE for both the dry (72.21%, 3,658/5,066) and rainy seasons (71.45%, 11,337/15,865) [F (2, 27) = 6.03, P = 0.019, 95% CI 135.2691–1388.731] (Fig. 3).
Biting patterns of An. gambiae s.l according to site categories
Biting activity varied across site categories, with a non-significant trend of higher activity observed in the irrigated urban farming site category, particularly during the late evening, peaking between 02:00 and 03:00 h [F (4,10) = 1.36, P = 0.176, 95% CI −793.9493–3781.949].
The human-biting activity of An. gambiae s.l. displayed a similar pattern across all study sites, showing a bimodal distribution. The first and highest peak occurred between 02:00 and 03:00 h (15.52%, 3,248/20,931), while a secondary peak was observed in the early morning between 04:00 and 05:00 h (12.09%, 2,530/20,931). Although biting activity was higher in the early morning hours compared to the early evening hours, biting activity steadily declined from 05:00 to 06:00 h.
Blood meal sources of resting An. gambiae s.l. mosquitoes collected
Out of the 589 An. gambiae s.l. mosquitoes collected, 70.63% (416/589) were found to have fed on blood from various sources. The Human Blood Index (HBI) was consistently higher across all site categories and species at 86.30% (359/416), compared to the Bovine Blood Index (BBI), which was 29.33% (122/416). Hybrids collected indoors at the Peri-Urban (PU) site exhibited the highest HBI of 100% (2/2), while the highest BBI, 50% (5/10), was observed among hybrids collected outdoors at the Irrigated Urban Farming (IUF) site.
Mixed blood meal sources were recorded sporadically, with the highest occurrences at irrigated urban farming and peri-urban site categories. An. gambiae s.s. exhibited consistently high HBI across all site categories, particularly for indoor feeding: LS (indoor = 57%, outdoor = 57%), MS (indoor = 52%, outdoor = 100%), HS (indoor = 100%, outdoor = 0%), IUF (indoor = 54%, outdoor = 46%), and PU (indoor = 82%, outdoor = 79%).
Similarly, An. coluzzii demonstrated strong anthropophilic tendencies with high HBI across all site categories: low socioeconomic (indoor = 66%, outdoor = 65%), middle socioeconomic (indoor = 47%, outdoor = 0%), high socioeconomic (indoor = 100%, outdoor = 72%), irrigated urban farming (indoor = 52%, outdoor = 45%), and peri-urban (indoor = 88%, outdoor = 68%) (Table 4).
Sporozoite infection rate in host-seeking An. gambiae s.l.
A subsample of 2,472 [dry = 1,670; rainy = 802] An. gambiae s.l. mosquitoes collected through HLC were also tested for Plasmodium falciparum circumsporozoite protein (CSP). Overall, 2.1% (54/2,472) were positive for P. falciparum CSP. There was higher sporozoite rate in the rainy season compared to the dry season [dry = 1.25% (21/1,670); rainy = 4.11% (33/802)] (χ2 = 14.8, df = 13, P = 0.320). The highest sporozoite rate was recorded in Dansoman (MS) (5.8%, 7/121) and Medie (PU) (4.3%, 8/184), whereas the least was recorded in Tantra hill (HS) (0.5%, 1/216). Among the sites categories, sporozoite rates did not vary significantly (χ2 = 59.0, df = 52, P = 0.235) (Table 5).
Sporozoite infection rates in resting Anopheles gambiae s.l.
All the resting Anopheles gambiae s.l sampled (n = 589) were analyzed for the presence of Plasmodium falciparum circumsporozoite protein (CSP). Of the 4.24% (25/589) that tested positive, those collected from indoors had higher sporozoite infectivity (4.56%, 16/351) as compared to those collected from outdoors (3.78%, 9/238) (X2 = 6.7818, df = 1, P = 0.009). Tuba from the IUF site category recorded the highest frequency of CSP positive Anopheles gambiae s.l. (5.77%, 9/156), [indoor = 6.82% (6/88); outdoor = 5.88% (4/68)]. And the least was recorded in Dansoman (2.70%, 1/37) [indoor = 2.94% (1/34); outdoor = 0.00% (0/3)] (Table 6).
Entomological inoculation rate of host-seeking An. gambiae s.l.
The study identified significant variations in malaria transmission indices across different study sites. The overall average Entomological Inoculation Rate (EIR) was 1.526 infective bites per person per night (ib/p/n). Seasonal differences were observed, with EIR values of 0.191 ib/p/n during the dry season and 2.862 ib/p/n during the rainy season (χ2 = 18.80, df = 15, P = 0.331).
During the rainy season, Tuba, an irrigated urban farming (IUF) site, recorded the highest transmission risk, with an estimated 12.787 infective bites per person per night (ib/p/n), while Chorkor a lower socioeconomic (LS) site had no infective bite. In the dry season, Oyarifa, a peri-urban (PU) site, had the highest entomological inoculation rate (EIR) with 0.602 ib/p/n, whereas Nima (LS), Teshie and Dansoman (MS), and Tantra Hill (HS) had no infective bites.
Overall, the highest malaria transmission risk was recorded in Tuba (IUF), with the highest Human Biting Rate (HBR) of 176.458 and EIR of 2.647 ib/p/n, followed by the two Peri-urban (PU) sites, Medie (HBR: 30.792, EIR:1.324 ib/p/n,) and Oyarifa (HBR:47.083, EIR:1.224 ib/p/n). In contrast, Nima (HBR: 6.854, EIR: 0.110 ib/p/n) and Chorkor (HBR: 5.542, EIR: 0.188 ib/p/n) in the low socioeconomic (LS) category had the lowest malaria transmission risk (Table 5).
Insecticide resistance mutation genotypes in resting Anopheles gambiae s.l.
All Anopheles gambiae s.l. samples (n = 589) collected from indoor and outdoor environments were genotyped to detect insecticide resistance mutations: L1014F, L1014S, and G119S Ace-1.
The L1014F allele was present at a frequency of 100% across indoor and outdoor environments and all An. gambiae s.l. species, except in An. gambiae s.s., where it was observed at a slightly lower frequency of 90%. The L1014S mutation was detected at a very low frequency (10%) and was limited to An. gambiae s.s. and hybrids.
For the G119S Ace-1 mutation, the allele frequency was 70% in indoor samples and 60% in outdoor samples. Among the species, G119S was observed at a frequency of 70% in both An. coluzzii and An. gambiae s.s., and 50% in hybrids (Table 7).