This study reveals that the two main malaria vectors in Zambia An. funestus s.l. and An. gambiae s.l. were present in all four sites, and they were molecularly identified as An. funestus s.s. and An. gambiae s.s., respectively. In Zambia, these mosquitoes have been implicated as the main vectors responsible for malaria transmission and have been found to exist in sympatry [20, 21, 29, 30]. Surprisingly, Anopheles funestus was found to be the most abundant malaria vector in Ndola. Historically, the province has been dominated by An. gambiae s.s but we are now seeing a shift to An. funestus s.s as the dominant malaria vector in Ndola other districts within the Copperbelt province [9, 15]. However, An. gambiae s.s. remained the more dominant malaria vector in peri-urban areas, whereas An. funestus s.s. was more abundant in rural areas, consistent with earlier studies conducted in sub-Saharan Africa [18, 21]. The disparity in vector abundance could be attributed to variations in ecological habitats. Anopheles gambiae prefers to breed in man-made water habitats such as drainages, tire tracks, small pools and agriculture sites, whilst An. funestus s.s. prefers to breed in permanent and semi-permanent water habitats with some vegetative cover [31, 32]. An earlier study identified An. funestus s.s as the primary driver of malaria transmission in the dry season whereas An. gambiae s.s as the primary driver in the wet season [30]. Nonetheless, the existence of breeding grounds for Anopheles gambiae s.s in peri-urban areas implies that even during the dry season, An. gambiae s.s will continue to be the primary driver of malaria transmission. The coexistence of these two malaria vectors pose an increased year round risk of malaria transmission in the area. The recent increase in the incidences of malaria reported in Ndola could be attributed to the changing vector bionomics that now includes An. funestus s.s. not reported previously in the area.
Mosquito diversity was observed to be higher in rural than peri-urban sites with the inclusion of An. gibbinsi, a potential secondary malaria vector. This vector has been reported in other parts of the country as a potential secondary malaria vector [33–35]. Secondary malaria vectors have not been adequately considered in most vector control programming yet they contribute to 5% of malaria transmission in the southern African region [36]. Their contribution to transmission is significant making the need to incorporate interventions targeting secondary malaria vectors into vector control toolkits inevitable.
The host-seeking behaviours of An. funestus s.s. and An. gambiae s.s. were different. The host-seeking behaviour of An. funestus s.s. was found to be homogeneous across the four sites, whereas the host-seeking behaviour of An. gambiae s.s. was found to be much higher in peri-urban sites with vast larval habitats. This heightened host-seeking behaviour of An. gambiae s.s. indicates an increased risk of disease transmission in peri-urban sites compared to rural sites [37]. As such the need for enhanced vector control methods in peri-urban settings with extensive larval habitats due to the elevated risk cannot be overemphasized.
The mean densities of An. funestus s.s found resting indoors were generally low across the rural and peri-urban sites. However, the indoor resting density of An. gambiae s.s in the peri-urban site was much higher than that in the rural site. Variations in the indoor resting behaviour of An. funestus s.s. and An. gambiae s.s. could be influenced by the presence of vast An. gambiae s.s breeding sites in peri-urban sites. Therefore, vector control interventions such as IRS and LLINs in such settings may not be sufficient but could be supplemented with larva source management [38].
The four predictors associated with reduced counts of malaria vectors in housing structures were; the number of people who slept in a housing structure, housing structures with a thatched roof, the number of LLINs used the previous night and sprayed housing structures. However, only sprayed housing structures were found to be statistically associated with reduced counts of malaria vectors, similar to what was found in Sao Tome and Principe [38]. Individuals who sleep in sprayed houses experience a lower vector-to-host contact, which entails reduced exposure to infectious mosquito bites unlike those sleeping in unsprayed houses. Additionally, maximum benefit is derived when at least 85% of houses are sprayed with an efficacious insecticide to kill host seeking mosquitoes that rest indoors [39]. On the other hand, number of animals in a housing structure, housing structures with mud wall surfaces and open eaves were associated with increased counts of malaria vectors but were not statistically significant. Elsewhere, a study conducted in Cameroon associated open eaves and holes in the walls to increased mosquito counts [40]. Another study in Gambia also found that closing the eaves reduces mosquitoes entering thatched houses but increases mosquito entry into metal-roofed houses [41].
The larval habitats that were active breeding sites were all from the two peri-urban sites adjacent to a dambo. The larval habitats identified included irrigation canals (or irrigation channels), garden ponds, tire marks, foundation trenches and blocked drainages. However, irrigation channels and garden ponds were found to be the main mosquito breeding sites, similar to studies conducted in Ghana, Tanzania, Cote d’Ivoire and China [42]. However, the larval densities found in this study were higher than that found in China, possibly due to differences in the climatic conditions, variations in the bacterial diversity and physicochemical composition of the larval habitats [43]. These factors have been found to influence mosquito oviposition, survival, and development into competent malaria vectors, thereby potentially impacting malaria incidence [3, 44]. Unfortunately, this study only identified the different types of larval habitats; future research is needed to fully characterize larval habitats in order to generate additional information valuable for an effective and targeted larval source management programme.
Susceptibility tests in this study reveal that An. gambiae s.s. was fully susceptible to organophosphates (malathion and pirimiphos-methyl) and neonicotinoids (clothianidin). This was also observed in several other districts in Zambia, where An. funestus s.s. and An. gambiae s.s. was found to be susceptible to these two classes of insecticides [20, 22]. In that regard, organophosphates and neonicotinoids could be effective at controlling mosquito populations of An. gambiae s.s in Ndola and several other districts in Zambia, with evidence of susceptibility. However, resistance of An. gambiae s.s. to pyrethroids (permethrin and deltamethrin) and carbamates (bendiocarb) was confirmed and this could be attributed to the extensive use of pesticides and insecticides for agriculture and public health purposes. These results align with previous studies that found extensive insecticide resistance to pyrethroids and carbamates in the Copperbelt province [9, 18]. The NMEC needs to develop a strategy that will delay the development of resistance against these insecticides in areas it has not developed yet. Previously, pyrethroids were the only insecticides used to impregnate mosquito bed nets until the introduction of LLINs with an insecticide synergist piperonyl butoxide (PBO). Piperonyl butoxide LLINs (PBO LLINs) have been found to enhance the impact of pyrethroids by inhibiting the enzymes responsible for detoxification in pyrethroid-resistant mosquitoes [45, 46]. However, recent studies found that the use of PBO LLINs in a structure sprayed with pirimiphos-methyl reduces the efficacy of the insecticide on pyrethroid-resistant mosquitoes [46]. As a result, use of a PBO LLIN in a pirimiphos-methyl sprayed structure may not be as beneficial as using a non-PBO LLIN in a pirimiphos-methyl sprayed structure.
Limitations of the study
Susceptibility testing for An. funestus s.s. was not performed due to limited numbers of adult An. funestus s.s collected to conduct forced oviposition and consequently establish F1 mosquitoes for use in the bioassay. However, the susceptibility of An. gambiae s.s was determined against seven different insecticides from five different classes. Another limitation of this study is that, since it was conducted in the dry season, the entomological indices determined may only be applicable to the dry season. However, the fact that the dry season that is usually not conducive for vector breeding and malaria transmission could be facilitating it, is enough reason to pay attention and plan additional measures.