Indoor mosquito sampling of rural houses in Namanolo and Ntaja in Balaka and Machinga Districts, respectively, of southeastern Malawi revealed three major malaria vector species. This finding was consistent with past studies in Malawi and southern Africa [34–36]. The results here provide key malariologic transmission indices (HBI, EIR) that demonstrate the vulnerability of humans to bites of vector Anopheles mosquitoes, despite the presence and use of LLINs as the primary anti-malaria intervention. Although no species was numerically dominant, An. funestus (s.s.) and An. gambiae (s.s.) were relatively more common in Ntaja and An. arabiensis was more common in Namanolo. By contrast, indoor collections of mosquitoes at other locations close to both Namanolo and Ntaja, Lindblade et al., (2015) found An. funestus (s.s.) to be dominant, while An. arabiensis was next in abundance and An. gambiae (s.s.) was uncommon. These populations exhibited resistance to the synthetic pyrethroid deltamethrin, with 38% mortality in WHO bioassays for An. funestus (s.l.) and 53% mortality for An. gambiae s.l. (probably, An. arabiensis) (Lindblade et al. 2015). Despite these variations in mosquito species abundances between sites, An. funestus (s.s.) and An. gambiae (s.s.) are generally considered epidemiologically more important than An. arabiensis due to their well-documented anthropophilic and endophilic behaviors [38–41]. This study found high rates of feeding on human blood by all three species, regardless of these variable phenotypes.
These findings are more evident in the fact that human blood comprised most blood meals in unmixed conditions, regardless of species or study site, and that blood meals from other potential sources (goats, dogs) were secondary, with goat blood meal being more frequent than dog. This observation is not surprising because it was commonly observed that people kept goats indoors in special rooms at night, probably for protection against theft, while dogs were left outside as guard dogs. This may explain the higher number of goat blood meals compared to dog blood meals. Killeen et al., (2001), in modeling mosquito populations of Tanzania and Kenya, demonstrated that there is a relationship between host availability and the amount of time that vectors spend seeking blood meals; by inference, hosts that require less time to locate will be fed upon more frequently. Orsborne et al., (2020), studying mosquito populations in Ghana, reached a similar conclusion, emphasizing that local host availability even for known anthropophilic malaria vectors, is a powerful driver for host selection. In Malawi, there have been no previous studies that consistently quantified relative availability of potential blood meal hosts. The high prevalence of human host blood-feeding by Anopheles species observed here is consistent with 2002 findings from southern Malawi, in which blood meals were nearly entirely from humans and secondarily from bovines . In northern and southern Zambia, similar high human host selection (> 90%) and comparatively lower goat selection (< 5%) by An. gambiae and An. funestus (s.s.) were observed [42, 43]. In contrast, the dominant blood meal of malaria vectors around Lake Victoria in western Kenya was humans for An. gambiae (s.s.) and An. funestus (s.s.), but for An. arabiensis was predominantly bovine  or equally bovine and human . Through application of the “ratio of ratios” method of host selection, this study approached the problem of variation in host selection semi-quantitatively in order to assess host selection tendencies of these often behaviorally stereotyped species.
The high frequency of human blood meals detected in this study can be attributed to several factors, in particular bed net use practices. The higher HBI and EIR in Namanolo compared to Ntaja (96.4% vs. 88.9% and 0.11 vs. 0.06, respectively) could be due to widespread use of PBO-containing Olyset Net Plus in Ntaja, which have been shown to be more effective than convention LLINs against pyrethroid-resistant Anopheles populations [11, 14]. Lindsay et al., (2021) have suggested that the underlying mechanism of PBO-containing LLINs may simply be that they are more toxic, rather than overcoming insecticide resistance. Regardless, other randomized field trials in Tanzania and Uganda have shown significantly lower human infection prevalence where LLINs with PBO were distributed [9, 10]. Although the entomological mediators of these reductions were not investigated, they are likely due to reduced transmission intensity. In the present study, the lower HBI of An. arabiensis and An. gambiae (s.s.) in Ntaja than Namanolo could also be explained by the use of LLINs with PBO in Ntaja, which apparently increases their susceptibility to the insecticide as explained above [14, 47].
In the only other study analyzing blood meals of Anopheles vectors in Malawi, conducted in Chikwawa district (southern Malawi) during 2002 prior to any mass distribution of insecticide-treated nets, most blood meals were from humans, with relatively few coming from bovine or mixed human-bovine feeding . The 2002 HBI estimates for the three dominant malaria vector species were similar to what was found in the present 2019–2020 study, despite there now being a long history of malaria control and LLIN use. The species-specific HBI estimates for 2002 vs. 2019–2020 were: An. arabiensis, 85.0% vs. 92.3%; Anopheles gambiae (s.s.), 99.2% vs. 93.5%; and An. funestus (s.s.), 99.2% vs. 96.5%. However, the estimated EIR in the present study (33 infectious bites per person per year) was lower than that reported by Mzilahowa et al., (2012) (183 infectious bites per person per year). Both studies used PCR-based detection of sporozoite infection in the head-thorax of individual mosquitoes, although prevalence was lower in the 2002 investigation (4.9%) compared to the present study (16.0%). However, indoor mosquito density was lower at the present study sites, thereby reducing the EIR. Another more recent study in Chikwawa, done during the implementation of a community-based control program, showed that 4 of 91 Anopheles (4.4%) tested by PCR were positive for P. falciparum infection during the rainy season, with an estimated EIR of 13.5 infectious bites per person per year , suggesting a reduced EIR in that region.
Molecular-based approaches to blood meal analysis to detect vertebrate host feeding have advanced since the review of this topic by [18, 25, 44, 49]). At the forefront of this advance has been development of qPCR methods using host-specific probes by either TaqMan or SYBR green detection [18, 50]. However, host species-specific probes in multiplex qPCR targeting Anopheles blood-meal hosts were developed only recently . The use of species-specific probes, designed within a qPCR format here, favored the detection of single and multiple (i.e., mixed) blood meals in this study. By screening all blood meals for human blood, and then analyzing by PCR, amplicon sequencing, and BLAST search matching those blood meals not reacting to the human probe, it was possible to reveal the narrow breadth of dominant hosts being utilized by the Anopheles community, and then using qPCR to reveal the extent of multiple feeding. This combinatorial approach indicated that ~ 12% of the blood meals were mixed feeding of human and goat (10.8%), human and dog (1%) or goat and dog (0.2%).
The host-selection analysis showed that, in both sites, two of the Anopheles species fed more often on goats than humans in proportion to availability of these hosts. An. arabiensis and An. gambiae (s.s.) over-selected goats and under-selected humans, while An. funestus (s.s.) selected the two hosts about equally (i.e., randomly) in proportion to their availability. These results are not surprising. Although An. arabiensis is reportedly more zoophilic, while An. funestus (s.s.) is more anthropophilic . Plasticity and or opportunistic tendencies have been observed in various Anopheles species [7, 38–41]. The explanation to the relatively higher goat feeding in indoor mosquito samples is consistent with goats being kept indoors at night, and malaria control interventions in the area (LLINs) (Malawi Malaria Operational Plan FY, 2018). These two activities provide easy accessibility to goat blood meal and makes it more difficult to access human blood meal due increased mosquito-goat contact and reduced mosquito-human contact . The switch in host utilization is indicated by the reduced access to human blood meal and increase in the utilization of the non-human hosts. The comparable increase in mixed blood meal may also suggest disruption in feeding either due to LLINs’ activity or otherwise, pushing the mosquito to get a full blood meal from other hosts. Either way this is one of the few studies in Malawi to report blood meal analysis including detection of mixed blood meals. This study will prompt more research in blood meal studies in Malawi to document the range of blood meal hosts, especially those involving goat blood meal which is rarely reported in literature.
The findings of this investigation suggest important implications for Plasmodium transmission and malaria control. Multiple host feeding by some Anopheles females might allow for increased survival and reproduction [49, 52]. The presence of multiple malaria vector species that successfully obtain human blood meals could lead to an increased Plasmodium transmission by increasing the basic reproductive number (Ro) [42, 53]. More widespread use of LLINs, particularly with PBO, could help reduce transmission, but this intervention alone is unlikely to reduce malaria incidence in this meso-endemic setting to acceptable levels where elimination can be contemplated. Residual Plasmodium transmission and weakened intervention efforts  are likely to persist into the future.