The WHO cone bioassay serves as a cornerstone for the evaluation of insecticide-based vector control tools. Despite its standardized nature, there is a critical need to comprehensively investigate how alteration to different parameters can impact the outcome of the tests. This study was conducted to investigate how alterations to parameters such as temperature, feeding status and mosquito density would affect the outcome of the bioassay.
Although the standard cone bioassay is conventionally conducted at a controlled temperature of 27 ± 2°C, our study deliberately explored temperature ranges both below and above this standard. Our findings revealed a notable influence of temperature on Anopheles funestus mortality rates for both pyrethroid and pyrethroid-PBO ITNs. High temperatures were associated with increased mosquito mortality, while lower temperatures led to decreased mortality. A similar trend was observed in tube assays where the effects of exposure to permethrin-impregnated nets were found to be greater at higher temperatures among susceptible and pyrethroid resistant malaria vectors [17]. This observation suggests the presence of a positive temperature coefficient which means an insecticide becomes more toxic as temperature increases [30].
Furthermore, the intensity of the impact of temperature on mosquito mortality appeared to vary across the different ITN classes examined. Notably, Veeralin consistently exhibited more pronounced and consequently, statistically significant effects at higher temperatures compared to MAGNet. This difference may be attributed to the inclusion of PBO in Veeralin, a synergist known to inhibit metabolic enzymes responsible for detoxifying insecticides, which in turn, restores the insecticidal potency of ITNs against metabolically pyrethroid-resistant malaria vectors [31]. In a related study by Glunt et al., [14] the interaction between PBO and temperature was investigated using the standard WHO tube test, however, their findings could not indicate that temperature influenced the restoration of pyrethroid susceptibility in resistant malaria vectors exposed to PBO because all mosquitoes exposed to PBO and subsequently to deltamethrin died [14]. The increased toxicity of PBO at higher temperatures in our study may potentially be attributed to an increased reaction rate of irreversible inhibition under these conditions [14]. An alternative hypothesis is that insecticides are more bioavailable at higher temperatures which may be the case for PBO that is a liquid (Skovmand, pers comm).
Data from this experiment adds to the body of evidence that temperature must be carefully considered when testing insecticides against biological systems. A bimodal temperature-activity distribution has been reported in several insecticides and mosquito species [32, 33, 34, 35] and 27 ± 2ºC gives a conservative measurement of mortality. Temperature affects the way in which pyrethroids work in insects. Toxicity of pyrethroids [30] and other insecticide classes [36] are positively correlated with temperature. There is some evidence that humidity can also affect mosquito mortality observed after insecticide exposure [37] and it is known to affect mosquito survival [38] and should therefore be carefully maintained during mosquito holding post-exposure.
Feeding status of mosquitoes is yet another dynamic factor that can affect their interaction with insecticides. In the standard bioassay, mosquitoes are sugar-fed before exposure to insecticides. However, this study evaluated bioassays conducted with starved and blood-fed mosquitoes on their interaction with ITNs. The study revealed that mosquito’s feeding status impacts the outcome of cone bioassay and this varied for the pyrethroid only and the pyrethroid-PBO ITNs. It was observed that with pyrethroid ITN, sugar-fed mosquitoes had similar mortality with blood-fed and lower mortality than starved mosquitoes, although the confidence intervals of the odds ratios were wide in all estimates, presumably because of low observed mortality in the pyrethroid ITN arm. Norris et al., [20] investigated the relationship between mosquito’s feeding status and susceptibility status in tube tests, similar to the current study they found that percentage mortality after permethrin exposure was significantly higher in starved mosquitoes than sugar-fed mosquitoes.
Interestingly, blood-fed mosquitoes had lower mortality rates than sugar-fed mosquitoes for pyrethroid-PBO net. It has been reported that blood meals stimulate insecticide detoxification mechanisms in pyrethroid-resistant An. funestus leading to increased insecticide tolerance in bottle bioassays [39]. Furthermore, a single blood meal significantly reduced insecticide-induced mortality to pyrethroid and DDT relative to sugar-fed resistant An. arabiensis in WHO susceptibility test [40]. Similarly, recent studies have observed that blood-feeding increases tolerance among blood-fed susceptible An. gambiae [41] and enhances the levels of resistance among resistant blood-fed An. funestus [15] to deltamethrin. The lower mortality rates observed with blood-fed mosquitoes in the present study in the pyrethroid-PBO ITN may suggest that blood meal ingestion triggers oxidative stress and boosts mosquitoes metabolic activity [42, 43], potentially resulting in increased detoxification enzyme expression [39] and affecting the toxic dose mosquitoes receive from insecticide exposure [44]. Nevertheless, additional research is needed to investigate the relationship between pyrethroid-PBO ITNs and blood meals.
It was also found that higher mosquito density was associated with increased mortality. This may be likely due to increased competition for available space within the cone, as mosquitoes crowd together, they interact more and are more likely to encounter and pick up insecticides from the treated surfaces. The effect of cage size and density has also been shown to influence the duration of insect repellent efficacy [45] presumably due to mosquito probability of encountering a host. Increased activity in cone tests is associated with increased mortality in video cone tests even in the control [46], and may be influenced by mosquito interactions with others in the confined cone.
It should be noted that our study focused exclusively on assessing the impact of temperature, mosquito feeding status and mosquito density within the scope of cone bioassays, therefore, the generalizability of these findings to broader contexts, such as susceptibility tests, may be limited. Additionally, the bioassays were conducted using only one mosquito strain, resistant An. funestus mosquitoes, and only alpha-cypermethrin ITNs. To enhance the comprehensiveness of our understanding, it is necessary to extend these investigations to include a broader spectrum of both susceptible and resistant malaria vectors, as well as a greater diversity of active ingredients. Similar studies are essential for gaining a more comprehensive perspective on the interplay between parameters that could influence insecticide susceptibility assessments and other types of bioassays routinely deployed for vector control product assessment such as tunnel tests.