Susceptibility to ivermectin has been shown to vary among mosquito species as well as among mosquito strains of the same species 27,42,43. Depending on the time frame which survival is monitored, An. gambiae have been shown to have an LC50 of 19.8, 15.9 and 22.4 ng/ml when survival is monitored for 9, 7 and 5 days, respectively 27,44,45. In our case by monitoring survival for 10 days we achieved 46.49% mortality with a concentration of 8 ng/ml. Though the levels of ivermectin in the blood drop rapidly, a concentration above 8 ng/ml can be maintained for close to 36 hours following an ivermectin dose in humans 14,27. Our results are aligned with the those of Smit et al. in which even very low ivermectin concentrations can increase mosquito mortality if the follow up period encompasses the usual lifespan 46.
At the used doses, ivermectin-induced mortality in mosquitoes is delayed by 2–3 days. One potential explanation is the time taken for ivermectin to be absorbed from the midgut, as faster lethality onsets has been observed when ivermectin is directly injected into the midgut than when it is taken as part of a blood meal 42. The second plausible explanation for the delayed mortality is the involvement of ivermectin metabolites rather than the parent compound in causing mosquito mortality. Presently, there is accumulating evidence suggesting the involvement of ivermectin metabolites in causing mosquito mortality though the specific metabolites are yet to be identified 46,47. However, even before the onset of lethality that is measurable with a 10-day follow up, ivermectin can potentially affect mosquito mortality in the wild due to its effects on locomotion 48. Simultaneously, a reduction in locomotion abilities would affect the vectorial capacity regardless of mosquito mortality, which can in turn further reduce malaria transmission.
Ivermectin-induced mortality is greatly dependent on attaining high systemic levels of ivermectin in the mosquito. The exposure to ivermectin is determined by the mosquito’s detoxification capacity. Generally, in insects, detoxification processes involve metabolic enzymes such as cytochrome P450, esterase and Glutathione-S-transferases (GSTs) together with efflux pumps like the P-gp 49. In this study, we investigated whether and how cytochrome P450s and P-gp transporters affected ivermectin-induced mortality in mosquitoes. Our results demonstrate that the simultaneous inhibition of cytochrome P450s and P-gp transporters enhances ivermectin-dependent mortality in a dose dependent manner indicating synergism. However, this only happened selectively when ritonavir or voriconazole was used. Unexpectedly, the use of cobicistat which is also a dual CYP/P-gp inhibitor rendered some protection from ivermectin. Cobicistat is a structural analogue of ritonavir but unlike ritonavir which is known to inhibit and induce multiple CYPs, cobicistat more selectively inhibits CYP3A4 50. Though, ritonavir and cobicistat are considered clinically equivalent, the small difference in ritonavir’s ability to induce CYPs could result in differences in drug to drug interaction 51,52. The induction of CYPs could possibly lead to ivermectin metabolism making ivermectin metabolites available. In addition to ivermectin parent compound, ivermectin metabolites have also been suggested to contribute to mosquito mortality 46,47.
Despite dual CYP/P-gp inhibitors showing an effect on ivermectin-induced mortality, P-gp selective inhibitors did not have a measurable effect. Taken together our results suggest that detoxification mechanisms mediated by CYPs are more important in ivermectin detoxification. This is contrary to what has been reported in mosquito larval stages where Buss et al. demonstrated that inhibition of P-gp using verapamil leads to increased toxicity in Culex mosquitoes 23. Collectively both findings suggest heterogeneity in detoxification mechanisms in different stages of mosquito development. As a holometabolous insect, the changes between the immature stages (larvae and pupae) and the adult stage are characterized by differences in diet, habitat, morphology, physiology and behavior. These differences could potentially lead to differences in evolution of protective mechanisms 53. Larval stages are known to be more prone to developing insecticide resistance compared to adults 53. Whether the P-gp mediated detoxification reported in Culex larvae is additive or alternative to CYP mediated detoxification in larvae warrants to be investigated. Larval habitats are often exposed to ivermectin through contamination of aquatic habitats with excreta from treated livestock. The stability of ivermectin in water for long periods increases the exposure of the larvae to ivermectin and could potentially accelerate the development of resistance 54. It is important to understand the mechanisms behind larval resistance to ivermectin and whether they contribute to ivermectin resistance in adults.
Our results warrant the investigation of selective CYP inhibitors for the ability to synergize ivermectin-induced mortality including piperonyl butoxide (PBO). This will help answer the question whether CYP inhibition independent of P-gp inhibition could still have practical implications.
The involvement of CYPs in ivermectin metabolism could potentially lead to cross-resistance between ivermectin and current insecticides used in vector control. Previously Deus et al. has demonstrated that pyrethroid resistant Ae. aegypti have higher tolerance to ivermectin 43. This could potentially affect ivermectin susceptibility in mosquito populations already showing metabolic resistance to insecticides and highlights the need to investigate the impact of insecticide resistance on susceptibility to ivermectin. Notably, the current recommendation to tackle metabolic resistance to insecticides is the use of piperonyl butoxide (PBO) 55. PBO which is an inhibitor of CYP450 enzymes and currently in use by incorporation into pyrethroid-LLINs, could also enhance ivermectin-induced mortality. However, there is needed to first evaluate whether it synergizes ivermectin-induced mortality.
Nevertheless, ivermectin remains a good alternative to mosquito populations whose mode of resistance is via point mutations since this resistance mechanism is not analogous between the current insecticides and ivermectin 56. While resistance to insecticides is caused by mutations in the sodium channel, acetylcholinesterase or GABA receptor genes, ivermectin resistance in other arthropods is associated with mutations on the Glutamate-gated chloride channels (GluCls) 57,58.
In the case ivermectin is to be used in mosquito populations with metabolic resistance to the current insecticides, our study provides insights on the possibility of ivermectin cross resistance with other insecticides. Our results suggest that detoxification mechanisms mediated by cytochrome P450 enzymes are more important in ivermectin resistance compared to detoxification mediated by efflux pumps.