Emerging mosquito resistance to piperonyl butoxide-synergized pyrethroid insecticide and its mechanism

Piperonyl butoxide (PBO)-synergized pyrethroid products are widely available for the control of pyrethroid-resistant mosquitoes. To date, no study has formally examined mosquito resistance to PBO-synergized insecticides. We used Culex quinquefasciatus as a model mosquito examined the insecticide resistance mechanisms of mosquitoes to PBO-synergized pyrethroid using modified World Health Organization tube bioassays and biochemical analysis of metabolic enzyme expressions prior- and post-PBO exposure. We measured mosquito mortalities and metabolic enzyme expressions in mosquitoes with/without pre-exposure to different PBO concentrations and exposure durations. We found that field Culex quinquefasciatus mosquitoes were resistant to all insecticides tested, including PBO-synergized pyrethroids (mortality ranged from 3.7{+/-}4.7% to 66.7{+/-}7.7%), except malathion. Field mosquitoes had elevated levels of carboxylesterase (COE, 3.8-fold) and monooxygenase (P450, 2.1-fold) but not glutathione S-transferase (GST) compared to susceptible mosquitoes. When the field mosquitoes were pre-exposed to 4% PBO, the 50% lethal concentration of deltamethrin was reduced from 0.22% to 0.10%, compare to 0.02% for susceptible mosquitoes. Knockdown resistance gene mutation (L1014F) rate was 62% in field mosquitoes. PBO pre-exposure suppressed P450 enzyme expression levels by 25[~]34%, GST by 11%, and had no impact on COE enzyme expression. Even with the optimal PBO concentration and exposure duration, field mosquitoes had significantly higher P450 enzyme expression levels after PBO exposure compared to laboratory controls. These results demonstrate that PBO alone may not be enough to control highly pyrethroid resistant mosquitoes due to the multiple resistance mechanisms. Mosquito resistance to PBO-synergized insecticide should be closely monitored. Authors SummaryMosquitoes are vectors of many major infectious diseases globally. Insecticides and related products are widely used for mosquito controls and disease preventions. Over time and following repeated use, mosquitoes (including Aedes, Anopheles and Culex) have developed very high resistance to multiple insecticides all over the world. Target site insensitivity due to mutations in the voltage-gated sodium channel gene and overproduction of metabolic detoxification enzymes such as cytochrome P450 (CYP) monooxygenases play critical role in insecticide resistance in mosquitoes. To enhance the killing power of insecticides, synergized insecticides were developed by mixing insecticide synergists with pyrethroids. Discovered in the 1940s, piperonyl butoxide (PBO) is one of the earliest and most commonly used insecticide synergists. Field application of PBO-synergized insecticides performed far better than mono-pyrethroids. PBO-treated long-lasting insecticidal nets (PBO-LLINs), which also use pyrethroids, outperformed regular LLIN for malaria control in many African countries. PBO-LLIN is soon to be rolled out on a large scale for malaria control in Africa. One important question regarding the use of synergized insecticides is whether they will select for vector population resistance to synergized insecticide products, in other words, are PBO-synergized pyrethroids effective against highly insecticide-resistant mosquitoes? To date, no study has formally examined mosquito resistance to PBO-synergized insecticides. Here, we used Culex quinquefasciatus as a model mosquito, we examined its resistance status to different insecticides including PBO-synergized pyrethrins and tested how PBO exposure affect mosquito mortality and the expressions of metabolic enzymes. We found that field Culex quinquefasciatus mosquitoes were resistant to multiple insecticides tested, including PBO-synergized pyrethroids. Field mosquitoes had elevated levels of carboxylesterase (COE) and monooxygenase (P450) but not glutathione S-transferase (GST) enzyme expressions compared to susceptible mosquitoes. Even with optimal PBO concentration and exposure duration, field mosquitoes had significantly higher P450 enzyme expression levels after PBO exposure compared to laboratory controls, and PBO exposure had no impact on COE enzyme expressions. The phenomena of the insecticide-resistant mosquitoes insensitivity to PBO exposure or PBO-synergized insecticides and multiple-resistance mechanisms have also been reported from Aedes and Anopheles mosquitoes in different countries. These results demonstrate that PBO alone is not enough to control highly pyrethroid resistant mosquitoes due to multiple resistance mechanisms. Mosquito resistance to PBO-synergized insecticide should be closely monitored