Standardization of a Polymerase Chain Reaction Protocol for Detecting Antimicrobial Peptides in Anopheles Darlingi (Diptera: Culicidae) Mosquitoes from the Brazilian Amazon

Background: Antimicrobial peptides (AMPs) are proteins of the innate immune system that can limit infections of the malaria-carrying parasite Plasmodium, which develops inside anopheline mosquitoes, the human malaria vectors. Despite this, studies on Brazilian Amazon species of anopheline mosquitoes are still needed. The aim of the study is to develop a standard molecular polymerase chain reaction (PCR) technique to detect the AMPs cecropin A (CecA) and defensin from Anopheles darlingi to support studies involving their detection and amplification and better understanding of the roles of these peptides in the Anopheles-Plasmodium interaction. Methods: The collection of anopheline mosquitoes was carried out in three municipalities in the Brazilian Amazon: Altamira and Peixe-Boi, in the state of Pará, and Cruzeiro do Sul, in the state of Acre. The primers were built based on the sequences available in GenBank, and PCR followed standard protocols with different annealing temperatures tested. The PCR products were purified and then sequenced by the dideoxy chain termination method. Results: CecA and defensin amplification were standardized with annealing temperatures of 59ºC and 55ºC, respectively. The amplified products and sequencing demonstrated the good quality of both primer sets. Conclusions: For the first time, a standardized molecular technique for detecting AMPs was described in An. darlingi, a mosquito species from the Brazilian Amazon, supporting future studies aiming to understand the interactions of this species and the action of these peptides during infection and providing important molecular markers for the control of human malaria.