This is the first study involving the standardization of a molecular technique for detecting peptides from the innate immune system of the main vector of human malaria in Brazil, An. darlingi. The role of these AMPs in the defence against pathogens makes these peptides possible targets for molecular studies for the control of human malaria, whether in studies that aim to explore their mechanisms of action, genetic structure, or pattern of gene expression under certain environmental conditions or during infection13,14.
We chose CecA and defensin AMPs because they are the main molecules of the mosquito immune system and have important activities against different pathogens, including Plasmodium11,15,16. Other studies have shown that cecropin and its derivatives have an action against pathogens such as Trypanosoma24, Leishmania25, Candida albicans26 and Brugia pahangi27. In anopheline mosquitoes, these AMPs, especially CecA, have a strong influence on the primary immune response in both pupae and adults, acting against infection of the malaria parasite11. In addition, the levels of gene expression of members of the CecA and CecB cecropin families have the potential to reduce the number of P. berghei oocysts in the midgut of An. gambiae by up to 60%15.
The defensin peptide also demonstrated an influence on An. gambiae, as evidenced by markers that showed high expression in the anterior midgut when females were fed blood infected with P. berghei, inducing a significant immune response in this vector28. Additionally, in An. gambiae, the antiparasitic activities of defensin showed an important role in the immune defence against sporozoites of P. gallinaceum16.
The search for new tools that can help in malaria control or eradication is still a challenge for those who aim to protect populations at risk of acquiring malaria. The lack of a Plasmodium vaccine, the parasite's resistance to drugs and the appearance of insecticide-resistant mosquitoes show the priority of searching for new methods that can provide understanding and elucidation of the parasite-host relationship, i.e., the parasite's biological cycle29.
In the case of Anopheles mosquitoes, the immune system tries to eliminate the parasites, mainly in the midgut and/or on the way to the salivary glands as a way to defend against invasion/infection by pathogens. Thus, effector molecules that are expressed in these locations, acting in defence of the host, can be important targets in controlling the parasite. In this context, the AMPs CecA and defensin can be used due to their fast action, solubility, and resistance to proteolytic digestion, in addition to being expressed in Plasmodium development sites, aiming for parasite elimination11,30,31.
Previously, most studies on AMPs have been in An. gambiae, the main vector of human malaria on the African continent, which highlights the importance of studies on the species An. darlingi, the main vector of human malaria in the Brazilian Amazon region. The epidemiological importance of An. darlingi is related to its behavioural characteristics, susceptibility to human malaria parasites and adaptation. It was found that this species has genetic and even phenotypic variations within the same population and between different populations, mainly due to climatic (seasonal variation) and environmental factors (degradation of the environment and geographical barriers such as biomes) 32,33. These issues are of concern for public health because the vector can maintain transmission in different regions in Brazil.
In this sense, the standardization of molecular techniques may assist studies exploring the functions of AMPs and their relationships with Plasmodium infection in the species An. darlingi, since AMPs are harmful molecules for a variety of microorganisms, causing lysis and cell death by integrating with the cell membrane of the pathogen11,30,31,34.