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 amplication 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 puried and then sequenced by the dideoxy chain termination method. Results: CecA and defensin amplication were standardized with annealing temperatures of 59ºC and 55ºC, respectively. The amplied products and sequencing demonstrated the good quality of both primer sets. Conclusions: For the rst 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.

with different annealing temperatures tested. The PCR products were puri ed and then sequenced by the dideoxy chain termination method.
Results: CecA and defensin ampli cation were standardized with annealing temperatures of 59ºC and 55ºC, respectively. The ampli ed products and sequencing demonstrated the good quality of both primer sets.
Conclusions: For the rst 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.

Background
The Brazilian Amazon is the site of almost all human malaria cases in Brazil, with approximately 98% of cases distributed in several municipalities that are endemic for the disease. This high percentage of malaria cases is related to the environmental changes that have occurred in the Amazon with agriculture, livestock, extraction activities and urban development works that resulted in a signi cant impact on its ecosystem and favour the development of vector mosquitoes 1,2,3 . The vector mosquitoes belong to the family Culicidae, subfamily Anophelinae, order Diptera, genus Anopheles. In Brazil, the most important subgenus is Nyssorhynchus, and its main representative is the species Anopheles (Nys.) darlingi Root, An. darlingi is considered the main vector of malaria given its behavioural characteristics, such as anthropophilia and endophagy, and because it is susceptible to human malaria parasites that circulate in Latin America. In addition, even when not abundant, this mosquito can maintain Plasmodium transmission locally 3,4,5 . Infection rates in this vector can be high, as has already been reported in the Brazilian Amazon, where an infection rate of more than 18% was observed in the state of Pará 6 , a value comparable to that of other anopheline mosquitoes of epidemiological importance such as Anopheles gambiae (11.1%) and Anopheles funestus (13.1%) 7.8 .
The vectorial competence of these mosquito species is in uenced by the vector's immune system. However, the molecular mechanism of immune system responses is still unclear 9 . In this context, studies have been performed to identify possible molecular markers for human malaria control. Included among the targets of interest are the main antimicrobial peptides (AMPs) of the innate immunity of mosquitoes, such as cecropins and defensins 10,11 .
AMPs are proteins that act in the protection against invading organisms such as bacteria, viruses, fungi and protozoa, forming the host's rst line of defence 12,13,14 . In mosquitoes, the involvement of these peptides has been demonstrated in studies involving Cecropin A (CecA) in An. gambiae against P. berghei 11,15 and defensin in Ae. aegypti against P. gallinaceum 16 .
Despite the importance of AMPs in reducing parasite development inside the mosquito, there are no exploratory studies on the mechanism of action of these peptides in species of anopheline mosquitoes in the Brazilian Amazon, such as An. darlingi. Thus, this work aims to standardize a molecular polymerase chain reaction (PCR) technique to detect the AMPs CecA and defensin from An. darlingi mosquitoes to support studies involving their detection and ampli cation and understanding of their role in the Anopheles-Plasmodium interaction.

Figure 1 Anopheline mosquito collection areas in three municipalities in the Brazilian Amazon.
The municipality of Peixe-Boi (PA) has an abundance of anopheline mosquitoes but no transmission of human malaria. Until August 2020, the number of cases of human malaria in the municipality of Altamira (PA) was 553 (4.1% of the total of Pará State), and the number of cases in Cruzeiro do Sul (AC) was 4482 (61.1% of the total of Acre State) 17 . The average annual rainfall in Peixe-Boi (PA), Altamira (PA) and Cruzeiro do Sul is 2,471 mm, 1,844 mm and 2,139 mm, respectively. The rainy season occurs mainly between November and April, as in other municipalities in the Brazilian Amazon, and the average annual temperature is 26ºC 18,19 .
Mosquito collection was conducted between July and December 2018 for four hours (6-10 pm) in the municipality of Peixe-Boi (PA) in the extra-home environment (carried out stably without negatively affecting the welfare of the animals) and 12 hours (6 pm-6 am) using the protected human attraction method in Altamira (PA) and Cruzeiro do Sul (AC), both in peri/intradomicile environments. For identi cation, the females were anaesthetized in a freezer (for approximately 5 minutes), at which point they were morphologically identi ed using entomological keys 20, 21 . Primer design Alignments were made to compare the sequences of CecA and defensina in all anopheline species available to verify regions that present greater similarity between species for primer design. The alignment of the sequences was performed in the Clustal Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/) 22 .
After comparing the sequences, primers were constructed based on the GenBank genetic sequence databases of An. darlingi for CecA (ADMH02001013.1) and defensin (ADMH02000296.1) ( Table 1). followed denaturation steps at 95ºC for 30 sec, and annealing temperatures that were tested between 54ºC and 61ºC for 40 sec and extension at 72ºC for 40 sec, repeated 35 times.
The PCR products were run on a 2% agarose gel (Ultra-pure agarose, BRL 155517-014) at 100 volts for 1 hour and 30 minutes, stained with GelRed and methylene blue to be viewed under ultraviolet light (Fluo-Link, FlowGen) and photographed in a Kodak® Edas 290 photo documentation system.
PCR products were puri ed by the PureLink® PCR Puri cation kit (Invitrogen, Carlsbad USA), sequenced by the dideoxy chain termination method using the ABI PRISM Dye Terminator kit (Applied Biosystems) and analysed using a 3130 Genetic Analyser (Thermo Fisher, Santa Clara, CA) according to the manufacturer's instructions. Afterwards, the samples were assembled in the BioEdit Sequence Alignment Editor program (version 7.0.5, Borland Software Corporation), and the sequence quality was veri ed by Chromas Lite software (version 2.6.5, Technelysium Pty. Ltd., Tewantin, Queensland, Australia).

Results
The PCR standardization of the two AMPs of interest ( Fig. 2-3) was described following the conditions in a thermocycler with annealing for 40 sec at 59ºC for CecA and 55ºC for defensin.
The proposed methodology allowed the detection of An. darlingi peptides from three different municipalities in the Brazilian Amazon. In addition, the samples showed good quality in relation to sequencing (Fig. 4).

Discussion
This is the rst 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 infection 13,14 .
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 Plasmodium 11,15,16 . Other studies have shown that cecropin and its derivatives have an action against pathogens such as Trypanosoma 24 , Leishmania 25 , Candida albicans 26 and Brugia pahangi 27 . In anopheline mosquitoes, these AMPs, especially CecA, have a strong in uence on the primary immune response in both pupae and adults, acting against infection of the malaria parasite 11 . 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 in uence 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 signi cant immune response in this vector 28 . Additionally, in An. gambiae, the antiparasitic activities of defensin showed an important role in the immune defence against sporozoites of P. gallinaceum 16 .
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 cycle 29 .
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 elimination 11,30,31 .
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 pathogen 11,30,31,34 .

Conclusion
For the rst time, the standardization of a molecular technique for detecting AMPs was described for 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 an infection and providing important molecular markers for the control of human malaria.

AVAILABILITY OF DATA AND MATERIALS
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Not applicable.

CONSENT FOR PUBLICATION
Not applicable.

COMPETING INTERESTS
The authors declare no con ict of interest.