Mosquitoes

The five anopheline species used in this study were collected in the Brazilian Amazon region (Fig. ​(Fig.1).1). The samples of A. darlingi were collected in two distinct localities: 1) in the District of Coração (N 0° 01′; W 51° 10′), located at km 13 of the Duca Serra Road, in the outskirts of the Macapá city, state of Amapá; 2) in the Ramal do Brasileirinho (S 3° 20′ 08.7″; W 59° 52′ 16.8″) located on the outskirts of city of Manaus, state of Amazonas. These localities are separated ~ 1100 km apart and in both localities there is malaria transmission. Samples of A. braziliensis were collected from the outskirts of Macapá city (N 0° 1′ 13.71″; W 51° 09′ 47.47″), whereas A. marajoara and A. triannulatus were collected in the Santa Bárbara Farm (N 0° 17′ 28.4″; W 50° 54′ 07.2″), municipality of Macapá, state of Amapá, Brazil. Specimens of A. nuneztovari were captured in the Ramal do Sampaio (S 03° 41′ 51.8″; W 59° 07′ 37.5″), municipality of Autazes, state of Amazonas, Brazil. The salivary glands of A. darlingi, A. braziliensis, A. marajoara, and A. triannulatus used in this study were collected from wild mosquitoes (females) captured from the field, whereas those from A. nuneztovari were female descendants from the F₁ generation reared in the insectary from wild mosquitoes (females) collected from field. The collections were authorized by the System of Authorization and Information in Biodiversity (SISBIO), with permanent license number 38440–1 awarded to VMS.

Adult mosquitoes were captured using a light trap, white Shannon-type, between 18:00 and 22:00 h, and then transferred into cups, properly labeled with locality and collection date. At the end of the captures, the cups containing the mosquitoes were covered with moistened towel paper and transported alive inside tightly closed isothermal boxes to the Laboratório de Genética de Populações e Evolução de Mosquitos Vetores at the Instituto Nacional de Pesquisas da Amazônia (INPA), in Manaus, Brazil. The following day, the specimens were identified using morphological keys of Forattini [15] and Faran and Linthicum [97]. Immediately after, the mosquitoes were cooled in a freezer at − 20 °C for a few minutes, transferred to an ice-chilled plate, when their salivary glands were dissected on a slide containing a drop of sterile Phosphate Buffered Saline (PBS) pH 7.4, under a stereomicroscope, SV11 model, Carl Zeiss. The salivary glands of each species were immediately transferred to an Eppendorf tube containing 200 μL of RNAlater (Thermo Fisher Scientific) solution. Depending on the species analyzed, pools ranging from 80 to 97 pairs of salivary glands were dissected, kept at 4^o C for 48 h and then stored at − 80 °C until the RNA extraction. For more details, see Additional file 1: Table S1.