2.1. Mosquito Sampling, Processing, Random PCR, and Sequencing

Mosquitoes were captured during two consecutive days in five plots of a Rapid Assessment Program and Long Term Ecological Research (RAPELD) grid in Pirizal, High Pantanal (16°14′06”S, 56°22′70”W). We used three Nasci aspirators (13:00–18:00 h) for 30 min and five CDC light traps (18:00–06:00 h) at 1.5 m high along a transect with 50 m intervals during tree climatic periods (Figure 1). The insect capture in preservation areas has been previously approved by the Brazilian Environmental and Natural Resource Institute (SISBIO/ICMBIO) under the number 43909-1.

Rapid Assessment Program and Long Term Ecological Research system and the respective location of the five sampled grids in High Pantanal, Mato Grosso State, Central-Western Brazil.

Briefly, captured specimens were kept alive under controlled temperature (24 °C), humidity, and artificial feeding with a 20% sucrose solution. Female mosquitoes were identified alive after immobilization (4 °C by 4 min) using dichotomy keys [22]; their dissected salivary glands [23] were pooled together (n = 3 to 117) according to date, place of collection, species, and gender; then homogenized in 0.4 mL of RNAse free phosphate saline buffer (pH 7.2) and centrifuged (5000× g for 4 min at 4 °C). RNA was extracted from the supernatant (0.2 mL) with a High Pure Viral RNA Kit (Roche) without RNA carrier, quantified (quantifluor RNA system; Quantus fluorometer, Promega, Madison, WI, USA), reverse transcribed (GoScript, Promega, Madison, WI, USA), and amplified in quintuplicates with a viral random PCR after double-strand cDNA synthesis (Klenow DNA polymerase I, New Englands BioLabs, Ipswich, MA, EUA) as previously described [19,21,24]. PCR products were purified with 20% polyethylene glycol, quantified with a quantifluor one dsDNA system (Quantus Fluorometer, Promega, Madison, WI, USA) and sequenced after genomic library preparation with the Truseq DNA PCR-free library kit (Illumina, San Diego, CA, USA) using 2 × 100 paired-end reads in two lanes with 60 GB on a Hiseq 2500 platform (Illumina, San Diego, CA, USA).