1.5 ml tube

Approximately 20 mg of muscle tissue was transferred to a 1.5 mL tube (Axygen, Silicon Valley, CA, USA) with 300 μL of lysis reagent (Promega, Madison, WI, USA) and then homogenized three times at 65 Hz for 30 s each. Then, 300 μL of RNA dilution liquid was added to the homogenate and incubated for 5 min until centrifugation at room temperature at 13,000 rpm, and total RNA was extracted, in accordance with the protocol of the manufacturer (Promega). The RNA concentration was detected by a NanoDrop spectrophotometer (Thermo, Waltham, MA, USA), and the quality was assessed by agarose gel electrophoresis. Total RNA was qualified and used for the next manipulation (Figure S1). cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan) in a 10 μL volume, in accordance with the instructions of the manufacturer.

Female adult Anophelessinensis mosquitoes were collected in 2020 (June 22 to 26) from 12 rural regions in ROK with the assistance of the regional center for vector surveillance against climate change supported by the Korea Centers for Disease Control and Prevention⁵⁴ . The collection sites were categorized as hyperendemic area if the number of malaria cases per 100,000 persons exceeds 1 patient. Otherwise, it was categorized as hypoendemic area (Fig. 1). The study regions were determined using statistics of malaria patient. The analysis could have improperly accounted for habitat or ecology since each region was not ecologically analyzed. Mosquitoes were sorted into genus using the morphological keys^{55 (link)}, and species identifications were confirmed using a diagnostic PCR assay based on DNA barcode analysis^{56 (link)}. Mosquito body parts such as legs and wings were transferred to a 1.5ml tube (Axygen, USA), after which DNA extraction was carried from mosquito parts using G-spin™ Total DNA Extraction kit (iNtRON’s, Korea) according to the manufacturer’s instructions. PCR cycle parameters involved an initial denaturation at 95 °C for 3 min, 35 cycles of 30 s at 95 °C, 30 s at 63 °C, and 2 min at 72 °C. A final extension at 72 °C for 10 min was completed. PCR products were subjected to electrophoresis on a 1.5% agarose gel and visualized under ultraviolet light.