1. DNA extraction
Genomic DNA was extracted from a single mosquito leg, which was identified morphologically using a simplified non-destructive method. Each leg was placed into a 1.5 mL microcentrifuge tube containing 50 µL of Buffer C (Tris 0.5 M, EDTA 0.5 M, Tween 20, Proteinase K 20 mg/mL, DEPC ddH2O), ensuring the tissue was fully submerged. The samples were incubated at 57¡C for 1 hour to facilitate lysis. Following incubation, the tubes were centrifuged at 14,000 rpm for 10 minutes. The resulting DNA extract was diluted 1:10 in DNase/RNase-free water and thoroughly mixed to ensure homogeneity before proceeding with downstream molecular analysis.

2. Molecular identification for multiplex PCR 1 (Barbirostris group)
The PCR master mix was prepared using the following components: 8.5 µL of PerfeCTa¨ Multiplex qPCR ToughMix (Quantabio), 0.5 µL of each forward primer (fBDS VW, fBar, fCamp), and 0.5 µL of each corresponding reverse primer (rBar and Van, rVan and Dis, rCamp) (Table S1). To complete the volume, 3.5 µL of DNase/RNase-free water was added, resulting in a total master mix volume of 15 µL. Subsequently, 2 µL of genomic DNA template was added to each reaction tube. The mixture was briefly centrifuged to ensure proper mixing and eliminate bubbles before amplification.
         PCR amplification was carried out under the following thermal cycling conditions: an initial denaturation step at 95¡C for 3 minutes, followed by 35 cycles of denaturation at 94¡C for 30 seconds, annealing at 50¡C for 30 seconds, and extension at 72¡C for 1 minute. A final extension step was performed at 72¡C for 6 minutes. Following PCR amplification, 1 µL of Purple (6X) loading dye (Catalog No. B7024S, New England Biolabs, USA) was added to each PCR product prior to gel electrophoresis. The amplified products 5 ?L were subjected to 2% agarose gel with 1x TBE for electrophoresis at 100 volts for 40 minutes. The resulting DNA bands were visualized under UV illumination using the Gel Docª XR+ Imaging System (Bio-Rad, USA), and images will be captured for documentation and analysis.
      
3. Molecular identification for multiplex PCR 2 (Barbirostris group)
Samples that did not yield visible bands in the first round of PCR (PCR 1) were subjected to a second round of amplification (PCR 2). The master mix was prepared using 8.5 µL PerfeCTa¨ Multiplex qPCR ToughMix (Quantabio) as the PCR reagent. The reaction mixture included 0.5 µL of the forward primer (F BDSVW) and 0.5 µL each of the reverse primers (rSaeu1, rSaeu2, and rWej) (Table S1). DNase/RNase-free water was added to bring the total volume of the master mix to 15 µL. Subsequently, 2 µL of genomic DNA template was added to each reaction tube. The mixture will be gently spun down to ensure complete mixing and removal of air bubbles.
         PCR amplification was performed using the same thermal cycling conditions as PCR 1: an initial denaturation at 95¡C for 3 minutes; followed by 35 cycles of denaturation at 94¡C for 30 seconds, annealing at 50¡C for 30 seconds, and extension at 72¡C for 1 minute; with a final extension at 72¡C for 6 minutes. The PCR products were analysed by agarose gel electrophoresis, as described in the previous section.
         
4. Molecular identification for self-developed multiplex PCR (Hyrcanus group and Minimus group)
A self-developed multiplex PCR assay was established to discriminate An. peditaeniatus, An. sinensis, and An. minimus using the universal ITS2A primer as the forward primer in combination with species-specific reverse primers (peditaeniatusR, sinensisR, and Minimus MIA)(Table S1). PCR amplification was performed in a total reaction volume of 25 µL, comprising 12.5 µL of PerfeCTa¨ Multiplex qPCR ToughMix (Quantabio), 1.5 µL of ITS2A primer, 0.75 µL each of the species-specific reverse primers, 6.25 µL of DNase/RNase-free water, and 2 µL of genomic DNA template. Thermal cycling conditions consisted of an initial denaturation at 95 ¡C for 2 minutes, followed by 35 cycles of denaturation at 95 ¡C for 30 seconds, annealing at 58 ¡C for 30 seconds, and extension at 72 ¡C for 30 seconds, with a final extension step at 72 ¡C for 5 minutes. PCR products were analyzed by 2% agarose gel electrophoresis prepared in 1? TBE buffer and stained with GelRed¨ (Biotium, USA). Electrophoresis was performed at 100 V for 35 minutes, and DNA fragments were visualized using a Gel Docª XR+ Imaging System (Bio-Rad, USA).
5. ITS2 PCR amplification
For the amplification of the ITS2 region, the PCR master mix was prepared using the following components: 12.5 µL of PerfeCTa¨ Multiplex qPCR ToughMix (Quantabio), 1 µL each of the ITS2A and ITS2B primers (10 µM) (Table S1), and 8.5 µL of DNase/RNase-free water, resulting in a total master mix volume of 23 µL. To each reaction tube, 2 µL of genomic DNA template was added. The mixture will be briefly centrifuged to ensure thorough mixing and eliminate air bubbles.
         PCR amplification was carried out with the following thermal cycling conditions: an initial denaturation at 95¡C for 3 minutes, followed by 35 cycles of denaturation at 94¡C for 30 seconds, annealing at 55¡C for 30 seconds, and extension at 72¡C for 1 minute. A final extension was performed at 72¡C for 6 minutes.
         The amplified products were analysed using agarose gel electrophoresis. A 1.5% agarose gel was prepared using 1? TBE buffer and stained with GelRed¨ nucleic acid stain (Biotium, USA). Following PCR amplification, 5 µL of Purple (6?) loading dye (Catalog No. B7024S, New England Biolabs, USA) was added to each PCR product prior to gel electrophoresis. 5 µL of each sample was loaded into the gel wells, and electrophoresis was conducted at 100 volts for 35 minutes. Visualization of DNA bands was performed using the Gel Docª XR+ Imaging System (Bio-Rad, USA), as described in the earlier section. The remaining 20 µL of each PCR product was preserved at -20¡C for subsequent Sanger sequencing.

6. cox1 PCR amplification
PCR amplification of cox1 gene was conducted using a 23 µL reaction volume consisting of 12.5 µL of PerfeCTa¨ Multiplex qPCR ToughMix (Quantabio), 1 µL each of the forward primer AnplCOXIF and the reverse primer AnplCOXIR (Table S1), and 8.5 µL of DNase/RNase-free water. To this mixture, 2 µL of genomic DNA template was added.
         The thermal cycling conditions included an initial denaturation at 95¡C for 3 minutes, followed by 35 cycles of denaturation at 94¡C for 30 seconds, annealing at 50¡C for 30 seconds, and extension at 72¡C for 1 minute. A final extension step was carried out at 72¡C for 6 minutes.
         The PCR products were analysed by agarose gel electrophoresis, following the procedure previously described.
      
7. cox2 PCR amplification
PCR amplification of the cox2 gene was carried out in a 23 µL reaction volume, consisting of 12.5 µL of PerfeCTa¨ Multiplex qPCR ToughMix (Quantabio), 1 µL of the forward primer AnplCOXIIF, 1 µL of the reverse primer AnplCOXIIR (Table S1), and 8.5 µL of DNase/RNase-free water. To this mixture, 2 µL of genomic DNA template was added.
         Thermal cycling conditions included an initial denaturation at 95¡C for 3 minutes, followed by 35 cycles of denaturation at 94¡C for 30 seconds, annealing at 50¡C for 30 seconds, and extension at 72¡C for 1 minute. A final extension was performed at 72¡C for 6 minutes.
         PCR products were analysed using agarose gel electrophoresis, following the procedure previously described.