objective Our previous transcriptome analysis of Anopheles dirus revealed upregulation of the An. dirus yellow-g gene upon ingestion of Plasmodium vivax-infected blood. This gene belongs to the yellow gene family, but its role regarding P. vivax infection is not known and remains to be validated. The aim of this study was to investigate the role of the An. dirus yellow-g gene in P. vivax infection.methods The qRT-PCR was used to detect the expression of the yellow-g gene in many organs of both male and female mosquitos. The yellow-g gene silencing was performed by dsRNA membrane feeding to An. dirus. These mosquitoes were later challenged by P. vivax-infected blood. The oocyst numbers were determined.results The yellow-g transcript was detected in several organs of both male and female An. dirus mosquitoes. Successful knockdown of yellow-g was achieved and resulted in reduced P. vivax infection in the mosquitoes. The decrease in yellow-g expression had no effect on the life span of the mosquitoes. conclusions These results support the yellow-g gene as having an important function in Plasmodium development in Anopheles mosquitoes. keywords dsRNA, malaria, oocyst development, yellow-g Sustainable Development Goals: Good Health and Wellbeing *Contributed equally.

Carboxypeptidase B (CPB) plays an important role in blood digestion in mosquitos, aiding the release of free amino acids. Anopheles CPB is a target to block malaria transmission because it facilitates Plasmodium invasion of the mosquito midgut. Our study aimed to discover inhibitors of Anopheles CPB to prevent Plasmodium development in the mosquito. The Anopheles gambiae cpb (Agcpb) gene without a signal sequence was cloned into the pET28b expression vector. The recombinant AgCPB protein was expressed in E. coli BL21(DE3) within inclusion bodies after induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 37°C for 4 h. The protein pellet was dissolved in 6 M urea, purified by affinity chromatography, and dialyzed in reaction buffer. The refolded recombinant AgCPB could digest the hippuryl-arginine substrate similarly to that of the commercial porcine pancreas CPB. The 20 top-scoring malaria box compounds from the virtual-screening results were then chosen for an in vitro inhibition assay against AgCPB. Four of the 20 malaria box compounds could inhibit AgCPB activity. The compound MMV007591 was the most potent inhibitor with an IC50 at 0.066 µM. The results indicate that these candidate compounds may be utilized in drug development against mosquito CPB activity to curb malaria transmission.