Bacterially expressed recombinant TMOF induces mortality and gut microbial alterations in Aedes albopictus larvae

Mosquitoes, as vectors of numerous diseases, pose significant threat to human health. Aedes mosquitoes transmit diseases such as dengue, yellow fever and chikungunya, with dengue fever alone responsible for approximately 40,000 deaths and more than 96 million symptomatic cases annually. Current mosquito control methods are inadequate and results in environmental health hazards and development of resistance. Therefore, targeted control strategies are essential. In this context, we cloned and expressed the Trypsin Modulating Oostatic Factor (TMOF), a decapeptide that inhibits trypsin biosynthesis in mosquitoes by binding to a receptor. The codon-optimized gene for the TMOF peptide was synthesised and cloned in to pFN29AHis6Halo vector and expressed in Escherichia coli. The supernatant from the bacterial lysate containing recombinant TMOF peptide exhibited larvicidal activity against Aedes albopictus mosquito larvae, with an LC50 (48 h) of 242.1 ± 6.04 µg/mL. However, lysate from BL21 cells alone or recombinant peptide expressed with a single base shift in reading frame did not cause any mortality. The recombinant TMOF peptide was purified using nickel affinity chromatography and showed an LC50 of 2.13 ± 0.02 µg/mL, exhibiting 113.6 times more efficacy than the bacterial lysate supernatant. The LC90 (48 h) for bacterial lysate and affinity purified TMOF was 340.41 ± 6.04 µg/mL and 4.39 ± 0.20 µg/mL, respectively. TMOF peptide released from the recombinant protein by trypsin digestion also showed larvicidal activity. Exposure of larvae to TMOF fusion protein resulted in inhibition of trypsin biosynthesis in-vivo. Metagenomic analysis of the gut microbiota from TMOF-treated larvae resulted in reduction in abundance of bacteria belonging to Pseudomonadota and Bacillota compared to that of untreated larvae. Recombinant TMOF is also effective against Culex mosquito larvae, but shows no effects on non-target organisms such as Drosophila melanogaster, Luprops tristis, and Aplocheilus lineatus. Thus, the use of TMOF expressed in E. coli offers a promising eco-friendly method of mosquito control. (Patent number: 554267).