Bioactivity and Changes in Gene Expression Profile Mediated by Tea (Camellia sinensis) extract on Anopheles gambiae sensu stricto Larvae

Abstract

Malaria, a deadly disease caused by Plasmodium falciparum, is vectored to humans by infected female Anopheline mosquitoes. Malaria intervention tools and specifically those regulating mosquito populations have reduced malaria incidences by 42% in Africa. However, residual transmission accounts for 214 million new cases, > 80% fatalities and 269 million people at risk of contracting the disease. The inexhaustive coverage of these vector controls, widespread insecticide resistance, and shifts in feeding habits of mosquito vectors prompts the need for novel interventions to reduce residual malaria transmission. The targeting of immature mosquito stages affects both vectorial capacity and vector competence. In this study, the effects of tea (Camellia sinensis) leaf extract and its bioactive constituents on An. gambiae s.s larvae physiology were investigated. Late third/early fourth instar larvae were challenged with increasing doses of C. sinensis extract and survival rates recorded every 24 h for 72 h continuously. Following bioassay-guided fractionation, the bioactive ingredients were analyzed using liquid chromatography electron spray ionization quadruple time of flight coupled with mass spectrometry (LC/ESI-Qtof/MS). Hormetic responses to the sublethal doses were transcriptionally assessed by quantifying gene expression levels of AgamJHAMT, AgamILP1, Hsp70 and CYP6M2 with RT-qPCR. Generally, the extract exhibited toxicity to mosquito larvae in a dose-dependent relationship. At 250 and 500 ppm, the extract elicited > 90% mortality after 24 h post exposure. Although only moderate larval mortality was observed with 100 ppm of the crude extract, this exposure induced growth disruption effects on mosquito larvae. No effects were observed at 25 ppm and 50 ppm. The bioactive polar fraction elicited 100% larval mortality at 25 ppm. The LC50 of the crude extract at 24 h was 117.15 ppm (95% CI 112.86-127.04) while active fraction was 5.52 ppm (95% CI 2.68-9.65). UPLC/ESI-Qtof/MS analysis revealed that proanthocyanidins were the toxicity-mediating compounds in the bioactive fraction. Gene expression analysis showed that, relative to controls, Hsp70 (AGAP004581) was significantly up-regulated by 159.7863 fold while the other three larval gene AGAP005256 (AgamJHAMT), AGAP010605 (AgamILP1) and AGAP008212 (CYP6M2) were down-regulated by 265.0278, 9.7136 and 621.6678 fold respectively (t-test, P < 0.001). The study showed that, tea leaf extract and its constituents have potential of controlling An. gambiae s.s larvae and inspires new larviciding chemistry for control of malaria mosquitoes.