Purification and structural elucidation of mosquito larvicidal compound(s) from cultures of an Ascomycete species Jo5035 against Aedes aegypti

Abstract

Mosquito transmitted diseases such as yellow fever, elephantiasis, dengue and malaria are some of the most deadly vector-borne diseases, affecting millions of people mainly in the tropics. In spite of major efforts undertaken for control of the diseases, through drug treatment and vector control, an increase in mosquito borne disease incidences has been witnessed in the last decades. Africa suffers most from mosquito borne diseases since mosquito vectors are distributed almost throughout the whole continent, and the parasite reservoir is big and continuously increasing. The search for new strategies or natural products to control destructive insects and vectors of diseases is desirable. Thus in this study secondary metabolites from higher fungi were investigated for possible mosquito larvicidal activity to control mosquitoes. An ascomycete serialized JO5035 was collected from undisturbed habitat in Mount Kenya forest. The ascomycete JO5035 was sub-cultured onto potato dextrose agar (PDA) to form a well grown culture. The strain was preserved as agar slant and the corresponding herbarium material kept in a fungal culture collection in the Integrated Biotechnology Research Laboratory at Egerton University. On initial screening it was found to produce active compounds in culture against Aedes aegypti larvae. It was cultured in sterile submerged nutrient liquid malt media. From the culture two sets of crude extracts were prepared with intracellular secondary metabolites prepared from mycelium (Mex) while extracellular secondary metabolites prepared from the cultured filtrate (Kex). The crude extracts were tested for larvicidal activities against late third instar larvae and early fourth instar larvae of Ae. aegypti before activity guided purification of the active compounds was carried out. The crude extracts were then fractionated, guided by mosquito larvicidal activity and which were subjected to NMR experiments (both 1D and 2D). The chemical structures of the compounds were determined using the NMR spectral data. A mosquito larvicidal compound, 2-para-tolylethanol was purified, with LC50 of 279.6 ppm and LC90 of 624.8 ppm. The compound was produced from cultures of an ascomycete JO5035 and reported to have mosquito larvicidal activity for the first time. During this study it was demonstrated that the ascomycete JO5035 had a potential as a source of mosquito larvicidal compounds despite heavy limitations of necessary research equipment. It is the recommendation of this work that these compounds can be investigated further for product development.