Larvicidal efficacy of Croton bonplandianus baill. Extract and its synergistic effects with different conventional insecticides against Aedes aegypti (Linnaeus, 1762) , a dengue fever vector

Abstract

Mosquitoes, found ubiquitously across various regions of the world, play a pivotal role in transmitting many devastating diseases. Their significance as vectors has stimulated the alarming rise in mosquito-borne diseases as they are responsible for carrying and transmitting pathogens to humans and animals. The most significant health risks associated with mosquito vectors include dengue, malaria, chikungunya, yellow fever, zika virus infection, filariasis, and West Nile virus infection. These diseases collectively contribute to extensive global disease and mortality rates, with millions of cases reported annually. Accurate identification of mosquito vectors in specific regions is crucial for strategizing effective disease management and resource allocation as it enables early detection and targeted response measures, reducing disease impact. For decades, Kerala has faced persistent intimidation of mosquito-borne diseases. Therefore, it becomes imperative to precisely identify, map, and document the vector mosquito population and the factors that drive their proliferation. The first objective of this study was to identify the important vector mosquito species within selected areas that represented urban and semi-urban regions of the Thrissur district in Kerala, India, using molecular identification techniques. According to the molecular data, a phylogenetic tree was constructed, comparing the genetic relations between the collected vector species with the other NCBI-deposited species from various regions around the globe. This objective presented detailed information on local vector populations, facilitating more focused and effective vector control strategies. This research combined traditional and molecular techniques to recognize mosquito species and utilised GIS technology for species mapping according to their habitat geographic region. The study also evaluated different diversity indices, including alpha, beta and gamma diversity indices of the collected vector populations. These diversity indices provided an overall awareness of the diversity of selected vector mosquito species within the study area. The molecular identification of mosquito vectors confirmed the existence of 11 vector species of primary and crucial mosquito-borne diseases. The collected vector mosquito species were identified and documented under four different genera, Anopheles, Aedes, Armigeres and Culex, within the study area. The selection of Ae. aegypti, as the experimental species for larvicidal activity studies was influenced by understanding the burden of disease associated with this vector mosquito and its role in disease transmission. The next objective of the study involved screening various locally available plant extracts against Ae. aegypti vector using organic solvents with increasing polarity. It comprised the identification and isolation of bioactive compound within effective phyto-extract, and the evaluation of the susceptibility of fourth-instar larvae of Ae. aegypti to the plant extract. This assessment utilised the standard larval bioassay procedure outlined by the World Health Organization (WHO). The following part of the study involved the larval susceptibility assay, where different conventional insecticides and the plant isolate were tested against Ae. aegypti larvae. The bioactive compound, identified as eicosane and termed CB1, and four conventional insecticides, lambda-cyhalothrin, cypermethrin, temephos, and malathion, were chosen for examination. The WHO protocol was followed for assessing larval susceptibility, and plant isolate bioassay, with modifications as needed to meet the specific requirements of the study. The results revealed the susceptibility status of Ae. aegypti towards all the tested compounds. Antimicrobial activities were also considered to determine the efficiency of CB1 to limit the growth of microorganisms. Of the four different bacterial strains tested, the growth of all except one was limited by the plant isolate. This result indicated an add- on advantage of CB1 as it could inhibit certain microbial growth when released to the environment as a larvicide. The fundamental objective of this study was to evaluate the synergistic impact, as synergy can enhance the efficacy of insecticides when combined with natural compounds like the plant isolate, thus improving the prospects of successful vector control. This cooperative approach also holds the potential to reduce the dependence on chemical insecticides alone, thereby promoting more sustainable vector management practices. Two distinct experimental approaches were established to examine the interplay between the insecticide and plant extract, yielding the Co- toxicity coefficient, CTC and the Synergistic factor, SF. These experiments aimed to gain a deeper understanding of the combined effects of each chemical insecticide with the plant's bioactive compound on Ae. aegypti larvae. The CTC analysis assessed the combined mixture- influenced mortality rates in comparison to expected outcomes, while the evaluation of the SF aimed to explain the degree of synergism or antagonism observed between the insecticide and the plant isolate. These investigations provided a better perceptive of the interactions among these substances and their aptitude for successful vector control tactics. The results demonstrated that all combinations exhibited a synergistic effect on the test species, with the eicosane- lambda cyhalothrin combination, SC4, displaying the most pronounced impact. SC4 was chosen for further analysis to investigate the possibility of resistance development in the laboratory reared Ae. aegypti. This involved quantitative assay of the detoxifying enzymes of the selected generations of Ae. aegypti, which had been raised through exposure to this compound for five consecutive generations and comparing their detoxifying enzyme activity with that of the susceptible strain. Bioassay experiments adhered to the standard WHO method and the Resistance Ratio (RR) assessment was also conducted following the WHO protocol. The derived lethal concentration (LC50) values indicated that although there was a minor rise in LC50 values with the progression of generations, it did not reach a level indicative of resistance development against SC4, and the Ae. aegypti strain remained susceptible even after five generations. Quantitative enzyme assays were also performed to analyse the mode of action of crucial detoxifying enzymes in a laboratory-reared susceptible strain of Ae. aegypti when exposed to the synergistic compound, SC4 and the plant isolate CB1 over different time intervals of 24, 48, and 72 hours. Specific enzyme activities of Acetylcholinesterase, Carboxylesterase, Glutathione S-transferase and Cytochrome P450 were evaluated along with the total protein concentration. The results consistently demonstrated a reduction in the activity of the tested detoxifying enzymes throughout all treated generations, implying a potential barrier to the rapid development of resistance to these compounds.