Biodiversity of Mosquitoes (Diptera) in Punjab, Pakistan and Screening of Bioactive Phytochemicals as a Larvicidal Potential against Culex quinquefasciatus

Abstract

Mosquitoes population are widely diverse in the whole Pakistan. Among all the Provinces of Pakistan, Punjab is the most populated area and constitutes highly favorable environment regarding temperature, rainfall and humidity for up breeding of insects like mosquitoes. The mosquitoes play a key role in causing number of diseases such as malaria, dengue fever, filariasis, yellow fever etc. being vector. Therefore, the current study mainly focused on biodiversity and control measures of mosquitoes using plants. For biodiversity assessment, mosquitoes were collected from eleven districts of Punjab from water containers, flowerpots, standing water, tires, sweep nets, aspirators etc. from April-November (2016-2019), and preserved in 70% ethanol for morphological study and in 95% ethanol for molecular analysis. A stereoscopic trinocular microscope was used to examine and measure all main body parts and identified with the help of key “The fauna of British India” (1934). The results revealed the presence of 27 mosquitoes spp. belonging to two subfamilies (Anophelinae and Culicinae) and five genera (Anopheles, Aedes, Culex, Armigeres, and Mansonia). The molecular identification was based upon the amplification of gene Cytochrome oxidase I (COI) which confirmed the presence of three species belonging to two genera Anopheles (An. gambiae) and Aedes (Ae. aegypti and Ae. albopictus). For control measures, the ethanol and water dried leaves extract were evaluated from six plants (Ocimum basilicum, Polygonum glabrum, Mentha piperita, Mallotus philippensis, Citrus aruntium, Fumaria officinalis) for their larvicidal potential against Culex quinquefasciatus larvae. The cytotoxicity assay was applied on brine shrimps using five concentrations multiple of 80. The bioassay was applied in triplicates in 1000ml plastic containers with extracts, added 30 larvae of each four instars separately and fed with dog biscuits. A control excluding extract was setup parallel. Observations were made after every 12hrs till 72. By using probit analysis, the LC50 and LC90 values were determined. The DPPH radical scavenging activity, ferric reducing power, and total antioxidant capacity was estimated taken ascorbic acid as standard. The ethanolic extract of M. piperita, found to be the most effective (LC50=208.98ppm) ones, it was characterized by preliminary qualitative and quantitative phytochemicals, UV-VIS spectroscopy, FT-IR, GC-MS, and molecular docking analysis. Biochemical analysis of dead mosquito larvae treated with ethanolic M. piperita extract showed the presence of significantly less value of proteins, carbohydrates, cholesterol, triglycerides and high-density lipid as compared with control. xx Maximum total antioxidant capacity (125.46 ± 3.55, 115.63±1.76 (µg/mg), ferric reducing power (378.17± 1.04, 365.12±0.31 (µg/mg), and DPPH radical scavenging activity was demonstrated by M. piperita and O. basilicum leaf extracts (85.43±0.22, 68.64±0.41 (μg/ml). Except for F. officinalis (LC50=569.31ppm), all of the experimental plants demonstrated cytotoxicity in a protective array >550ppm. The qualitative phytochemical study showed that M. piperita had the highest concentration of organic components (such as phenols, alkaloids, flavonoids, carbohydrates, and flavonoids etc.) compared to the other five plants used in the experiment. The quantitative phytochemical study showed that M. piperita had the highest levels of total phenolic (6.44±0.0018 µgGAE/mgDW) and flavonoid contents (8.47±0.0017 µgQA/mg DW). UV-VIS spectroscopy revealed two absorption peaks with absorption values of (2.34, 0.79) at 209.51 and 282.81nm, respectively. FT-IR results revealed the presences of alcohols, alkanes, aldehydes, aromatic rings, ether linkage, ester, and halo-compounds. The GC-MS analysis identified the compounds t-Butyl hydrogen phthalate (13.9%), 2H-Pyran-2,4(3H)-dione, 3-ethyl-5,5-dimethyl 6-phenyl (13.15%), and 1,2-Benzenedicarboxylic acid, mono-(2-ethylhexyl) ester (12.45%) with high % peak area 1,2-Benzenedicarboxylic acid and 3-ethyl-5,5-dimethyl-6-phenyl were the two compounds that best bound to the NS3 protease domain with PDB ID: 2FOM out of all the others. To isolate bioactive secondary metabolites in the current study, the crude leaves extract of M. piperita was fractionated by column chromatography and further characterized by thin-layer chromatography using various solvents with multiple ratios, including n-hexane, ethyl acetate, chloroform, and methanol. The column chromatography showed that fractions F3, F4, F12, and F13 had the high potential of larvicides. The cytotoxic, antilarval and antioxidant properties (total antioxidant capacity, ferric reducing power, and DPPH) of 15 fractions were tested against Cx. quinquefasciatus. In the dose-response bioassay, various concentrations (80, 160, 240, 320, and 400ppm) were used. The highest antilarval activity was shown by fractions 3, 4, 12, and 13 against all four instars larvae, with LC50 values of (127.9, 121.7, 105.1, 112.5), (131.5, 128.1, 118.6, 95.1), (136.6, 112.5, 102.4, 52.6), and (104.1, 110.9, 130.7, 46.9) respectively. Maximum ferric reducing power (250±0.85, 255±0.65, 265±0.69, 275±0.94), total antioxidant capacity (70±0.54, 72±0.34, 77±0.21, 85±0.25), and DPPH radical scavenging (90±0.13, 96±0.11, 97±0.87, 98±0.34) activity were demonstrated by F3, F4, F12, and F13. The fraction (F13) had much higher antilarval activity, with an IC50 of 7.65±0.19ug/ml as compared with control 7.23±0.55ug/ml. xxi The active fractions were further put for chromatographic techniques preliminary phytochemical tests, UV-VIS, FT-IR, GC-MS analyses, and molecular docking. The phytochemical analysis executed the presences of 20 organic compounds. The UV-VIS profile exhibited peaks ranging from 200 to 800nm with a range of absorptions. The FT-IR spectrum of four active fractions demonstrated the presence of alcohol, primary amine, aliphatic primary amine, Alkyl aryl ether, secondary amine, alkane, carboxylic acid, alkene, anhydride, nitro-compound etc. The GC-MS analysis results showed the presence of various phytochemical, including ethanol, ammonium oxalate, carbazic acid, propanamide, isobornylthiocyanoacetate, 1, 2-Benzenedicarboxylic acid, N [5-(5-phenyl-3,4-dihydro-2H-pyrrol-4-yl) nonan-5-yl] benzamide, eicosanoic acid. Among all identified compounds from GC-MS, in silico molecular docking experiments, the 1, 2 benzenedicarboxylic acid of F3, eicosanoic acid of F4, phytol of F12, and N-[5-phenyl-3, 4 dihydro-2H-pyrrol-4-yl] nonan-5-yl] benzamide of F13 had the best binding affinity for the target NS3 protease. The results showed that all fractions F3, F4, F12, and F13 were effective against Cx. quinquefasciatus. It is suggested that the M. piperita could be a good source of larvicides.