Seaweed Sargassum wightii mediated preparation of TiO2 nanoparticles, larvicidal activity against malaria and filariasis vectors, and its effect on non-target organisms.

Malaria and Lymphatic filariasis are considered significant public health concerns in several countries. As a researcher, controlling those mosquitos using safe and eco-friendly insecticides is essential. Thus, we aimed to explore the potential use of seaweed Sargassum wightii for the biosynthesis of TiO2 NPs and evaluate its efficiency in controlling disease-transmitting mosquito larvae (using Anopheles subpictus and Culex quinquefasciatus larvae as model systems (in vivo)) as well as its potential effect on non-target organisms (using Poecilia reticulata fish as an experimental model). XRD, FT-IR, SEM-EDAX, and TEM carried out the characterization of TiO2 NPs. It evaluated the larvicidal activity against the fourth instar larvae of A. subpictus and C. quinquefasciatus. The larvicidal mortality was observed after 24 h of exposure to S. wightii extract and TiO2 NPs. S. wightii synthesized TiO2 NPs show excellent activity against A. subpictus and C. quinquefasciatus (LC50 = 4.37 and 4.68; LC90 = 8.33 and 8.97; χ2 = 5.741 and 4.531) mg/L respectively. The GC-MS results indicate the presence of some important long-chain phytoconstituents like linoleic acid, palmitic acid, oleic acid methyl ester, and stearic acid, among others. Furthermore, when testing the possible toxicity of biosynthesized NPs in a non-target organism, no adverse effects were observed in Poecilia reticulata fish exposed for 24 h, considering the evaluated biomarkers. Thus, overall, our study results reveal that biosynthesized TiO2 NPs are an effective and exciting eco-friendly approach to controlling the A. subpictus and C. quinquefasciatus.

Characterizations of Larval Gut Bacteria of Anopheles subpictus Grassi (1899) and their Role in Mosquito Development in Hooghly, West Bengal, India.

Malaria is a serious vector borne disease transmitted by different species of Anopheles mosquitoes. The present study was aimed to isolate and characterize the bacterial flora from the gut of larvae of An. subpictus Grassi (1899) prevalent in Hooghly and explore their roles in host survival and development. Mosquito larvae and adults were collected from field and were maintained in laboratory. Bacterial load in the larval mid-gut was determined, and predominant strains were isolated and characterized by polyphasic approach. Role of these bacteria in larval survival and development were assayed. Bacterial load in the gut of larvae was found to vary in field-collected and lab-reared mosquitoes in different seasons. Morphological, bio-chemical, and molecular analyses explored four common bacterial isolates, namely Bacillus subtilis, Bacillus pumilus, Bacillus cereus, and Proteus vulgaris in the larval gut throughout the year. Larval survival rate was greatly reduced (0.06) and time of pupation was prolonged (17.8 ± 0.57) [days] in the absence of their gut bacteria. Total tissue protein (7.78 ± 0.56) [µg/mg], lipid (2.25 ± 0.19) [µg/mg] & carbohydrate (16.5 ± 0.79) [µg/mg] contents of larvae, and body weight & wing length of adult male (0.17 ± 0.02 & 1.74 ± 0.43) [mm] & female (0.19 ± 0.02 & 1.99 ± 0.46) [mm] mosquitoes were also found to be greatly reduced in the absence of gut bacteria. Developmental characteristics were restored with the introduction of culture suspension of all four resident gut bacterial isolates. Present study indicates that the mosquitoes largely depend on their gut bacteria for their survival and development. So, manipulation or control of this gut bacterial communities might inhibit survival and development of vector mosquitoes.

Compensatory Base Changes Reveal Sexual Incompatibility among Members of the Anopheles subpictus Sensu Lato (Diptera: Culicidae) Species Complex in Sri Lanka.

The mosquito Anopheles (Cellia) subpictus sensu lato (s.l.) is a major secondary vector of malaria in Sri Lanka. The sibling species composition in this species complex in Sri Lanka remains debatable. Compensatory base changes (CBCs) in the secondary structures of internal transcribed spacer 2 (ITS2) are reliable sources to predict sexual incompatibility among closely related species. The objective of the present study was to investigate the An. subpictus s.l. populations in Sri Lanka using the CBC analysis. Mosquito DNA was amplified and sequenced for the ITS2 region. The sequences were annotated using ITS2 Database. ITS2 secondary structures were constructed and analyzed for CBCs using various bioinformatics tools. The ITS2 regions consisted of two different lengths, 575 bp and 480 bp. The two CBCs and three hemi CBCs identified in the present study suggest that there may be at least two sexually incompatible sibling species. In conclusion, it is likely that there may be only two reproductively isolated sibling species in the An. subpictus species complex in Sri Lanka. However, due to high divergence of ITS2 in these species, it is reasonable to assume that they may be undergoing a speciation event to separate as a distinct species.

Molecular forms of Anopheles subpictus and Anopheles sundaicus in the Indian subcontinent.

BACKGROUND: Anopheles subpictus and Anopheles sundaicus are closely related species, each comprising several sibling species. Ambiguities exist in the classification of these two nominal species and the specific status of members of these species complexes. Identifying fixed molecular forms and mapping their spatial distribution will help in resolving the taxonomic ambiguities and understanding their relative epidemiological significance. METHODS: DNA sequencing of Internal Transcribed Spacer-2 (ITS2), 28S-rDNA (D1-to-D3 domains) and cytochrome oxidase-II (COII) of morphologically identified specimens of two nominal species, An. subpictus sensu lato (s.l.) and An. sundaicus s.l., collected from the Indian subcontinent, was performed and subjected to genetic distance and molecular phylogenetic analyses. RESULTS: Molecular characterization of mosquitoes for rDNA revealed the presence of two molecular forms of An. sundaicus s.l. and three molecular forms of An. subpictus s.l. (provisionally designated as Form A, B and C) in the Indian subcontinent. Phylogenetic analyses revealed two distinct clades: (i) subpictus clade, with a single molecular form of An. subpictus (Form A) prevalent in mainland India and Sri Lanka, and (ii) sundaicus clade, comprising of members of Sundaicus Complex, two molecular forms of An. subpictus s.l. (Form B and C), prevalent in coastal areas or islands in Indian subcontinent, and molecular forms of An. subpictus s.l. reported from Thailand and Indonesia. Based on the number of float-ridges on eggs, all An. subpictus molecular Form B were classified as Species B whereas majority (80%) of the molecular Form A were classified as sibling species C. Fixed intragenomic sequence variation in ITS2 with the presence of two haplotypes was found in molecular Form A throughout its distribution. CONCLUSION: A total of three molecular forms of An. subpictus s.l. and two molecular forms of An. sundaicus s.l. were recorded in the Indian subcontinent. Phylogenetically, two forms of An. subpictus s.l. (Form B and C) prevalent in coastal areas or islands in the Indian subcontinent and molecular forms reported from Southeast Asia are members of Sundaicus Complex. Molecular Form A of An. subpictus is distantly related to all other forms and deserve a distinct specific status.

Integrated systematics of Anopheles subpictus (Diptera: Culicidae) in the Oriental Region, with emphasis on forms in Thailand and Sulawesi, Indonesia.

The Anopheles subpictus complex consists of four species informally designated, based on fixed inversions of polytene chromosomes and morphology, as species A, B, C and D in India. However, recent studies revealed the presence of only species A and B in Sri Lanka. Little is known about the specific identity of the taxon in other countries in Asia. This paper reports the results of a molecular and morphological study of An. subpictus in Thailand and South Sulawesi, Indonesia. The maxillary palpi of most females from Thailand have the apical pale band longer than the subapical dark band, seta 7-I of pupae branched and short, and eggs with 18-25 float ridges. These characters do not agree with those described for species A, B, C and D in India. The females of An. subpictus from South Sulawesi usually have the subapical dark band of the maxillary palpus equal in length to the apical pale band. Phylogenetic analyses of sequences of the internal transcribed spacer 2 (ITS2) region of rDNA and the cytochrome c oxidase subunit I (COI) gene of mtDNA of specimens from Thailand, and South Sulawesi, and from various localities in GenBank, were conducted. ITS2 sequences of specimens from all localities in Thailand were identical, except for a small divergence in specimen from Phang Nga Province. Three distinct COI clades were detected in specimens from Chiang Mai Province in northern Thailand. However, crossing experiments between the three clades revealed no genetic incompatibility, suggesting that they were conspecific. ITS2 and COI sequences of most specimens from Thailand fell in clades other than those of An. subpictus species A and B and An. subpictus from Indonesia (East Nusa Tenggara, Java, South Sulawesi) and the Philippines. ITS2 sequences from South Sulawesi and East Nusa Tenggara were very similar, and fell in a clade consisting of specimen from Phang Nga in southern Thailand and sequences of some specimens from Cambodia and Vietnam, but their COI sequences were distinct. DNA sequences and morphological differences suggest the presence of two species within An. subpictus in Thailand, and more than one species in Indonesia.

Insecticide susceptibility status and major detoxifying enzymes activity in Anopheles subpictus from Kasur, Pakistan.

Anopheles subpictus s.l. Grassi (Diptera: Culicidae) is a malaria vector in South Asia, where insecticides are the mainstay for vector control interventions. Information on any variation in metabolic enzyme levels in mosquitoes is helpful with respect to adapting alternative strategies for vector control. The scarce data on the biochemical basis of insecticide resistance in malaria vectors of Pakistan limit the available information for vector control interventions within the country. The insecticide susceptibility status and its biochemical basis against dichlorodiphenyltrichloroethane (DDT) (4%), deltamethrin (0.05%) and permethrin (0.75%) in An. subpictus s.l. collected from all Tehsils of district Kasur were evaluated. For this purpose, a World Health Organization susceptibility bioassay was performed followed by the detection of altered metabolic enzyme activity using biochemical assays. Similarly, a significant difference in knock-down effect was observed among field collected and susceptible strain against all insecticides 24 h post exposure. The overall mean mortality rates of DDT, deltamethrin and permethrin were 27.86% [95% confidence interval (CI) = 29.65-26.06], 44.89% (95% CI = 46.23-43.54) and 78.82% (95% CI = 80.16-77.47), respectively. The biochemical assays revealed an elevated level of metabolic enzymes in the field population. The results provide evidence of resistance against organochlorine and pyrethroid groups in a field population of An. subpictus s.l. from district Kasur mediated by multiple metabolic mechanisms, including acetylcholinesterases, esterases, cytochrome P450 and glutathione S-transferases.

Alterations and Interchange of Morphometric Characters in Different Life Cycle Stages with Reference to Genomic Variations of Anopheles subpictus (Diptera; Culicidae) Sibling Species Complex in Sri Lanka.

The species complex of the mosquito Anopheles subpictus is designated by the sibling species A⁻D, depending on morphological characters of life cycle stages and variations in polytene chromosomes. However, morphological aberrations in the life cycle stages make the identification of sibling species uncertain and imprecise. The objective of the present study is to determine the suitability of morphological variations of sibling species and their genomic variations to identify the sibling species status of an An. subpictus population in Sri Lanka. Life cycle stages of larvae, pupal exuviae, and adults were examined for previously reported distinctive morphological features. Five nuclear and mitochondrial genome regions, including the Internal transcribed spacer 2 (ITS2) region, D3 region, white gene, cytochrome c oxidase I (COI), and Cytochrome b (Cyt-b), were sequenced and analyzed for variations. The eggs changed their distinct sibling morphological characters during metamorphosis (89.33%). The larvae, pupal exuviae, and adult stages showed deviation from their sibling characters by 26.10%, 19.71%, and 15.87%, respectively. However, all the species from the analysis shared two distinct sequence types for all regions, regardless of the morphological variations. In conclusion, the An. subpictus sibling species complex in Sri Lanka is not identifiable using morphological characters due to variations, and the genomic variations are independent from the morphological variations.

Genetic diversity and population structure of malaria vector mosquitoes Anopheles subpictus, Anopheles peditaeniatus, and Anopheles vagus in five districts of Sri Lanka.

BACKGROUND: Although Sri Lanka is considered as a malaria-free nation, the threat of re-emergence of outbreaks still remains due to the high prevalence and abundance of malaria vectors. Analysis of population genetic structure of malaria vectors is considered to be one of the vital components in implementing successful vector control programmes. The present study was conducted to determine the population genetic structure of three abundant malaria vectors; Anopheles subpictus sensu lato (s.l.), Anopheles peditaneatus and Anopheles vagus from five administrative districts in two climatic zones; intermediate zone (Badulla and Kurunegala districts) and dry zone (Ampara, Batticoloa and Jaffna districts) of Sri Lanka using the mitochondrial gene, cytochrome c oxidase subunit I (COI). METHODS: Adult mosquitoes of An. subpictus s.l., An. peditaeniatus, and An. vagus were collected from five study sites located in five districts using cattle baited traps and backpack aspirators. Representative samples of each species that were morphologically confirmed were selected from each locality in generating COI sequences (> 6 good quality sequences per species per locality). RESULTS: Anopheles subpictus s.l. specimens collected during the study belonged to two sibling species; An. subpictus 'A' (from all study sites except from Jaffna) and An. subpictus 'B' (only from Jaffna). The results of haplotype and nucleotide diversity indices showed that all the three species are having high genetic diversity. Although a high significant pairwise difference was observed between An. subpictus 'A' and 'B' (Fst> 0.950, p < 0.05), there were no significant genetic population structures within An. peditaeniatus, An. vagus and An. subpictus species A (p > 0.05), indicating possible gene flow between these populations. CONCLUSIONS: Gene flow among the populations of An. peditaeniatus, An. vagus and An. subpictus species A was evident. Application of vector control measures against all mosquito species must be done with close monitoring since gene flow can assist the spread of insecticide resistance genes over a vast geographical area.

Larvicidal activity of phytosteroid compounds from leaf extract of Solanum nigrum against Culex vishnui group and Anopheles subpictus.

BACKGROUND: Vector control is facing a menace due to the appearance of resistance to synthetic insecticides. Insecticides of plant origin may provide appropriate substitute biocontrol techniques in the future. The present study was carried out to investigate the bio control potentiality of active ingredient isolated from chloroform: methanol (1:1 v/v) extract of mature leaves of Solanum nigrum L. (Solanaceae) against early 3rd instar larvae of Culex vishnui group (comprising of Cx. vishnui Theobald, Cx. pseudovishnui Colless and Cx. tritaeniorhynchus Giles) and Anopheles subpictus Grassi. S. nigrum is a common plant distributed in many parts of India with medicinal properties. METHODS: Bioactive compound isolated from chloroform: methanol (1:1 v/v) extract of mature leaves of S. nigrum was (25, 45, 60 mg/L) tested against early 3rd instar larvae of Cx. vishnui group and An. subpictus. The lethal concentration was determined by log probit analysis. The chemical nature of the active substance was also evaluated following gas chromatography-mass spectroscopy (GC-MS) and infrared (IR) analysis. The compound was also studied on non target organisms such as Daphnia sp. and Diplonychus annulatum. RESULTS: TLC spot having Rf value of 0.94 (Rf = 14.1/15 = 0.94) showed larvicidal activity. In a 72 h bioassay experiment, mortality rate at 60 mg/L was significantly higher (P < 0.05) than those at 25 and 45 mg/L against early 3rd instar. Result of log-probit analysis (at 95% confidence level) revealed that LC50 and LC90 values gradually decreased with the exposure period showing the lowest value at 72 h of exposure. A clear dose-dependent mortality was observed, as the rate of mortality (Y) was positively correlated with the concentration (X) having regression coefficient value close to one in each case. The compound was found to be eco-friendly as it did not show any adverse effect to the studied non target organisms. Chemical characterization (GC-MS and IR analyses) of the active ingredient revealed the presence of phytosteroid compounds responsible for mosquito larvicidal activity. CONCLUSION: Leaf extract of S. nigrum has great potential as bio control agent against Cx. vishnui group and An. subpictus. In near future the isolated bioactive phytochemical could be used as a source of an effective mosquitocidal agent.

Facile fabrication of eco-friendly nano-mosquitocides: Biophysical characterization and effectiveness on neglected tropical mosquito vectors.

Mosquito (Diptera: Culicidae) vectors are solely responsible for transmitting important diseases such as malaria, dengue, chikungunya, Japanese encephalitis, lymphatic filariasis and Zika virus. Eco-friendly control tools of Culicidae vectors are a priority. In this study, we proposed a facile fabrication process of poly-disperse and stable silver nanoparticles (Ag NPs) using a cheap leaf extract of Ichnocarpus frutescens (Apocyanaceae). Bio-reduced Ag NPs were characterized by UV-vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The acute toxicity of I. frutescens leaf extract and green-synthesized Ag NPs was evaluated against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Compared to the leaf aqueous extract, Ag NPs showed higher toxicity against A. subpictus, A. albopictus, and C. tritaeniorhynchus with LC50 values of 14.22, 15.84 and 17.26µg/mL, respectively. Ag NPs were found safer to non-target mosquito predators Anisops bouvieri, Diplonychus indicus and Gambusia affinis, with LC50 values ranging from 636.61 to 2098.61µg/mL. Overall, this research firstly shed light on the mosquitocidal potential of I. frutescens, a potential bio-resource for rapid, cheap and effective synthesis of poly-disperse and highly stable silver nanocrystals.

Characterization and control of symbiotic Bacillus cereus isolated from the mid gut of Anopheles subpictus Grassi.

An investigation was done to find out the role of gut bacterium on the larval development and survival of Anopheles subpictus, the vector responsible for the transmission of malaria. An. subpictus mosquitoes breed exclusively in stagnant water, including shrimp/fish ponds with high nutrient level. One bacterial strain (CX2) was isolated from the midgut of late third instar of mosquito larvae. The bacterial isolate was sensitive to recommended doses of tetracyclin (30 µg/disc), doxycycline (30 µg/disc), kanamycin (30 µg/disc), gentamycin (10 µg/disc), streptomycin (10 µg/disc), vancomycin (30 µg/disc), ofloxacin (5 µg/disc), levofloxacin (5 µg/disc), gatifloxacin (10 µg/disc), but resistant to ampicillin (10 µg/disc).The larvae which were fed with the mixture of two antibiotics tetracylin (30 µg/disc) and doxycyclin (30 µg/disc) (1:1) could not survive in rice-field water. In the control experiments without any antibiotic treatment, 95-100 % survival and 95 % adult emergence were observed. The study indicates that the elimination of gut bacteria suppressed larval growth. Phylogenetic analysis of the 16S rRNA gene sequence was also done. Based on the morphological, biochemical, FAME analysis and phylogenetic analysis, the bacterial isolate CX2 was identified as Bacillus cereus. Poly acrylamide gel electrophoresis analysis revealed that the isolate showed discrete bands ranging from 24.272 to 60.049 kDa proteins. Water extract and methanol extract of Tamarindus indica showed inhibitory effect against B. cereus.

Single-step biosynthesis and characterization of silver nanoparticles using Zornia diphylla leaves: A potent eco-friendly tool against malaria and arbovirus vectors.

Mosquitoes (Diptera: Culicidae) are vectors of important pathogens and parasites, including malaria, dengue, chikungunya, Japanese encephalitis, lymphatic filariasis and Zika virus. The application of synthetic insecticides causes development of resistance, biological magnification of toxic substances through the food chain, and adverse effects on the environment and human health. In this scenario, eco-friendly control tools of mosquito vectors are a priority. Here single-step fabrication of silver nanoparticles (AgNP) using a cheap aqueous leaf extract of Zornia diphylla as reducing and capping agent pf Ag(+) ions has been carried out. Biosynthesized AgNP were characterized by UV-visible spectrophotometry, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDX) and X-ray diffraction analysis (XRD). The acute toxicity of Z. diphylla leaf extract and biosynthesized AgNP was evaluated against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Both the Z. diphylla leaf extract and Ag NP showed dose dependent larvicidal effect against all tested mosquito species. Compared to the leaf aqueous extract, biosynthesized Ag NP showed higher toxicity against An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus with LC50 values of 12.53, 13.42 and 14.61µg/ml, respectively. Biosynthesized Ag NP were found safer to non-target organisms Chironomus circumdatus, Anisops bouvieri and Gambusia affinis, with the respective LC50 values ranging from 613.11 to 6903.93µg/ml, if compared to target mosquitoes. Overall, our results highlight that Z. diphylla-fabricated Ag NP are a promising and eco-friendly tool against larval populations of mosquito vectors of medical and veterinary importance, with negligible toxicity against other non-target organisms.

Eugenol, α-pinene and ß-caryophyllene from Plectranthus barbatus essential oil as eco-friendly larvicides against malaria, dengue and Japanese encephalitis mosquito vectors.

Mosquito-borne diseases represent a deadly threat for millions of people worldwide. Eco-friendly mosquitocides are a priority. In Ayurvedic medicine, Plectranthus species have been used to treat heart disease, convulsions, spasmodic pain and painful urination. In this research, we evaluated the acute toxicity of essential oil from Plectranthus barbatus and its major constituents, against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. The chemical composition of P. barbatus essential oil was analyzed by gas chromatography-mass spectroscopy. Nineteen components were identified. Major constituents were eugenol (31.12%), α-pinene (19.38%) and ß-caryophyllene (18.42%). Acute toxicity against early third-instar larvae of An. subpictus, Ae. albopictus and Cx. tritaeniorhynchus was investigated. The essential oil had a significant toxic effect against larvae of An. subpictus, Ae. albopictus and Cx. tritaeniorhynchus, with 50% lethal concentration (LC50) values of 84.20, 87.25 and 94.34 µg/ml and 90% lethal concentration (LC90) values of 165.25, 170.56 and 179.58 µg/ml, respectively. Concerning major constituents, eugenol, α-pinene and ß-caryophyllene appeared to be most effective against An. subpictus (LC50 = 25.45, 32.09 and 41.66 µg/ml, respectively), followed by Ae. albopictus (LC50 = 28.14, 34.09 and 44.77 µg/ml, respectively) and Cx. tritaeniorhynchus (LC50 = 30.80, 36.75 and 48.17 µg/ml, respectively). Overall, the chance to use metabolites from P. barbatus essential oil against mosquito vectors seems promising, since they are effective at low doses and could be an advantageous alternative to build newer and safer mosquito control tools.

Green synthesis and characterization of silver nanoparticles fabricated using Anisomeles indica: Mosquitocidal potential against malaria, dengue and Japanese encephalitis vectors.

Mosquitoes (Diptera: Culicidae) represent a key threat for millions of people worldwide, since they act as vectors for devastating parasites and pathogens. In this scenario, eco-friendly control tools against mosquito vectors are a priority. Green synthesis of silver nanoparticles (AgNP) using a cheap, aqueous leaf extract of Anisomeles indica by reduction of Ag(+) ions from silver nitrate solution has been investigated. Bio-reduced AgNP were characterized by UV-visible spectrophotometry, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDX) and X-ray diffraction analysis (XRD). The acute toxicity of A. indica leaf extract and biosynthesized AgNP was evaluated against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Both the A. indica leaf extract and AgNP showed dose dependent larvicidal effect against all tested mosquito species. Compared to the leaf aqueous extract, biosynthesized AgNP showed higher toxicity against An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus with LC50 values of 31.56, 35.21 and 38.08 µg/mL, respectively. Overall, this study firstly shed light on the mosquitocidal potential of A. indica, a potential bioresource for rapid, cheap and effective AgNP synthesis.

L1014F-kdr Mutation in Indian Anopheles subpictus (Diptera: Culicidae) Arising From Two Alternative Transversions in the Voltage-Gated Sodium Channel and a Single PIRA-PCR for Their Detection.

Leucine-to-phenylalanine substitution at residue L1014 in the voltage-gated sodium channel, target site of action for dichlorodiphenyltrichloroethane (DDT) and pyrethroids, is the most common knockdown resistance (kdr) mutation reported in several insects conferring resistance against DDT and pyrethroids. Here, we report presence of two coexisting alternative transversions, A>T and A>C, on the third codon position of L1014 residue in malaria vector Anopheles subpictus Grassi (species A) from Jamshedpur (India), both leading to the same amino acid substitution of Leu-to-Phe with allelic frequencies of 19 and 67%, respectively. A single primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) was devised for the identification of L1014F-kdr mutation in An. subpictus resulting from either type of point mutation. Genotyping of samples with PIRA-PCR revealed high frequency (82%) of L1014F-kdr mutation in the study area.

Mosquito larvicidal and ovicidal properties of Pemphis acidula Frost. (Lythraceae) against Culex tritaeniorhynchus Giles and Anopheles subpictus Grassi (Diptera:Culicidae)

Objective: This study was undertaken to assess the larvicidal and ovicidal potential of the crude methanol, benzene and acetone solvent extracts from the medicinal plant Pemphis acidula (Pe. acidula) against the medically important mosquito vectors, Culex tritaeniorhynchus (Cx. tritaeniorhynchus) and Anopheles subpictus (An. subpictus) were exposed to various concentrations and (Diptera: Culicidae). Methods: Twenty five late third instar of Cx. tritaeniorhynchus and An. subpictus were exposed to various concentrations and were assayed in the laboratory by using the protocol of WHO 2005. The larval mortality was observed 24 h of treatment. Hundred eggs of Cx. tritaeniorhynchus and An. subpictus were exposed to various concentrations and were assayed in the laboratory by using the protocol of Su and Mulla 1998. The ovicidal activity was observed 48 h of treatment. Results: The LC50 and LC90 values being 10.81and 20.64 and 22.10 and 43.71 ppm and hundred percent of egg mortality was observed at 350 and 400 ppm methanol extract of Pe. acidula against Cx. tritaeniorhynchus and An. subpictus, respectively. Conclusion: These results suggest that the leaf extracts have the potential to be used as an ideal ecofriendly approach for the control of mosquitoes.