Malarial Vector Research Articles

Identification of Anopheles daciae in Germany through ITS2 sequencing

Until the middle of the twentieth century, malaria was frequently endemic in parts of Germany; Anopheles maculipennis complex species were considered the primary vectors. Three species of this complex have been identified in Germany: A. maculipennis s.s., Anopheles messeae and Anopheles atroparvus; the last predominantly from the coastal regions of Northern Germany. Anopheles daciae is a recently described member of the A . maculipennis complex and resembles the well-characterised species A. messeae, although the two species can be distinguished through their egg morphology and sequencing of the internal transcribed spacer 2 (ITS2) region of their nuclear rDNA. In this study, we harvested larval and adult mosquito samples from five breeding sites and ten CO(2) trap collection sites in the Upper Rhine Valley of Southwestern Germany to analyse the complement of anopheline species present. Mosquito ITS2 DNA was extracted and polymerase chain reaction (PCR)-amplified using established protocols. Genomic analysis was performed by a species-diagnostic restriction fragment length polymorphism assay as well as by sequencing of PCR products; the data obtained were aligned against nucleic acid sequences from English mosquitoes retrieved from GenBank. Additionally, the larval breeding sites of A. messeae were characterised through water quality measurement. Forty-seven samples were successfully processed, of which 6 were identified as A. daciae and 41 as A. messeae. All samples of A. daciae, which has not previously been found in Central Europe, originated from one CO(2) trap collection site in Dettenheim, close to Karlsruhe, Southwestern Germany. The identification of this malarial vector in a novel area may have implications for the re-emergence of disease subsequent to climatic changes.

Potential evidence of parasite avoidance in an avian malarial vector

Epidemiological studies of malaria or other vector-transmitted diseases often consider vectors as passive actors in the complex life cycle of the parasites, assuming that vector populations are homogeneous and vertebrate hosts are equally susceptible to being infected during their lifetime. However, some studies based on both human and rodent malaria systems found that mosquito vectors preferentially selected infected vertebrate hosts. This subject has been scarcely investigated in avian malaria models and even less in wild animals using natural host–parasite associations. We investigated whether the malaria infection status of wild great tits, Parus major , played a role in host selection by the mosquito vector Culex pipiens . Pairs of infected and uninfected birds were tested in a dual-choice olfactometer to assess their attractiveness to the mosquitoes. Plasmodium -infected birds attracted significantly fewer mosquitoes than the uninfected ones, which suggest that avian malaria parasites alter hosts' odours involved in vector orientation. Reaction time of the mosquitoes, that is, the time taken to select a host, and activation of mosquitoes, defined as the proportion of individuals flying towards one of the hosts, were not affected by the bird's infection status. The importance of these behavioural responses for the vector is discussed in light of recent advances in related or similar model systems.

Larvicidal activity of Morinda citrifolia L. (Noni) (Family: Rubiaceae) leaf extract against Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti

Morinda citrifolia leaf extract was tested for larvicidal activity against three medically important mosquito vectors such as malarial vector Anopheles stephensi, dengue vector Aedes aegypti, and filarial vector Culex quinquefasciatus. The plant material was shade dried at room temperature and powdered coarsely. From the leaf, 1-kg powder was macerated with 3.0 L of hexane, chloroform, acetone, methanol, and water sequentially for a period of 72 h each and filtered. The yield of extracts was hexane (13.56 g), chloroform (15.21 g), acetone (12.85 g), methanol (14.76 g), and water (12.92 g), respectively. The extracts were concentrated at reduced temperature on a rotary vacuum evaporator and stored at a temperature of 4°C. The M. citrifolia leaf extract at 200, 300, 400, 500, and 600 ppm caused a significant mortality of three mosquito species. Hexane, chloroform, acetone, and water caused moderate considerable mortality; however, the highest larval mortality was methanolic extract, observed in three mosquito vectors. The larval mortality was observed after 24-h exposure. No mortality was observed in the control. The third larvae of Anopheles stephensi had values of LC(50) = 345.10, 324.26, 299.97, 261.96, and 284.59 ppm and LC(90) = 653.00, 626.58, 571.89, 505.06, and 549.51 ppm, respectively. The Aedes aegypti had values of LC(50) = 361.75, 343.22, 315.40, 277.92, and 306.98 ppm and LC(90) = 687.39, 659.02, 611.35, 568.18, and 613.25 ppm, respectively. The Culex quinquefasciatus had values of LC(50) = 382.96, 369.85, 344.34, 330.42, and 324.64 ppm and LC(90) = 726.18, 706.57, 669.28, 619.63, and 644.47 ppm, respectively. The results of the leaf extract of M. citrifolia are promising as good larvicidal activity against the mosquito vector Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. This is a new eco-friendly approach for the control of vector control programs. Therefore, this study provides first report on the larvicidal activities against three species of mosquito vectors of this plant extracts from India.

Effect of climate on malarial vector distribution in Monsoon Asia: coupled model for Ecophysiological and Climatological Distribution of mosquito generations (ECD-mg)

The relationship between the life cycle and climate conditions of the malaria vector mosquito Anopheles was modeled in order to describe its temporal and geographic distribution at a fine resolution. Since the mosquito grows through immature life stages in an aquatic environ- ment, the model was designed to express the surface moisture conditions conducive to the mos- quito's growth. The development of the mosquito was estimated using either air or water temper- ature, depending on the developmental stage of the mosquito's life cycle. To achieve this, the previous model was modified in order to improve the estimation of the water conditions of its habi- tat. As a result, the modified model was able to predict seasonal patterns of occurrence of Anophe- les at representative sites with a more reasonable degree of accuracy than the previous model. This model was then applied using simple climate data in order to obtain the geographic distrib- ution of the occurrence of various species of Anopheles in Monsoon Asia. The most notable results of the simulated spatio-temporal emergence of the Anopheles mosquito is that although the mod- ified model did not include unique parameters of each species, distribution was clearly divided into sub-regions for each dominant species that corresponded to the climate conditions.

Biolarvicidal and pupicidal potential of silver nanoparticles synthesized using Euphorbia hirta against Anopheles stephensi Liston (Diptera: Culicidae)

Vector control is a critical requirement in epidemic disease situations, as is an urgent need to develop new and improved mosquito control methods that are economical and effective yet safe for nontarget organisms and the environment. Mosquitoes transmit serious human diseases, causing millions of deaths every year. Use of synthetic insecticides to control vector mosquitoes has caused physiological resistance and adverse environmental effects in addition to high operational cost. Insecticides of synthesized natural products for vector control have been a priority in this area. In the present study, activity of silver nanoparticles (AgNPs) synthesized using Euphorbia hirta (E. hirta) plant leaf extract against malarial vector Anopheles stephensi (A. stephensi) was determined. Range of concentrations of synthesized AgNPs (3.125, 6.25, 12.5, 25, and 50 ppm) and methanol crude extract (50, 100, 150, 200, and 250 ppm) were tested against larvae of A. stephensi. The synthesized AgNPs from E. hirta were highly toxic than methanolic crude extract against malarial vector, A. stephensi. The synthesized AgNPs were characterized by UV-vis spectrum, scanning electron microscopy (SEM), and X-ray diffraction. SEM analyses of the synthesized showed that AgNPs, measuring 30-60 nm in size, were clearly distinguishable. The synthesized AgNPs showed larvicidal effects after 24 h of exposure; however, the highest larval mortality was found in the synthesized AgNPs against the first to fourth instar larvae and pupae of values LC(50) (10.14, 16.82, 21.51, and 27.89 ppm, respectively), LC(90) (31.98, 50.38, 60.09, and 69.94 ppm, respectively), and the LC(50) and LC(90) values of pupae of 34.52 and 79.76 ppm, respectively. Methanol extract exhibited the larval toxicity against the first to fourth instar larvae and pupae of values LC(50) (121.51, 145.40, 169.11, and 197.40 ppm, respectively), LC(90) (236.44, 293.75, 331.42, and 371.34 ppm, respectively), and the LC(50) and LC(90) values of pupae of 219.15 and 396.70 ppm, respectively. No mortality was observed in the control. These results suggest that synthesized silver nanoparticles are a rapid, eco-friendly, and single-step approach; the AgNPs formed can be potential mosquito larvicidal agents.

Effect of Vetiveria zizanioides L. Root extracts on the malarial vector, Anopheles stephensi Liston

ObjectiveTo evaluate the ovicidal and oviposition deterrent potential of the ethanolic extract from Vetiveria zizanioides (V. zizanioides) roots against the malarial vector, Anopheles stephensi (A. stephensi). MethodsThe dried clean V. zizanioides roots were powerdered and extracted with ethanol for 8 h in a soxhlet apparatus. After evaporation, the residue was dissolved in acetone. One hundred freshly laid eggs of A. stephensi were exposed to the extract at differnt concentrations for 48 h, and the hatch rate was calculated to evaluate the ovicidal activity. Those exposed to actone aqueous solution were used as control. The egg laying behavior of gravid female A. stephensi was also observed using oviposition deterrent test. Effective repellency (ER) was used to evaluate the oviposition deterrent activity. ResultsExposure to the crude ethanol extract of V. zizanioides reduced the hatchability rate of A. stephensi eggs, and zero hatchability was exerted at 375 ppm. In the oviposition deterrent test, the extract alleviated the egg laying with an ER of 78.9% at the highest concentration of 375 ppm and even 53.7% at the lowest concentration of 125 ppm. Moreover, the negative values of oviposition active index also suggests the extract was a good deterrent agent. ConclusionsThe ethanolic extract of V. zizanioides roots may be used an alternative pesticide to control A. stephensi at the early stage of life history, possibly due to the presence of various active chemical compounds.

Larvicidal efficacy of the Citrus limetta peel extracts against Indian strains of Anopheles stephensi Liston and Aedes aegypti L.

The extracts from the peels of Citrus limetta were prepared using hexane and petroleum ether as the solvents. The larvicidal potential of each extract was assessed against dengue fever vector, Aedes aegypti, and malarial vector, Anopheles stephensi, by evaluating the toxicity effects on early fourth instars. Both the extracts were found effective against both the species. The bioassay with hexane extracts resulted in LC(50) values of 132.45 and 96.15 ppm against A. stephensi and A. aegypti, respectively; while the petroleum ether extracts from the C. limetta peels showed LC(50) values of 244.59 and 145.50 ppm, respectively. It revealed that the hexane extracts possessed 1.9-fold more larvicidal potential against A. stephensi and 1.5-fold more efficacy against A. aegypti as compared to the extracts obtained using petroleum ether as solvent. The data further revealed that the extracts were 1.4-1.7 times more effective against A. aegypti as compared to A. stephensi. The qualitative phytochemical study of the extracts showed the presence of terpenoids and flavonoids as the common phytochemical constituents in the extracts suggesting their possible role in the toxicity. Other constituents tested were not detected except alkaloids which were found to be present only in the petroleum ether extract. Further studies are needed to isolate and identify the active principles involved, their mode of action, formulated preparations for enhancing potency and stability, toxicity, and effects on non-target organisms and the environment. This could help in formulating efficient strategies for mosquito control.

Evaluation of larvicidal and pupicidal activity of Morinda citrifolia L. (Noni) (Family: Rubiaceae) against three mosquito vectors

ObjectiveTo evaluate the mosquito larvicidal and pupicidal activity against three important medically mosquito vector such as malarial vector, Anopheles stephensi (An. stephensi), dengue vector, Aedes aegypti (Ae. aegypti) and filarial vector Culex quinquefasciatus (Cx. quinquefasciatus). MethodsMorinda citrifolia (M. citrifolia) leaf was collected in and around Alleppy districts, Kerala, India. M. citrifolia leaf was washed with tap water and shade dried at room temperature. An electrical blender powdered the dried plant materials (leaves). From the leaf, 1 kg powdered was macerated with 3.0 L of methanol sequentially for a period 72 h and filtered. The crude plant extracts were evaporated to dryness in rotary vacuum evaporator. The larvicidal and pupicidal activity was assayed at various concentrations ranging from (100–500 ppm) under the laboratory as well as field conditions. The LC50and LC90value of the M. citrifolia leaf extract was determined by Probit analysis. ResultsThe plant extract showed larvicidal and pupicidal effects after 24 and 48 hrs of exposure; All larval instars and pupae have considerably moderate mortality; however, the highest larval and pupal mortality was methanolic extract of M. citrifolia observed in three mosquito vectors at 48 h. The LC50and LC90of M. citrifolia against the first to fourth instar larvae and pupae against mosquito vectors. An. stephensi had values of LC50=146.08, 159.07, 172.16, 185.08 and 202.68 ppm and LC90=322.12, 363.48, 388.56, 436.51 and 513.56 ppm, respectively. The Ae. aegypti had values of LC50=181.27, 210.40, 229.80, 256.73 and 292.01 ppm and LC90=407.99, 485.65, 534.14, 624.16 and 756.79 ppm, respectively. The Cx. quinquefasciatus had values of LC50=226.70, 256.97, 290.05, 316.33 and 358.11 ppm and LC90=560.35, 652.07, 733.03, 797.09 and 875.25 ppm, respectively at 24 h. ConclusionsThe results of the leaf extract of M. citrifolia are promising as good larvicidal and pupicidal activity against the mosquito vector, An. stephensi, Ae. aegypti, Cx. quinquefasciatus. This is a new eco-friendly approach for the control of vector control programs. Therefore, this study provides first report on the larvicidal and pupicidal activities against three species of mosquito vectors of this plant extract from India.

Biolarvicidal and pupicidal activity of Acalypha alnifolia Klein ex Willd. (Family: Euphorbiaceae) leaf extract and Microbial insecticide, Metarhizium anisopliae (Metsch.) against malaria fever mosquito, Anopheles stephensi Liston. (Diptera: Culicidae)

The present study was to establish of Acalypha alnifolia leaf extract and microbial insecticide, Metarizhium anisopliae on larvicidal and pupicidal properties of against the malaria fever mosquito, Anopheles stephensi. The leaf extract showed larvicidal and pupicidal effects after 24 h of exposure; however, the highest larval and pupal mortality was found in the leaf extract of ethanol A. alnifolia against the 1st to 4th instar larvae and pupae of values LC(50) value of 1st instar was 5.388%, 2nd instar was 6.233%, 3rd instar was 6.884%, 4th instar was 8.594%, and pupae was 10.073%, respectively, and microbial insecticide, M. anisopliae against the 1st to 4th instar larvae and pupae of values LC(50) value of 1st instar was 7.917%, 2nd instar was 10.734%, 3rd instar was 17.624%, 4th instar was 26.590%, and pupae was 37.908%, respectively. Moreover, combined treatment of values of LC(50) value of 1st instar was 3.557%, 2nd instar was 4.373%, 3rd instar was 5.559%, 4th instar was 7.223%, and pupae was 8.542%, respectively. No mortality was observed in the control. The results that the leaves extract of A. alnifolia and microbial insecticide, M. anisopliae is promising as good larvicidal and pupicidal properties of against malaria fever mosquito, A. stephensi. This is an ideal eco-friendly approach for the control of malarial vector, A. stephensi as a vector control programs. Therefore, this study provides first report on the combined effect of mosquitocidal efficacy of this plant extract and fungal pathogen of M. anisopliae against A. stephensi from Southern India.

Anopheles atroparvus density modeling using MODIS NDVI in a former malarious area in Portugal

Malaria is dependent on environmental factors and considered as potentially re-emerging in temperate regions. Remote sensing data have been used successfully for monitoring environmental conditions that influence the patterns of such arthropod vector-borne diseases. Anopheles atroparvus density data were collected from 2002 to 2005, on a bimonthly basis, at three sites in a former malarial area in Southern Portugal. The development of the Remote Vector Model (RVM) was based upon two main variables: temperature and the Normalized Differential Vegetation Index (NDVI) from the Moderate Resolution Imaging Spectroradiometer (MODIS) Terra satellite. Temperature influences the mosquito life cycle and affects its intra-annual prevalence, and MODIS NDVI was used as a proxy for suitable habitat conditions. Mosquito data were used for calibration and validation of the model. For areas with high mosquito density, the model validation demonstrated a Pearson correlation of 0.68 (p<0.05) and a modelling efficiency/Nash-Sutcliffe of 0.44 representing the model's ability to predict intra- and inter-annual vector density trends. RVM estimates the density of the former malarial vector An. atroparvus as a function of temperature and of MODIS NDVI. RVM is a satellite data-based assimilation algorithm that uses temperature fields to predict the intra- and inter-annual densities of this mosquito species using MODIS NDVI. RVM is a relevant tool for vector density estimation, contributing to the risk assessment of transmission of mosquito-borne diseases and can be part of the early warning system and contingency plans providing support to the decision making process of relevant authorities.

Mosquito adulticidal and repellent activities of botanical extracts against malarial vector, Anopheles stephensi Liston (Diptera: Culicidae)

Objective To determine the adulticidal and repellent activities of different solvent leaf extracts of Eclipta alba ( E. alba) and Andrographis paniculata ( A. paniculata) against malarial vector, Anopheles stephensi ( An. stephensi). Methods Adulticidal efficacy of the crude leaf extracts of E. alba and A. paniculata with five different solvents like benzene, hexane, ethyl acetate, methanol and chloroform was tested against the five to six day old adult female mosquitoes of An. stephensi. The adult mortality was observed after 24 h under the laboratory conditions. The repellent efficacy was determined against An. stephensi mosquito species at three concentrations viz., 1.0, 2.5 and 5.0 mg/cm 2 under laboratory conditions. Results Among the tested solvents the maximum efficacy was observed in the methanol extract. The LC 50 and LC 90 values of E. alba and A. paniculata against adults of An. stephensi were 150.36, 130.19 ppm and 285.22, 244.16 ppm, respectively. No mortality was observed in controls. The chi-square values were significant at P<0.05 level. Methanol extract of E. alba and A. paniculata was produce maximum repellency against An. stephensi. Conclusions From the results it can be concluded the crude extract of E. alba and A. paniculata was an excellent potential for controlling An. stephensi mosquitoes.

Spinosad and neem seed kernel extract as bio–controlling agents for malarial vector, Anopheles stephensi and non–biting midge, Chironomus circumdatus

Objective Midge egg masses are reported to support non-pathogenic strains of the cholera pathogen, Vibrio cholera (V. cholera). Mosquito born diseases have been reported to cause millions of death worldwide. The present research reveals the toxicity effect of spinosad and neem seed kernel extract (NSKE) against different larval stages of Anopheles stephensi ( An. stephensi) and Chironomus circumdatus (Ch. circumdatus). Methods The neem seeds were collected from Marudamalai hills, Bharathiar University, Coimbatore, India. Neem seed kernels were powdered, extracted and diluted for different concentrations (2 ppm to 10 ppm). Spinosad was purchased from Kalpatharu pesticide Limited, Coimbatore, Tamil Nadu, India and thoroughly mixed with distilled water to prepare various concentrations (0.01 to 0.08 ppm) and used for bioassay. Results The results depict that spinosad is more toxic in lower concentrations when compared to NSKE and mosquitoes are more susceptible than chironomids. Lethal concentrations were evaluated using the observed mortality. The lowest LC 50 value obtained from spinosad against An. stephensi and Ch. circumdatus were 0.002 05 ppm and 0.008 91 ppm. This study investigated on effect of Spinosad and NSKE on the biology of mosquito. The immature stages of both species were susceptible to Spinosad and NSKE. Spinosad and NSKE in individual as well as combined treatment provided additional days in development for mosquitoes. Conclusions The results conclude that Spinosad and NSKE are potential larvicides against An. stephensi and Ch. circumdatus.

Larvicidal and pupicidal activity of spinosad against the malarial vector Anopheles stephensi

Objective To investigate the larvicidal and pupicidal activity of spinosad against Anopheles stephensi Listen. Methods Spinosad from the actinomycete, Saccharopolyspora spinosa was tested against Anopheles stephensi at different concentrations (0.01, 0.02, 0.04, 0.06 and 0.08 ppm.), and against first to fourth instar larvae and pupae. Results The larval mortality ranged from 36.1±1.7 in (0.01 ppm) to 79.3±1.8 (0.08 ppm) the first instar larva. The LC 50 and LC 90 values of first, second, third and fourth instar larva were 0.001, 0.031, 0.034, 0.036 and 0.0113, 0.102, 0.111, 0.113, respectively. The pupal mortality ranged from 33.0±2.0 (0.01 ppm) to 80.0±0.9 (0.08 ppm). The LC 50 and LC 90 values were 0.028 and 0.1020, respectively. The reduction percentage of Anopheles larvae was 82.7%, 91.4% and 96.0% after 24, 48, 72 hours, respectively, while more than 80% reduction was observed after 3 weeks. Conclusions In the present study spinosad effectively caused mortality of mosquito larvae in both the laboratory and field trial. It is predicted that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.

Evaluation of larvicidal activity of Acalypha alnifolia Klein ex Willd. (Euphorbiaceae) leaf extract against the malarial vector, Anopheles stephensi, dengue vector, Aedes aegypti and Bancroftian filariasis vector, Culex quinquefasciatus (Diptera: Culicidae)

The leaf extract of Acalypha alnifolia with different solvents - hexane, chloroform, ethyl acetate, acetone and methanol - were tested for larvicidal activity against three important mosquitoes such as malarial vector, Anopheles stephensi, dengue vector, Aedes aegypti and Bancroftian filariasis vector, Culex quinquefasciatus. The medicinal plants were collected from the area around Kallar Hills near the Western Ghats, Coimbatore, India. A. alnifolia plant was washed with tap water and shade dried at room temperature. The dried leaves were powdered mechanically using commercial electrical stainless steel blender. The powder 800 g of the leaf material was extract with 2.5 litre of various each organic solvents such as hexane, chloroform, ethyl acetate, acetone, methanol for 8 h using Soxhlet apparatus, and filtered. The crude plant extracts were evaporated to dryness in a rotary vacuum evaporator. The yield of extracts was hexane (8.64 g), chloroform (10.74 g), ethyl acetate (9.14 g), acetone (10.02 g), and methanol (11.43 g). One gram of the each plant residue was dissolved separately in 100 ml of acetone (stock solution) from which different concentrations, i.e., 50, 150, 250, 350 and 450 ppm, was prepared. The hexane, chloroform, ethyl acetate, acetone was moderate considerable mortality; however, the highest larval mortality was methanolic extract observed in three mosquito vectors. The larval mortality was observed after 24 h exposure. No mortality was observed in the control. The early fourth-instar larvae of A. stephensi had values of LC(50) = 197.37, 178.75, 164.34, 149.90 and 125.73 ppm and LC(90) = 477.60, 459.21, 435.07, 416.20 and 395.50 ppm, respectively. The A. aegypti had values of LC(50) = 202.15, 182.58, 160.35, 146.07 and 128.55 ppm and LC(90) = 476.57, 460.83, 440.78, 415.38 and 381.67 ppm, respectively. The C. quinquefasciatus had values of LC(50) = 198.79, 172.48, 151.06, 140.69 and 127.98 ppm and LC(90) = 458.73, 430.66, 418.78, 408.83 and 386.26 ppm, respectively. The results of the leaf extract of A. alnifloia are promising as good larvicidal activity against the mosquito vector, A. stephensi, A. aegypti, C. quinquefasciatus. Therefore, this study provides first report on the larvicidal activities against three species of mosquito vectors of this plant extracts from Southern India.

Studies on larvicidal and pupicidal activity of Leucas aspera Willd. (Lamiaceae) and bacterial insecticide, Bacillus sphaericus, against malarial vector, Anopheles stephensi Liston. (Diptera: Culicidae)

The efficacy of whole plant extracts of Leucas aspera and Bacillus sphaericus has been proven against larvicidal and pupicidal activities of the malarial vector, Anopheles stephensi. The present study investigated the larvicidal and pupicidal activity against the first to fourth instar lavae and pupae of the laboratory-reared mosquitoes, A. stephensi. The medicinal plants were collected from the area around Maruthamalai hills, Coimbatore, Tamil Nadu, India. L. aspera whole plant was washed with tap water and shade dried at room temperature. The dried plant materials were powdered by an electric blender. From the powder, 100g of the plant materials was extracted with 300ml of organic solvents of ethanol for 8h using a Soxhlet apparatus. The extracts were filtered through a Buchner funnel with Whatman number 1 filter paper. The crude plant extracts were evaporated to dryness in a rotary vacuum evaporator. The plant extract showed larvicidal and pupicidal effects after 24h of exposure. All larval instars and pupae have considerably moderate mortality; however, the highest larval mortality was the ethanolic extract of whole plant L. aspera against the first to fourth instar larvae and pupae values of LC(50) = I instar was 9.695%, II instar was 10.272%, III instar was 10.823%, and IV instar was 11.303%, and pupae was 12.732%. B. spaericus against the first to fouth instar larvae and pupae had the following values: I instar was 0.051%, II instar was 0.057%, III instar was 0.062%, IV instar was 0.066%, and for the pupae was 0.073%. No mortality was observed in the control. The present results suggest that the ethanolic extracts of L. aspera and B. sphaericus provided an excellent potential for controlling of malarial vector, A. stephensi.

Chemical composition and larvicidal activity of Blumea densiflora essential oils against Anopheles anthropophagus: a malarial vector mosquito

Blumea densiflora, an edible and medicinal plant, is chiefly distributed in Southeast Asia and South Asia. Essential oils extracted by steam distillation from B. densiflora were investigated for their chemical composition and larvicidal activity against Anopheles anthropophagus, the primary vector of malaria in China and other East Asian countries. Totally, 46 compounds were identified by gas chromatography and mass spectroscopy. The major chemical compounds identified were borneol (11.43%), germacrene D (8.66%), β-caryophyllene (6.68%), γ-terpinene (4.35%), sabinene (4.34%), and β-bisabolene (4.24%). A series of concentrations of essential oil (that ranged from 6.25 to 150 ppm) were tested against A. anthropophagus fourth-instar larvae according to WHO recommendation. In general, larval mortality increased as concentration and exposure time increased, indicating a dose-dependent effect, and high insecticidal activity showed that 100% mortality occurred within 6 h at 150 ppm, 10 h at 100 ppm, 30 h at 50 ppm, and 30 h at 25 ppm essential oil concentration. The LC(50) values were 22.32 (after 12 h) and 10.55 ppm (after 24 h), and the LC(90) values were 54.04 (after 12 h) and 33.56 ppm (after 24 h). Pylarvex, the reference standard, had better larvicidal activity, causing 100% mortality within 2 h at 150 ppm and within 6 h at 6.25 ppm. The results clearly reveal that the essential oil of B. densiflora served as a potential, eco-friendly mosquito larvicide against the malarial vector mosquito A. anthropophagus.

An Expression of an Insect Membrane-bound Cytochrome P450 CYP6AA3 in the Escherichia coli in Relation to Insecticide Resistance in a Malarial Vector

This laboratory investigation was carried out at the Faculty of Sciences, Mahidol University, Thailand during October 2007 to May 2009. The objectives of this study include: the search for heterologous expression of the cytochrome P450 CYP6AA3 enzyme of the Anopheles minimus mosquitoes in relation to Malaria disease and to provide some information on molecular mechanism of insects' pyrethroid resistance. The polymerase chain reaction aided by the Pfu DNA polymerase and some specific generated primers were used to modify the CYP6AA3 gene. The PCR product was ligated with a predigested pET-3a at the NdeI and BamHI restriction sites. The modified CYP6AA3 enzyme was expressed in the Escherichia coli BL21 (DE3) pLysS in order to achieve a high amount of soluble form of its expression. The results showed that the use of the isopropyl-beta-D-thiogalactopyranoside (IPTG) and incubation together with ferric chloride and delta-aminolevulinic acid did not increase any soluble form of the CYP6AA3 enzyme. A significant amount of soluble enzyme was produced upon the replacement of the 30 N-terminal residues with a short peptide where it gave Ldelta30CYP6AA3 protein and after purification process was taken place, it yielded up to 10.64 mg 10 L(-1) or approximately 1 mg L(-1) of the homogenous Ldelta30CYP6AA3. When this purified Ldelta30CYP6AA3 protein was used in a metabolizing process with the cypermethrin, deltamethrin and permethrin substrates, it gave their apparent Km values for cypermethrin and deltamethrin of 12.5 and 23.5 microM, respectively. The heterologous expression carried out with the use of the E. coli gave a high amount of soluble CYP6AA3 enzyme of the An. minimus mosquitoes hence the modified technique being used was successfully achieved.

Larvicidal and repellent potential of Moringa oleifera against malarial vector, Anopheles stephensi Liston (Insecta: Diptera: Culicidae)

Objective To evaluate the larvicidal and pupicidal potential of the methanolic extracts from Moringa oleifera ( M. oleifera) plant seeds against malarial vector Anopheles stephensi ( A. stephensi) mosquitoes at different concentrations (20, 40, 60, 80 and 100 ppm). Methods M. oleifera was collected from the area of around Bharathiar University, Coimbatore. The dried plant materials were powdered by an electrical blender. From each sample, 100 g of the plant material were extracted with 300 mL of methanol for 8 h in a Soxhlet apparatus. The extracts were evaporated to dryness in rotary vacuum evaporator to yield 122 mg and 110 mg of dark greenish material (residue) from Arcang amara and Ocimum basilicum, respectively. One gram of the each plant residue was dissolved separately in 100 mL of acetone (stock solution) from which different concentrations, i.e., 20, 40, 60, 80 and 100 ppm were prepared. Results Larvicidal activity of M. oleifera exhibited in the first to fourth instar larvae of the A. stephensi, and the LC 50 and LC 90 values were 57.79 ppm and 125.93 ppm for the first instar, 63.90 ppm and 133.07 ppm for the second instar, 72.45 ppm and 139.82 ppm for the third instar, 78.93 ppm and 143.20 ppm for the fourth instar, respectively. During the pupal stage the methanolic extract of M. oleifera showed that the LC 50 and LC 90 values were 67.77 ppm and 141.00 ppm, respectively. Conclusions The present study indicates that the phytochemicals derived from M. oleifera seeds extracts are effective mosquito vector control agents and the plant extracts may be used for further integrated pest management programs.

Evaluation of indigenous plant extracts against the malarial vector, Anopheles stephensi (Liston) (Diptera: Culicidae)

Since ancient times, plant and microbial products were used in various aspects. However, their use against insects decreased when chemical products became developed. Recently, concerns increased with respect to public health and environmental security requiring detection of natural products that may be used against insects. In this study, mosquito Larvicidal and ovicidal activity of crude hexane, ethyl acetate, benzene, chloroform, and methanol extracts of the leaf of three plants, Eclipta alba, Cardiospermum halicacabum, and Andrographis paniculata, were tested against the early third-instar larvae of Anopheles stephensi (Liston) (Diptera: Culicidae). The larval mortality was observed after 24 h of exposure. All extracts showed moderate larvicidal effects; however, the highest larval mortality was found in methanol extract of A. paniculata, E. alba, and C. halicacabum against the larvae of A. stephensi (LC(50) = 79.68, 112.56, and 133.01 ppm; LC(90) = 154.66, 220.68, and 270.72 ppm), respectively. Mean percent hatchability of the ovicidal activity was observed 48 h post-treatment. The percent hatchability was inversely proportional to the concentration of extract and directly proportional to the eggs. Mortality of 100% with methanol and ethyl acetate extract of A. paniculata and methanol extract of E. alba were exerted at 200 ppm and methanol and benzene extract of C. halicacabum exerted at 150 ppm. This is an ideal eco-friendly approach for the control of the malaria vector, A. stephensi. Therefore, this study provides first report on the larvicidal and ovicidal activities against malaria vector, A. stephensi of E. alba plant extracts.

Efficacy of medicinal plant extracts against malarial vector, Anopheles subpictus Grassi

Insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. The aim of this study was to evaluate the adulticidal activity and adult emergence inhibition (EI) of leaf hexane, chloroform, ethyl acetate, acetone, and methanol extracts of Aegle marmelos (Linn.) Correa ex Roxb, Andrographis lineata Wallich ex Nees., Andrographis paniculata (Burm.f.) Wall. ex Nees., Cocculus hirsutus L. Diels, Eclipta prostrata L., and Tagetes erecta L. tested against malarial vector, Anopheles subpictus Grassi (Diptera: Culicidae). All plant extracts showed moderate adulticidal activity and EI effects after 24 h of exposure at 1,000 ppm; however, the highest adulticidal activity was observed in ethyl acetate extract of A.lineata, chloroform extract of A. paniculata, acetone extract of C. hirsutus, and methanol extract of T. erecta (LD(50) = 126.92, 95.82, 109.40, and 89.83 ppm; LD(90) = 542.95, 720.82, 459.03, and 607.85 ppm); and effective EI was found in leaf acetone extract of the A. marmelos, ethyl acetate extract of A. lineata, methanol extracts of C. hirsutus, and T. erecta, (EI(50) = 128.14, 79.39, 143.97, and 92.82 ppm; EI(90) = 713.53, 293.70, 682.72, and 582.59 ppm), respectively, against A. subpictus. These results suggest that the leaf methanol extract of C. hirsutus and T. erecta have the potential to be used as an ideal eco-friendly approach for the control of A. subpictus. Therefore, this study provides first report on the mosquito adulticidal activity and EI of plant extracts against malaria vector.