Imd pathway is involved in the interaction of Drosophila melanogaster with the entomopathogenic bacteria,Xenorhabdus nematophila and Photorhabdus luminescens

Xenorhabdus nematophila/Steinernema carpocapsae and Photorhabdus luminescens/Heterorhabditis bacteriophora are nemato-bacterial complexes highly pathogenic for insects. Using a syringe as artificial vector, we have analyzed the effects of the two bacteria, X. nematophila and P. luminescens on the genetic tool insect, Drosophila melanogaster. Both bacteria were found to kill adult flies in a dose dependent manner with X. nematophila being the fastest. On the other hand, when an injection of non-pathogenic bacteria, Escherichia coli, is performed 1 day before challenge with the entomopathogenic bacteria, then the survival of Drosophila flies was prolonged by at least 20 h. After injection of entomopathogenic bacteria, Drosophila mutant Dif¹, affected on the Toll pathway, showed a similar phenotype than wild-type flies whereas Drosophila mutant Dredd^D55, affected on the imd pathway, was not protected by a prior injection of E. coli. This suggested that members of the imd pathway might be targets of these entomopathogenic bacteria albeit synthesis of antimicrobial peptides through this signaling pathway was induced by X. nematophila as well as P. luminescens. Finally, P. luminescens phoP mutant, an avirulent mutant in the Lepidopteran insect, Spodoptera littoralis, was found poorly virulent for D. melanogaster. phoP mutant partially protected D. melanogaster flies if injected 1 day before the injection of P. luminescens wild-type TT01 to the same extent than the E. coli-induced protection. However, phoP recovered a level of pathogenicity comparable to P. luminescens wild-type TT01 when injected to Drosophila flies affected on the imd pathway.     

Myco-synthesis of silver nanoparticles using Beauveria bassiana against dengue vector,Aedes aegypti (Diptera: Culicidae)

The efficacy of silver synthesized biolarvicide with the help of entomopathogenic fungus, Beauveria bassiana, was assessed against the different larval instars of dengue vector, Aedes aegypti. The silver nanoparticles were observed and characterized by a scanning electron microscope (SEM) and energy-dispersive X-ray (EDX). A surface plasmon resonance band was observed at 420 nm in UV-vis spectrophotometer. The characterization was confirmed by shape (spherical), size 36.88–60.93 nm, and EDX spectral peak at 3 keV of silver nanoparticles. The synthesized silver nanoparticles have been tested against the different larval instars of Ae. aegypti at different concentrations for a period of 24 h. Ae. aegypti larvae were found more susceptible to the synthesized silver nanoparticles. The LC₅₀ and LC₉₀ values are 0.79 and 1.09 ppm with respect to the Ae. aegypti treated with B. bassiana (Bb) silver nanoparticles (AgNPs). First and second instar larvae of Ae. aegypti have shown cent percent mortality while third and fourth instars found 50.0, 56.6, 70.0, 80.0, and 86.6 and 52.4, 60.0, 68.5, 76.0, and 83.3 % mortality at 24 h of exposure in 0.06 and 1.00 ppm, respectively. It is suggested that the entomopathogenic fungus synthesized silver nanoparticles would be appropriate for environmentally safer and greener approach for new leeway in vector control strategy through a biological process.     

Ovicidal,larvicidal and adulticidal properties of Asparagus racemosus (Willd.) (Family: Asparagaceae) root extracts against filariasis (Culex quinquefasciatus), dengue (Aedes aegypti) and malaria (Anopheles stephensi) vector mosquitoes (Diptera: Culicidae)

Several diseases are associated to the mosquito–human interaction. Mosquitoes are the carriers of severe and well-known illnesses such as malaria, arboviral encephalitis, dengue fever, chikungunya fever, West Nile virus and yellow fever. These diseases produce significant morbidity and mortality in humans and livestock around the world. The present investigation was undertaken to study the ovicidal, larvicidal and adulticidal activities of crude hexane, ethyl acetate, benzene, chloroform and methanol extracts of root of Asparagus racemosus were assayed for their toxicity against three important vector mosquitoes, viz., Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi (Diptera: Culicidae). The mean percent hatchability of the eggs was observed after 48 h post-treatment. The percent hatchability was inversely proportional to the concentration of extract and directly proportional to the eggs. All the five solvent extracts showed moderate ovicidal activity; however, the methanol extract showed the highest ovicidal activity. The methanol extract of Asparagus racemosus against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi exerted 100 % mortality (zero hatchability) at 375, 300 and 225 ppm, respectively. Control eggs showed 99–100 % hatchability. 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 root of Asparagus racemosus against the larvae of Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi with the LC₅₀ and LC₉₀ values were 115.13, 97.71 and 90.97 ppm and 210.96, 179.92, and 168.82 ppm, respectively. The adult mortality was observed after 24 h recovery period. The plant crude extracts showed dose-dependent mortality. At higher concentrations, the adult showed restless movement for some times with abnormal wagging and then died. Among the extracts tested, the highest adulticidal activity was observed in methanol extract against Anopheles stephensi followed by Aedes aegypti and Culex quinquefasciatus with the LD₅₀ and LD₉₀ values were 120.44, 135.60, and 157.71 ppm and 214.65, 248.35, and 290.95 ppm, respectively. No mortality was recorded in the control. The finding of the present investigation revealed that the root extract of Asparagus racemosus possess remarkable ovicidal, larvicidal and adulticidal activity against medically important vector mosquitoes and this is the low cost and ideal eco-friendly approach for the control of mosquitoes. This is the first report on the mosquito ovicidal, larvicidal and adulticidal activities of the reported Asparagus racemosus root.     

Sublethal effects of atrazine and glyphosate on life history traits of Aedes aegypti and Aedes albopictus (Diptera: Culicidae)

Although exposure of mosquito larvae to agricultural chemicals such as herbicides is common and widespread, our understanding of how these chemicals affect mosquito ecology and behavior is limited. This study investigated how an environmentally relevant concentration of two herbicides, atrazine and glyphosate, affects mosquito life history traits. One hundred and fifty (150) first instar Aedes (Stegomyia) aegypti (L.) or Aedes (Stegomyia) albopictus (Skuse) larvae were reared in 1.6 L of live oak leaf (Quercus virginiana) infusion in the presence (5 mg/L) or absence (0 mg/L) of atrazine or glyphosate. The containers were monitored daily to determine the emergence rates, sex ratio, male and female emergence times, and female body size. Emergence rates of A. aegypti from atrazine treatment were significantly higher relative to either glyphosate or control treatments (A. aegypti: atrazine?=?93?±?6 % (±95 % CI), glyphosate?=?82?±?5 %, control?=?78?±?5 %), while emergence rates of A. albopictus in atrazine treatments were significantly higher than in glyphosate treatments but not in controls (A. albopictus: atrazine?=?84?±?5 %, glyphosate?=?76?±?4 %, control?=?78?±?4 %). For both mosquito species, a sex ratio distortion with male bias was observed in control and glyphosate treatments, but not in atrazine treatments (A. aegypti: atrazine?=?0.90?±?0.17 (±SE), glyphosate?=?1.63?±?0.21, control?=?1.69?±?0.26; A. albopictus: atrazine?=?1.09?±?0.08, glyphosate?=?1.88?±?0.12, control?=?1.37?±?0.11). Emergence times for both sexes of the two mosquito species were significantly longer in atrazine treatments compared to glyphosate or control treatments (A. aegypti: females: atrazine?=?11.20?±?0.50 (days?±?95 % CI), glyphosate?=?9.71?±?0.23, control?=?9.87?±?0.21; males: atrazine?=?9.46?±?0.27, glyphosate?=?8.80?±?0.25, control?=?8.85?±?0.24; A. albopictus: females: atrazine?=?17.40?±?1.70, glyphosate?=?12.4?±?0.40, control?=?12.5?±?0.30; males: atrazine?=?12.96?±?0.41, glyphosate?=?10.48?±?0.24, control?=?10.64?±?0.37). For A. albopictus but not A. aegypti, adult females from atrazine treatment had significantly longer wing lengths compared to those from glyphosate or control treatments (A. albopictus: atrazine?=?3.06?±?0.07 (mm?±?95 % CI), glyphosate?=?2.80?±?0.07, control?=?2.83?±?0.06). These results demonstrate the potential for atrazine, a widely used herbicide, to influence epidemiologically relevant life history traits of mosquitoes.     

Green synthesis of silver nanoparticles using Sida acuta (Malvaceae) leaf extract against Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti (Diptera: Culicidae)

Mosquitoes act as a vector for most of the life-threatening diseases like malaria, yellow fever, dengue fever, chikungunya fever, filariasis, encephalitis, West Nile Virus infection, etc. Under the Integrated Mosquito Management, emphasis was given on the application of alternative strategies in mosquito control. The continuous application of synthetic insecticides causes development of resistance in vector species, biological magnification of toxic substances through the food chain, and adverse effects on environmental quality and nontarget organisms including human health. Application of active toxic agents from plant extracts as an alternative mosquito control strategy was available from ancient times. These are nontoxic, easily available at affordable prices, biodegradable, and show broad-spectrum target-specific activities against different species of vector mosquitoes. In the present study, the larvicidal activity of silver nanoparticles (AgNPs) synthesized using Sida acuta plant leaf extract against late third instar larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti was determined. Range of concentrations of synthesized AgNPs (10, 20, 30, 40, and 50 μg/mL) and aqueous leaf extract (50, 100, 150, 200, and 250 μg/mL) were tested against the larvae of C. quinquefasciatus, A. stephensi and A. aegypti. The synthesized AgNPs from S. acuta leaf were highly toxic than crude leaf aqueous extract in three important vector mosquito species. The results were recorded from UV–Vis spectrum, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and energy-dispersive X-ray spectroscopy analysis. Larvae were exposed to varying concentrations of aqueous crude extract and synthesized AgNPs for 24 h. Considerable mortality was evident after the treatment of S. acuta for all three important vector mosquitoes. The LC₅₀ and LC₉₀ values of S. acuta aqueous leaf extract appeared to be most effective against A. stephensi (LC₅₀, 109.94 μg/mL and LC₉₀, 202.42 μg/mL) followed by A. aegypti LC₅₀ (119.32 μg/mL and LC₉₀, 213.84 μg/mL) and C. quinquefasciatus (LC₅₀, 130.30 μg/mL and LC₉₀, 228.20 μg/mL). Synthesized AgNPs against the vector mosquitoes of A. stephensi, A. aegypti, and C. quinquefasciatus had the following LC₅₀ and LC₉₀ values: A. stephensi had LC₅₀ and LC₉₀ values of 21.92, and 41.07 μg/mL; A. aegypti had LC₅₀ and LC₉₀ values of 23.96, and 44.05 μg/mL; C. quinquefasciatus had LC₅₀ and LC₉₀ values of 26.13 and 47.52 μg/mL. These results suggest that the use of S. acuta synthesized silver nanoparticles can be a rapid, environmentally safer biopesticide which can form a novel approach to develop effective biocides for controlling the target vector mosquitoes. This is the first report on the mosquito larvicidal activity of the plant aqueous extract and synthesized nanoparticles.     

Larvicidal,ovicidal, and adulticidal efficacy of Erythrina indica (Lam.) (Family: Fabaceae) against Anopheles stephensi,Aedes aegypti,and Culex quinquefasciatus (Diptera: Culicidae)

Mosquitoes are the major vector for the transmission of malaria, dengue fever, yellow fever, filariasis, schistosomiasis, and Japanese encephalitis. Mosquito control is facing a threat because of the emergence of resistance to synthetic insecticides. Insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. In view of the recently increased interest in developing plant origin insecticides as an alternative to chemical insecticide, this study was undertaken to assess the larvicidal, ovicidal, and adulticidal potential of the crude hexane, benzene, chloroform, ethyl acetate, and methanol solvent extracts from the medicinal plant Erythrina indica against the medically important mosquito vectors, Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus (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 leaf of E. indica against the larvae of A. stephensi, A. aegypti, and C. quinquefasciatus with the LC₅₀ and LC₉₀ values of 69.43, 75.13, and 91.41 ppm and 125.49, 134.31, and 167.14 ppm, respectively. The mean percent hatchability of the eggs was observed after 48 h post treatment. The percent hatchability was inversely proportional to the concentration of extract and directly proportional to the eggs. All the five solvent extracts showed moderate ovicidal activity; however, the methanol extract showed the highest ovicidal activity. The methanol extract of E. indica against A. stephensi, A. aegypti, and C. quinquefasciatus exerted 100 % mortality (zero hatchability) at 150, 200, and 250 ppm, respectively. Control eggs showed above 99.3–100 % hatchability. The adult mortality was observed after 24 h recovery period. The plant crude extracts showed dose-dependent mortality. At higher concentrations, the adult showed restless movement for some times with abnormal wagging and then died. Among the extracts tested, the highest adulticidal activity was observed in methanol extract against A. stephensi followed by A. aegypti and C. quinquefasciatus with the LD₅₀ and LD₉₀ values of 88.76, 94.09, and 119.64 ppm and 160.83, 169.01, and 219.77 ppm, respectively. No mortality was recorded in the control. Our data suggest that the crude hexane, benzene, chloroform, ethyl acetate, and methanol solvent extracts of E. indica have the potential to be used as an eco-friendly approach for the control of the A. stephensi, A. aegypti, and C. quinquefasciatus.     

Optimization and synthesis of silver nanoparticles using Isaria fumosorosea against human vector mosquitoes

The efficacy of silver generated larvicide with the help of entomopathogenic fungi, Isaria fumosorosea (Ifr) against major vector mosquitoes Culex quinquefasciatus and Aedes aegypti. The Ifr-silver nanoparticles (AgNPs) were characterized structurally and functionally using UV-visible spectrophotometer followed by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy and Fourier transform infrared (FTIR) spectra. The optimum pH (alkaline), temperature (30 °C) and agitation (150 rpm) for AgNP synthesis and its stability were confirmed through colour change. Ae. aegypti larvae (I–IV instars) were found highly susceptible to synthesized AgNPs than the larvae of Cx. quinquefasciatus. However, the mortality rate was indirectly proportional to the larval instar and the concentration. The lethal concentration that kills 50 % of the exposed larvae (LC₅₀) and lethal concentration that kills 90 % of the exposed larvae (LC₉₀) values of the tested concentration are 0.240, 0 0.075.337, 0.430, 0.652 and 1.219, 2.210, 2.453, 2.916; 0.065, 0.075, 0.098, 0.137 and 0.558, 0.709, 0.949, 1.278 ppm with respect to 0.03 to 1.00 ppm of Ifr-AgNPs against first, second, third and fourth instars of Cx. quinquefasciatus and Ae. aegypti, respectively. This is the first report for synthesis of AgNPs using Ifr against human vector mosquitoes. Hence, Ifr-AgNPs would be significantly used as a potent mosquito larvicide.     

Mosquito larvicidal properties of silver nanoparticles synthesized using Heliotropium indicum (Boraginaceae) against Aedes aegypti,Anopheles stephensi,and Culex quinquefasciatus (Diptera: Culicidae)

Mosquitoes transmit dreadful diseases to human beings wherein biological control of these vectors using plant-derived molecules would be an alternative to reduce mosquito population. In the present study activity of aqueous leaf extract and silver nanoparticles (AgNPs) synthesized using Helitropium indicum plant leaves against late third instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. The range of varying concentrations of synthesized AgNPs (8, 16, 24, 32, and 40 μg/mL) and aqueous leaf extract (30, 60, 90, 120, and 150 μg/mL) were tested against the larvae of Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. The synthesized AgNPs from H. indicum were highly toxic than crude leaf aqueous extract in three important vector mosquito species. The results were recorded from UV–Vis spectrum, Fourier transform infrared spectroscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy analysis, transmission electron microscopy, and histogram. The synthesized AgNPs showed larvicidal effects after 24 h of exposure. Considerable mortality was evident after the treatment of H. indicum for all three important vector mosquitoes. The LC₅₀ and LC₉₀ values of H. indicum aqueous leaf extract appeared to be effective against A. stephensi (LC₅₀, 68.73 μg/mL; LC₉₀, 121.07 μg/mL) followed by A. aegypti (LC₅₀, 72.72 μg/mL; LC₉₀, 126.86 μg/mL) and C. quinquefasciatus (LC₅₀, 78.74 μg/mL; LC₉₀, 134.39 μg/mL). Synthesized AgNPs against the vector mosquitoes of A. stephensi, A. aegypti, and C. quinquefasciatus had the following LC₅₀ and LC₉₀ values: A. stephensi had LC₅₀ and LC₉₀ values of 18.40 and 32.45 μg/mL, A. aegypti had LC₅₀ and LC₉₀ values of 20.10 and 35.97 μg/mL, and C. quinquefasciatus had LC₅₀ and LC₉₀ values of 21.84 and 38.10 μg/mL. No mortality was observed in the control. These results suggest that the leaf aqueous extracts of H. indicum and green synthesis of silver nanoparticles have the potential to be used as an ideal ecofriendly approach for the control of A. stephensi, A. aegypti, and C. quinquefasciatus. This is the first report on the mosquito larvicidal activity of the plant extracts and synthesized nanoparticles.     

Low-cost and eco-friendly green synthesis of silver nanoparticles using Feronia elephantum (Rutaceae) against Culex quinquefasciatus,Anopheles stephensi,and Aedes aegypti (Diptera: Culicidae)

Mosquitoes transmit serious human diseases, causing millions of deaths every year. The 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, the larvicidal activity of silver nanoparticles (AgNPs) synthesized using Feronia elephantum plant leaf extract against late third-instar larvae of Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus was determined. The range of concentrations of synthesized AgNPs (5, 10, 15, 20, and 25 μg mL^?1) and aqueous leaf extract (25, 50, 75, 100, and 125 μg mL^?1) were tested against the larvae of A. stephensi, A. aegypti, and C. quinquefasciatus. Larvae were exposed to varying concentrations of aqueous crude extract and synthesized AgNPs for 24 h. Considerable mortality was evident after the treatment of F. elephantum for all three important vector mosquitoes. The synthesized AgNPs from F. elephantum were highly toxic than crude leaf aqueous extract to three important vector mosquito species. The results were recorded from UV–visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy analysis (EDX). Synthesized AgNPs against the vector mosquitoes A. stephensi, A. aegypti, and C. quinquefasciatus had the following LC₅₀ and LC₉₀ values: A. stephensi had LC₅₀ and LC₉₀ values of 11.56 and 20.56 μg mL^?1; A. aegypti had LC₅₀ and LC₉₀ values of 13.13 and 23.12 μg mL^?1; and C. quinquefasciatus had LC₅₀ and LC₉₀ values of 14.19 and 24.30 μg mL^?1. No mortality was observed in the control. These results suggest that the green synthesis of silver nanoparticles using F. elephantum has the potential to be used as an ideal eco-friendly approach for the control of A. stephensi, A. aegypti, and C. quinquefasciatus. This is the first report on the mosquito larvicidal activity of the plant extracts and synthesized nanoparticles.     

Biosynthesized silver nanoparticles from Pedilanthus tithymaloides leaf extract with anti-developmental activity against larval instars of Aedes aegypti L. (Diptera; Culicidae)

Mosquitoes transmit dreadful diseases to human beings wherein biological control of these vectors using plant-derived molecules would be an alternative to reduce mosquito population. Aqueous leaf extract and green synthesized silver nanoparticles (Ag NPs) from Pedilanthus tithymaloides (L.) Poit. were investigated for their efficacy against the dengue vector Aedes aegypti L. (Diptera; Culicidae). The biologically synthesized Ag NPs were characterized by UV–vis spectrum, X-ray diffraction, Fourier transform infrared, and surface characteristics by atomic force microscopy. Further, on exposure of the larvae to varying concentrations of aqueous leaf and Ag NPs for 24 h, these Ag NPs showed 100 % mortality from first to fourth instars and pupae of A. aegypti at 0.25 %, which is the highest concentration, tested, wherein it was the lowest concentration of aqueous leaf extract alone which showed only 10–18 % of mortality. Lethal concentration (LC₅₀) values of Ag NPs against the larval and pupal stages were 0.029, 0.027, 0.047, 0.086, and 0.018 % with no mortality in control. These results suggest that the use of P. tithymaloides silver nanoparticles can be a rapid, environmentally safer bio-pesticide which can form a novel approach to develop effective biocides for controlling the target vector.     

Chemical composition and larvicidal activity of plant extracts from Clausena dentata (Willd) (Rutaceae) against dengue,malaria, and filariasis vectors

Mosquitoes in the larval stage are attractive targets for pesticides because mosquitoes breed in water, and thus, it is easy to deal with them in this habitat. The use of conventional pesticides in the water sources, however, introduces many risks to people and/or the environment. Natural pesticides, especially those derived from plants, are more promising in this aspect. Aromatic plants and their essential oils are very important sources of many compounds that are used in different respects. Insecticides of botanical origin may serve as suitable alternative to chemical insecticides. Acetone, chloroform, ethyl acetate, methanol, and petroleum benzine leaf extracts of Clausena dentata were tested against the fourth instar larvae of Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti (Diptera: Culicidae). Larval mortality was observed after 24 h of exposure. The highest larval mortality was found in acetone leaf extract, C. quinquefasciatus (LC₅₀?=?0.150278 mg/ml; LC₉₀?=?7.302613 mg/ml), A. aegypti (LC₅₀?=?0.169495 mg/ml; LC₉₀?=?1.10034 mg/ml), and A. stephensi (LC₅₀?=?0.045684 mg/ml; LC90?=?0.045684 mg/ml). GC–MS analysis of plant extracts of acetone solvent revealed 16 compounds, of which the major compounds were benzene,1,2,3-trimethoxy-5-(2-propenyl) (14.97 %), Z,Z-6,28-heptatriactontadien-2-one (6.81 %), 2-allyl-4-methylphenol (28.14 %), 2-allyl-4-methylphenol (17.34 %), and 2,6,10,14,18,22-tetracosahexaene, 2,6,10,15,19,23-hexamethyl (10.35 %). Our result shows acetone leaf extracts of C. dentata have the potential to be used as an ideal eco-friendly approach for mosquito control.     

Strong larvicidal potential of Artemisia annua leaf extract against malaria (Anopheles stephensi Liston) and dengue (Aedes aegypti L.) vectors and bioassay-driven isolation of the marker compounds

Malaria and dengue are the two most important vector-borne human diseases caused by mosquito vectors Anopheles stephensi and Aedes aegypti, respectively. Of the various strategies adopted for eliminating these diseases, controlling of vectors through herbs has been reckoned as one of the important measures for preventing their resurgence. Artemisia annua leaf chloroform extract when tried against larvae of A. stephensi and A. aegypti has shown a strong larvicidal activity against both of these vectors, their respective LC50 and LC90 values being 0.84 and 4.91 ppm for A. stephensi and 0.67 and 5.84 ppm for A. aegypti. The crude extract when separated through column chromatography using petroleum ether-ethyl acetate gradient (0–100 %) yielded 76 fractions which were pooled into three different active fractions A, B and C on the basis of same or nearly similar R _f values. The aforesaid pooled fractions when assayed against the larvae of A. stephensi too reported a strong larvicidal activity. The respective marker compound purified from the individual fractions A, B and C, were Artemisinin, Arteannuin B and Artemisinic acid, as confirmed and characterized through FT-IR and NMR. This is our first report of strong mortality of A. annua leaf chloroform extract against vectors of two deadly diseases. This technology can be scaled up for commercial exploitation.     

Biological control of the tick Ixodes ricinus with entomopathogenic fungi and nematodes: Preliminary results from laboratory experiments

In Central Europe, Lyme disease and tick-borne encephalitis (TBE) are transmitted by the tick Ixodes ricinus. In the last years, the prevalence of TBE virus in I. ricinus and also the number of human TBE infections have increased considerably in south-western Germany. A similar development is presumed for Borrelia burgdorferi s.l. For biocontrol of the vector I. ricinus, several options are presently evaluated. Among potential biocontrol agents, entomopathogenic fungi, nematodes, and parasitic wasps are the most promising candidates. In first laboratory experiments, different strains of the fungi Metarhizium anisopliae (five strains), Beauveria bassiana (three strains), and Paecilomyces fumosoroseus (two strains) were tested by placing I. ricinus larvae (fed and unfed) and nymphs (fed and unfed) in Petri dishes with filter paper soaked with conidial or blastospore suspensions (2.4×10³ to 9.6×10⁶ per cm²). Ticks were exposed to the different fungi for at least 35 days in the dark at temperatures of 21–24 °C. The best LT₅₀ values for unfed larvae (14 days) and unfed nymphs (21 days) were obtained with blastospores of M. anisopliae 97 at 1.4×10⁶ per cm². In order to evaluate the potential of entomopathogenic nematodes, the efficacies of three different species, Steinernema carpocapsae, S. feltiae, and Heterorhabditis bacteriophora, were investigated in the laboratory. I. ricinus nymphs (fed and unfed) and unfed females were placed in Petri dishes containing a wet filter paper with 300 and 600 infective nematode larvae per cm². Ticks were exposed to the nematodes for at least 15 days in the dark at an average temperature of 25 °C. The highest mortality was observed in unfed female ticks treated with S. carpocapsae. Results show that among the fungi tested the most promising control efficacy was obtained with two isolates of M. anisopliae, whereas S. carpocapsae was most successful among the nematodes tested against I. ricinus.     

Trypsin inhibitor from Moringa oleifera flowers interferes with survival and development of Aedes aegypti larvae and kills bacteria inhabitant of larvae midgut

Moringa oleifera flower extract, with trypsin inhibitor activity, is a larvicidal agent on Aedes aegypti. This work reports the isolation of trypsin inhibitor (M. oleifera flower trypsin inhibitor (MoFTI)) and its effect on A. aegypti egg hatching, viability of newly hatched larvae, survival of pupae, and growth of inhabitant bacteria from midgut of fourth-instar larvae (L₄). MoFTI (K _i, 2.4 μM), isolated by affinity chromatography on trypsin–agarose column, was an 18.2 kDa polypeptide on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Flower extract (at concentrations of 8.5–17.0 mg/mL) reduced egg hatchability while MoFTI (0.05–0.5 mg/mL) did not affect the hatching rate. Mortality of newly hatched larvae ranged from 3.5 to 19.1 % in the presence of the extract (4.0–17.0 mg/mL) and was also promoted by MoFTI (LC₅₀, 0.3 mg/mL). After 72 h, larvae incubated with extract at 13.0 and 17.0 mg/mL were at stages L₂ and L₁, respectively, while in control they reached L₃ instar. In the presence of MoFTI, at all concentrations tested, the larvae did not pass the first instar. Flower extract and MoFTI did not interfere on pupae survival. The extract and MoFTI inhibited the growth of L₄ gut bacteria (minimum inhibitory concentrations of 3.47 and 0.031 mg/mL, respectively) but only the inhibitor showed bactericide effect (minimum bactericidal concentration of 1.0 mg/mL). The findings reported herein indicate that MoFTI constitutes a larvicidal principle from M. oleifera flowers against A. aegypti newly hatched larvae and is an antibacterial agent active against the microbiota from L₄ gut.     

Larvicidal potential of silver nanoparticles synthesized from Leucas aspera leaf extracts against dengue vector Aedes aegypti

Vector-borne diseases caused by mosquitoes are one of the major economic and health problems in many countries. Aedes aegypti mosquito is a vector of several diseases in humans like yellow fever and dengue. Vector control methods involving use of chemical insecticides are becoming less effective due to the development of insecticides resistance, biological magnification of toxic substances through the food chain, and adverse effects on environmental quality and nontarget organisms including human health. Application of active toxic agents from plant extracts as an alternative mosquito control strategy was available from ancient times. These are nontoxic, easily available at affordable prices, biodegradable, and show broad-spectrum target-specific activities against different species of vector mosquitoes. Today, nanotechnology is a promising research domain which has a wide ranging application in vector control programs. The present study investigates the larvicidal potential of solvent leaf extracts of Leucas aspera and synthesized silver nanoparticles using aqueous leaf extract against fourth instar larvae of A. aegypti. Larvae were exposed to varying concentrations of plant extracts and synthesized AgNPs for 24 h. The results were recorded from UV–Vis spectra, X-ray diffraction (XRD), Fourier transform infrared (FTIR), and scanning electron microscopy (SEM), and were used to characterize and support the biosynthesis of silver nanoparticles. The formation of the synthesized AgNPs from the XRD spectrum compared with Bragg reflections can be indexed to the (111) orientations, respectively, confirmed the presence of AgNPs. The FTIR spectra of AgNPs exhibited prominent peaks at 3,447.77, 2,923.30, and 1,618.66 cm^?1. The spectra showed sharp and strong absorption band at 1,618.66 cm^?1 assigned to the stretching vibration of (NH) C═O group. The band 1,383 developed for C═C and C═N stretching, respectively, and was commonly found in the proteins. SEM analysis of the synthesized AgNPs clearly showed the clustered and irregular shapes, mostly aggregated and having the size of 25–80 nm. Energy-dispersive X-ray spectroscopy (EDX) showed the complete chemical composition of the synthesized AgNPs. In larvicidal activity, the results showed that the maximum efficacy was observed in synthesized AgNP from leaf extracts against the fourth instar larvae of A. aegypti with LC₅₀ values of 8.5632, 10.0361, 14.4689, 13.4579, 17.4108, and 27.4936 mg/l and LC₉₀ values of 21.5685, 93.03928, 39.6485, 42.2029, 31.3009, and 53.2576 mg/l respectively. These results suggest that the synthesized AgNP from leaf extracts have a higher larvicidal potential as compared to crude solvent extracts thus making them an effective combination for controlling A. aegypti.     

Larvicidal potential of silver nanoparticles synthesized from Leucas aspera leaf extracts against dengue vector Aedes aegypti

Vector-borne diseases caused by mosquitoes are one of the major economic and health problems in many countries. The Aedes aegypti mosquito is a vector of several diseases in humans like yellow fever and dengue. Vector control methods involving the use of chemical insecticides are becoming less effective due to development of insecticides resistance, biological magnification of toxic substances through the food chain, and adverse effects on environmental quality and non-target organisms including human health. Application of active toxic agents from plant extracts as an alternative mosquito control strategy was available from ancient times. These are nontoxic, easily available at affordable prices, biodegradable, and show broad-spectrum target-specific activities against different species of vector mosquitoes. Today, nanotechnology is a promising research domain which has wide-ranging application vector control programs. The present study investigates the larvicidal potential of solvent leaf extracts of Leucas aspera and synthesized silver nanoparticles using aqueous leaf extract against fourth instar larvae of Aedes aegypti. Larvae were exposed to varying concentrations of plant extracts and synthesized AgNPs for 24 h. The results were recorded from UV–Vis spectra, x-ray diffraction (XRD), Fourier transform infrared (FT-IR), and scanning electron microscopy (SEM), and were used to characterize and support the biosynthesis of silver nanoparticles. The formation of the AgNPs synthesized from the XRD spectrum compared with Bragg reflections can be indexed to the (111) orientations, respectively, confirmed the presence of AgNPs. The FT-IR spectra of AgNPs exhibited prominent peaks at 3,447.77; 2,923.30; and 1,618.66 cm^?1. The spectra showed sharp and strong absorption band at 1,618.66 cm^?1 assigned to the stretching vibration of (NH) C═O group. The band 1,383 developed for C═C and C═N stretching, respectively, and was commonly found in the proteins. SEM analysis of the synthesized AgNPs clearly showed the clustered and irregular shapes, mostly aggregated, and having the size of 25–80 nm. Energy-dispersive x-ray spectroscopy showed the complete chemical composition of the synthesized AgNPs. In larvicidal activity, the results showed that the maximum efficacy was observed in synthesized AgNPs leaf extracts against the fourth instar larvae of A. aegypti (LC₅₀ values of 8.5632, 10.0361, 14.4689, 13.4579, 17.4108, and 27.4936 mg/l) and (LC₉₀ values of 21.5685, 93.03928, 39.6485, 42.2029, 31.3009, and 53.2576 mg/l), respectively. These results suggest that the synthesized AgNPs leaf extracts have a higher larvicidal potential as compared to crude solvent extracts thus making them an effective combination for controlling A. aegypti.     

Mosquito attractant blends to trap host seeking Aedes aegypti

Aedes aegypti is the key vector of three important arboviral diseases -dengue, yellow fever and chikungunya. To identify volatile chemicals which could be used in odour based traps for Aedes mosquito surveillance, a few synthetic compounds and compound blends have been evaluated in an indigenously designed olfactometer. A total of 24 compounds and seven compound blends were screened against unfed adult female Ae. aegypti mosquitoes for attraction and compared with control group. The attractancy or repellency index of the test material to mosquitoes was calculated and rated them as class-1, class-2 and class-3 with rating values ranging 1–15, 16–33 and 34–100 respectively. Out of the 24 compounds tested, six were showing significant attractancy (P?<?0.05) and among that 1-octene-3-ol showed maximum attractancy with a rating value of 57.81. Sixteen compounds showed significant repellency (P?<?0.05) and among that with a rating value of 72.47, 1-hexene-3-ol showed strong repellent action against Ae. aegypti. All the seven blends showed significant mosquito attractancy (P?<?0.05) and among that with a rating of 62.08 Myristic acid, Lactic acid and CO₂ blend exhibited first-rate mosquito attractancy.     

Repellency of essential oils extracted from Thai native plants against Aedes aegypti (Linn.) and Culex quinquefasciatus (Say)

Repellent activity of essential oils derived from 10 Thai native plants, belonging to three families were evaluated against female Aedes aegypti and Culex quinquefasciatus and to compare them with a commercial chemical repellents (DEET; N,N-diethyl-3-methylbenzamide 20 % w/w; Sketolene Shield®). Each test repellent was applied at 1, 5, and 10 % concentrations for testing by arm in cage method. The results showed significant differences in repellency among the repellents by mosquito species. The protection time of the essential oils against Ae. aegypti ranged from 3 to 30 min. According to the Culex mosquito, it showed the protection time ranged from 3 to 260 min. 10 % Boesenbergia rotunda essential oil provided the best efficiency, in which protection time was 4.3 h as equal as DEET. The essential oils which exhibited protection time more than 2 h were those of 10 % Zingiber zerumbet, Litsea petiolata, Curcuma zedoaria, and Zingiber cassumunar essential oils (3.1, 2.8, 2.6, and 2.3 h, respectively). The biting percentage ranged from 0.9 to 18.0 % and 0.8 to 3.6 % against Ae. aegypti and Cx. quinquefasciatus, respectively. The results revealed that the potential of essential oil extracted from B. rotunda, Z. zerumbet, L. petiolata, C. zedoaria, and Z. cassumunar had attributes of good repellent and deterred biting. We recommend the five essential oils for further study to develop as commercial repellents.     

Larvicidal, oviposition, and ovicidal effects of Artemisia annua (Asterales: Asteraceae) against Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus (Diptera: Culicidae)

This study focuses on the larvicidal, oviposition, and ovicidal effects of a crude extract of Artemisia annua against Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus. Dried cells of Artemisia annua from cell suspension cultures were extracted using hexane. The extract showed moderate larvicidal effects against mosquitoes. At 24-h post treatment, the LC₅₀ values for Anopheles sinensis, Aedes aegypti, and Culex quinquefasciatus were recorded as 244.55, 276.14, and 374.99 ppm, respectively. The percentage mortality of larvae was directly proportional to the tested concentration. Anopheles sinensis was found to be the most susceptible species, whereas Culex quinquefasciatus was the most tolerant to the Artemisia annua extract. The results indicated that the Artemisia annua extract showed concentration-dependent oviposition deterrent activity and had a strong deterrent effect. At 500 ppm, the percentage effective repellency was more than 85 % compared with the control group for all the species, with oviposition activity index values of ?0.94, ?0.95, and ?0.78 for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. In the ovicidal assay, the percentage hatchability of eggs after treatment with 500 ppm of Artemisia annua extract was significantly lower than the control, with values of 48.84?±?4.08, 38.42?±?3.67, and 79.35?±?2.09 % for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. Artemisia annua was found to be more effective against Aedes aegypti and Anopheles sinensis compared with Culex quinquefasciatus. This study indicated that crude extract of A. annua could be a potential alternative for use in vector management programs.     

Are fish paratenic natural hosts of the caiman haemoparasite Hepatozoon caimani?

The susceptibility of two fish and four mosquito species to the Caiman yacare haemoparasite Hepatozoon caimani was experimentally investigated. Mosquitoes belonging to four species (Aedes fluviatilis, Aedes albopictus, Aedes aegypti and Culex quinquefasciatus) were blood-fed on two naturally infected C. yacare from the Central-West Region of Brazil that exhibited distinct levels of parasitaemia: caimans A (11.05 %) and B (1.25 %). None of the engorged A. fluviatilis, A. albopictus or A. aegypti mosquitoes fed on caiman A survived for the duration of the sporogonic cycle; the great majority of the engorged mosquitoes died within 48 h of the blood meal. All A. aegypti fed on caiman B were negative, whereas 91.3 % of dissected C. quinquefasciatus fed on the same caiman contained oocysts. Characid fish—Metynnis sp. and Astyanax sp.—were individually fed with C. quinquefasciatus females previously engorged (21–23 days) on caiman B. No parasite was found in the Astyanax fish. By contrast, 100 % of the Metynnis fish depicted numerous cysts harbouring cystozoites identical to those of H. caimani, even more than 8 months after the ingestion of the infected mosquitoes. The cysts were located near the veins of the liver and, in some cases, close to the tunica intima of these vessels. No inflammatory reaction was observed. Gametocytes were observed in the blood smears of juvenile caimans that had ingested infected fish 9–12 weeks earlier. The potential role of fish as paratenic vertebrate hosts of H. caimani in nature is discussed.