Evaluation of Moringa oleifera seed lectin in traps for the capture of Aedes aegypti eggs and adults under semi-field conditions

The water-soluble lectin isolated from Moringa oleifera seeds (WSMoL) is a larvicidal, ovicidal, and oviposition-stimulating agent against Aedes aegypti under laboratory conditions. This study investigated the effect of WSMoL in traps for the capture of A. aegypti eggs and adult females under semi-field conditions and determined whether gravid females could detect WSMoL by an olfactory response. WSMoL was isolated according to a previously described procedure using chitin chromatography. The bioassays were performed in large cages (12.5 m³). Two traps for collection of eggs (ovitrap) or adult mosquitoes (MosquiTRAP^TM) were placed in a cage. One was filled with WSMoL (0.1 mg/mL) and the other with tap water (negative control). An infusion of Panicum maximum leaves was used as a positive control. Forty gravid females were then released in each cage. After 2 (for oviposition) or 3 h (for female capture), the traps were removed, and the number of eggs or females was counted. An olfactometry assay was performed to investigate whether the effect of WSMoL on gravid females was linked to an olfactory response. WSMoL showed an oviposition-stimulating effect (65?±?14 %) that was similar (p?<?0.05) to that promoted by the P. maximum infusion (67?±?11 %). The efficiency of MosquiTRAP^TM in capturing gravid females was not increased by WSMoL. The olfactometry assay indicated that the response of females to WSMoL did not involve the stimulation of olfactory sensilla. WSMoL effectively captured eggs when used in ovitraps under semi-field conditions; this property, together with the ovicidal and larvicidal activities of this lectin, makes it an interesting candidate for A. aegypti control.     

Evaluation of novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae)

Insecticides are one of the major tools for controlling vector populations and for reducing the transmission of human pathogens. However, there are few new insecticides being developed and marketed for vector control. Herein, we report on the toxicity of six novel insecticides to both adult and larval Aedes aegypti (L). and the toxicity of three novel insect growth regulators (IGRs) to larvae. Four insecticides were highly or moderately toxic to larvae with LC50 values of 16 (chlorfenapyr), 70 (hydramethylnon), 79 (indoxacarb), and 84 ng/ml (imidacloprid). Diafenthiuron and chlorfenapyr were moderately toxic to adult mosquitoes with LC50 values of 13 and 92 ng/cm2, respectively. Imidacloprid was strongly synergized by piperonyl butoxide (PBO) in Ae. aegypti adults, suggesting that neonicotinoids are intrinsically very toxic to adult mosquitoes (in the absence of detoxification). The effect of PBO on the toxicity in adults and larvae was considerably different, both in terms of the insecticides that were synergized (or antagonized for chlorfenapyr versus adults) and in terms of the degree of synergism. This result implies that the cytochrome P450s involved in metabolism of these insecticides are different between adults and larvae. Pyriproxyfen was confirmed as a potent IGR (EC50 of 0.0017 ng/ml) for mosquitoes, although tebufenozide lacked activity. The potential for use of these materials in mosquito control is discussed.     

Toxicity of Brazilian plant seed extracts to two strains of Aedes aegypti (Diptera: Culicidae) and nontarget animals

Seed ethanolic extracts of 21 Brazilian plants were evaluated for ovicidal, larvicidal, and pupicidal activities against insecticide-susceptible (SS) and field-collected (FC) strains of Aedes aegypti (L.) (Diptera: Culicidae), as well as for their effects on nontarget organisms. Myracrodruon urundeuva Fr. Allemao extract was highly toxic to both mosquito strains. Schinopsis brasiliensis Engler extract showed low toxicity and was 38-68 times less toxic to Ae. aegypti larvae than was M. urundeuva extract. The pupicidal activity (LC50) of 14 plant seed extracts ranged between 9 and 433/g/ml, and toxicities were comparable to both mosquito strains. Piptadenia moniliformis Benth. and Luetzelburgia auriculata (Allemao) Ducke extracts showed the highest activities against pupae of FC and SS strains. None of the extracts showed 100% ovicidal activity. In addition, the active extracts did not show high acute toxicity to mice (LD50 > 1.5 g/kg), except that of Enterolobium contortisiliquum (Vell.) Morong. Most of the active extracts exhibited low toxicity against brine shrimp (Artemia sp.) nauplii. The extracts of M. urundeuva, P. moniliformis, and L. auriculata are promising sources of recognized classes of insecticidal compounds with good selectivity against immature stages of Ae. aegypti.     

Transduction of Aedes aegypti mosquitoes with vectors derived from Aedes densovirus

Aedes densovirus (AeDNV)-based constructs that express green fluorescent protein (GFP) from either the P7 or the P61 promoter were made. The construct in which GFP protein was expressed as a fusion protein to the C-terminus of NS1 (NS1-GFP) showed the highest level of GFP expression. This hybrid NS1-GFP protein preserved the biological functions of the parental proteins: it showed GFP fluorescence, it stimulated expression from the virus promoters, and it facilitated rescue and replication of the cloned AeDNV genome. Similar to NS1, the hybrid NS1-GFP localized in the nucleus predominantly in a punctate pattern. Transducing virus particles carrying the NS1-GFP gene infected mosquito larvae. Expression of GFP was detected as early as 48 h postinfection and in larval and pupal stages. Midgut, hindgut, and Malpighian tubule cells expressed GFP soon after transduction. However, the anal papillae were the most commonly infected organ system. The anal papillae are syncytia and regulate ion concentration in the hemolymph of mosquito larvae, and they might be a novel route of mosquito larvae infection with densoviruses.     

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.     

Molecular and morphological characterization of Aedes albopictus in northwestern Greece and differentiation from Aedes cretinus and Aedes aegypti

The presence of Aedes albopictus (Skuse) was recently confirmed for the first time in northwestern Greece. This location is within the distribution range of a morphologically similar species, Aedes cretinus Edwards, and is a potentially favorable region for the reintroduction of Aedes aegypti (L.). It was thus compelling to use methods in addition to morphology-based keys to correctly identify specimens badly damaged, rubbed, or otherwise altered in their external characteristics. It was decided to use molecular techniques as a novel and reliable method for differentiating the three Stegomyia species. The nuclear internal transcribed spacer 2 (ITS2) fragments from morphologically identified Ae. albopictus and Ae. cretinus specimens were amplified, and their sequences were compared with those in GenBank for Ae. albopictus, Ae. cretinus, and Ae. aegypti. Also, mitochondrial cytochrome oxidase I (COI) fragments were amplified for Ae. albopictus and Ae. cretinus (so far not available in GenBank) and compared with Ae. aegypti fragments. ITS2 and COI sequences generated in our study were deposited in GenBank and could be useful in future studies of mosquitoes by other research workers.     

Olfactometric evaluation of spatial repellents for Aedes aegypti

The spatial repellency responses of Aedes aegypti (L.) to deet, dehydrolinalool and linalool were evaluated using a dual port olfactometer. In the absence of human attractant mixture, each of the three chemicals resulted in activation and/or orientation of mosquitoes to the chemical source. Linalool was the most attractive compound. In the presence of human attractant mixture, activation and/or orientation of mosquitoes to each of the three chemicals was reduced. We compared reductions in mosquito responses to each of the three chemicals, in the presence of human attractant mixture, to estimate spatial repellency. As expected, lowest spatial repellency (7.3%) was observed using human attractant alone. Highest spatial repellency (33.6%) was observed using a combination of linalool and dehydrolinalool. Deet did not manifest spatial repellency, whereas linalool and dehydrolinalool alone, and in combination, exhibited spatial repellency.     

Assessment of combined tools and strategies for Aedes aegypti control with low environmental impact

The control of the mosquito vector Aedes aegypti L. (Diptera: Culicidae) is the main action against dengue, chikungunya, and Zika. The excessive use of conventional insecticides has promoted the development of other control methods and strategies with lower environmental impact. We evaluated the effectiveness of applying triflumuron 1 ppm and emptying water-filled containers in a field trial in temperate Argentina. Both control methods were implemented either individually or combined and regularly from the beginning of the mosquito reproductive season or once it reached peak abundance. The impact on a non-target midge of the genus Chironomus was also tested. The highest reductions of Ae. aegypti were achieved in treatments which included triflumuron. This effect was stronger when applied from the beginning of the reproductive season, with < 1.3% of positive containers throughout the entire season. No enhancing effects were obtained when combining both control methods. Treatments with triflumuron were not completely innocuous for the non-target species, with Chironomus sp. more susceptible to treatments including triflumuron applied from the beginning of the reproductive season than all others. Sharp reductions of mosquito populations in urban environments with high density of water-filled containers are possible with minimum container management efforts, by applying triflumuron 1 ppm every 6 weeks. In temperate urban settings, better results can be obtained when applications begin early in the reproductive season of the mosquito vector Aedes aegypti.

     

Blood feeding patterns of Aedes aegypti and Aedes albopictus in Thailand

Aedes aegypti (L.) and Aedes albopictus (Skuse) were collected with aspirators from Mae Sot, Nakhon Sawan, Nakhon Ratchasima, Surat Thani, and Phatthalung study sites in Thailand from July 2003 though April 2004. The sandwich-B enzyme-linked immunosorbent assay was used to analyze 1,021 blood-fed specimens. Ae. aegypti almost exclusively fed on humans (99%, 658/664) in single host species, and 97% (86/88) of multiple-host bloodmeals included at least one human host. A low frequency of other hosts, including bovine, swine, cat, rat, and chicken were detected, but they represented <1% of bloodmeals. An even higher percentage of human feeding was detected in Ae. albopictus. Hosts of Ae. albopictus collected from sites in southern Thailand were entirely human (100%, n = 105) from both single and mixed meals. In the small number of double-host meals from Ae. albopictus, we detected 3.8% as swine-human and <1% from dog-human and cat-human. Forage ratios for Ae. aegypti indicated that human, dog, and swine were preferred hosts in order of preference. In contrast, bovine and chicken were avoided hosts for this species in Thailand.     

Larvicidal activity against Aedes aegypti of pacharin from Bauhinia acuruana

The aim of the present study was to evaluate the activity of pacharin isolated from the ethanol extract from roots of Bauhinia acuruana on third-instar larvae of Aedes aegypti Linn. (Diptera: Culicidae). The crude ethanol extract showed larvicidal activity at the concentration of 500 μg/mL. Given this larvicidal activity, this extract was submitted to chromatographic fractionation on a silica gel column eluted with n-hexane, dichloromethane, ethyl ether, ethyl acetate, and methanol in order to isolate the active compound(s). Pacharin, obtained in pure form from fraction eluted with ethyl ether, was evaluated for their larvicidal effects against A. aegypti. In these bioassays, the larvae were exposed at concentrations of 500, 250, 100, 50, and 25 μg/mL of the crude ethanol extract or pacharin. After 24 h, the number of dead larvae was counted and the LC₅₀ values for larval mortality were calculated. Pacharin showed LC₅₀ value of 78.9?±?1.8 μg/mL. The structure of isolated compound was identified on the basis of their spectral data (IR, 1D- and 2D-NMR) and by comparison with literature spectral data. The results indicate pacharin as a potential natural larvicide.     

Impact of Parthenium hysterophorus leaf extracts on the fecundity, fertility and behavioural response of Aedes aegypti L

Different extracts of 1,000 ppm were prepared from the leaves of Parthenium hysterophorus using acetone, benzene, petroleum ether, diethyl ether and hexane as the solvents. The efficacy of each extract was assessed against dengue fever vector, Aedes aegypti by evaluating the variations in fecundity, fertility and behavioural response of the female adults. The leaf extracts could cause 70–100% repellency in the oviposition behaviour of the adults. The diethyl ether extract was found to be the most effective extract resulting in maximum effective repellency (99.7%) leading to the highest levels of reduced fecundity and 100% egg mortality followed by benzene extracts causing 93.8% reduced oviposition and 100% ovicidal effect. Hexane and acetone extracts with the least oviposition deterrence of 70–74% and negligible egg mortality (8–9%) proved to be the least effective extracts. The petroleum ether extract had a moderate impact resulting in 93.2% diminished fecundity and 41% ovicidal effect. The behavioural response of female adults of A. aegypti was evaluated by performing spatial repellency and contact irritancy assays. The most significant spatial repellency behaviour was elicited by acetone extracts leading to escape of 80% mosquitoes. Hexane and diethyl ether extracts could cause moderate response with 50–60% escape, while a slight and no reaction was observed on exposure to petroleum ether and benzene extracts, respectively. An interesting observation was the knocked-down activity caused by the hexane extracts with no recovery even after 24 h. A significant contact irritancy response was noticed in the mosquitoes on exposure to acetone leaf extracts resulting in first flight only after 4 s and a total of 12 flights during exposure. No irritancy behaviour was observed on exposure to diethyl ether and benzene leaf extracts. However, as against controls, a slight irritability response was noticed on exposure to hexane leaf extracts resulting in relative irritability of 1.2. Our results suggest the selective efficiency of Parthenium leaf extracts against A. aegypti, as the most effective oviposition deterrent and ovicidal agent was least effective as irritant extract and vice-versa. Further detailed research is needed to identify the active ingredient in the extracts and implement the effective mosquito management programme.     

Evaluation of a sampling methodology for rapid assessment of Aedes aegypti infestation levels in Iquitos, Peru

An epidemic of dengue during 2001 in Northwestern Peru reemphasized the need for efficient, accurate, and economical vector surveillance. Between November 1998 and January 1999, we carried out extensive entomological surveys in two neighborhoods of approximately 600 contiguous houses located in the Amazonian city of Iquitos, providing a unique opportunity to evaluate the Aedes aegypti (L.) rapid assessment survey strategy. Based on Pan American Health Organization recommendations, this strategy is used by the Peruvian Ministry of Health (MOH). In our analysis all household locations, including closed and unoccupied houses, were georeferenced and displayed in a geographic information system, which facilitated simulations of MOH surveys based on hypothetical systematic sampling transects. Larval, pupal, and adult mosquito indices were calculated for each simulation (n = 10) and compared with the indices calculated from the complete data set (n = 4). The range of indices calculated from simulations was moderately high, but included actual indices. For example, simulation ranges for house indices (HI, percentage of infested houses from complete survey) were 38-56% (45%), 36-42% (38%), 21-34% (30%), and 13-33% (27%) in four surveys. HI, Breteau index, pupae per hectare, adult index, and adults per hectare were more robust entomological indicators (coefficient of variation [CV]/mean = 0.1-2.9) than the container index, pupae per person, pupae per house, adults per person, and adults per house (CV/mean >20). Our results demonstrate that the MOH's Ae. aegypti risk assessment program provides reasonable estimates of indices based on samples from every house. However, it is critical that future studies investigate the association of these indices with rates of virus transmission to determine whether sampling variability will negatively impact the application of indices in a public health context.     

Field evaluation of a lethal ovitrap for the control of Aedes aegypti (Diptera: Culicidae) in Thailand

In 1999 and 2000 we evaluated a lethal ovitrap (LO) for the control of Aedes aegypti (L.) in three villages in Ratchaburi Province, Thailand. Two blocks of 50 houses (a minimum of 250 m apart) served as treatment and control sites in each village, with each house in the treatment area receiving 10 LOs. Thirty houses in the center of each treatment and control block were selected as sampling sites, with larval and adult mosquito sampling initiated when LOs were placed. Sampling was conducted weekly in 10 of the 30 houses at each site, with each block of 10 houses sampled every third week. Sampling continued for 30 wk. Efficacy of the LO was evaluated by determining number of containers with larvae and/or pupae per house and number of adult mosquitoes collected inside each house. In 1999, the LO had a negligible impact on all measures of Ae. aegypti abundance that were assessed; however, fungal contamination of insecticide-impregnated strips may have been responsible for the low efficacy. In 2000, significant suppression was achieved based on changes in multiple entomologic criteria (containers with larvae, containers with pupae, and number of adult Ae. aegypti); however, control was not absolute and neither immature nor adult Ae. aegypti were ever eliminated completely. We conclude that the LO can reduce adult Ae. aegypti populations in Thailand; however, efficacy of the LO is lower than desired due primarily to the high number of alternative oviposition sites. LO efficacy may be improved when used as part of an integrated control program that places emphasis on reduction of adjacent larval habitats. Further studies are required to assess this issue.     

The role of hemagglutinins in the midgut extracts of two lines of Aedes aegypti in their susceptibility to Dengue-2 virus

Hemagglutinin activity (HA) was studied in the midgut extracts from highly (h) and lowly susceptible strains of Aedes aegypti mosquitoes to Dengue-2 virus (DEN-2). HA in the midgut extracts from these two isofemale strains of mosquitoes was high in as compared to (h) mosquitoes. HA was found to be higher with chicken red blood cells (RBCs) than with rabbit and human RBCs of O group. Larval midgut extracts showed higher activity than those from adult female mosquitoes. Exposure of midgut extracts to 100 degrees C for 10 mins destroyed the activity. The activity was observed between pH 6 and pH 10. HA in midgut extracts was also studied using twenty different carbohydrates; five of them showed an inhibition of HA. The inhibitory carbohydrates, when incorporated into DEN-2-infected bloodmeal, showed a reduction in the susceptibility of mosquitoes to the virus as compared to the control ones fed on the virus alone. Similarly, when these carbohydrates were incorporated in the DEN-2-infected inoculum, the inoculated mosquitoes showed a reduction in the susceptibility to the virus. HA in the virus-infected midgut extracts was higher than that in the uninfected controls. These results suggest that the presence of HA in the midgut may be one of the factors that affect the susceptibility of Ae. aegypti mosquitoes to DEN-2.     

Toxicity of herbal extracts used in ethno-veterinary medicine and green-encapsulated ZnO nanoparticles against Aedes aegypti and microbial pathogens

Dengue and chikungunya are arboviral diseases mainly vectored by the mosquito Aedes aegypti. Presently, there is no treatment for these viral diseases and their prevention is still based on vector control measures. Nanopesticides fabricated using herbal extracts as reducing and capping agents currently represent an excellent platform for pest control. In this scenario, the present study assessed the acute toxicity of seven plants employed in ethno-veterinary medicine of southern India, as well as the green synthesis of zinc oxide nanoparticles, on third-instar larvae of A. aegypti. Larvae were exposed to extracts of the seven plants obtained with solvents of different polarity (acetone, ethanol, petroleum ether, and water) for 24 h. Maximum efficacy was observed for Lobelia leschenaultiana leaf extracts prepared using all the four solvent extracts (LC₅₀ = 22.83, 28.12, 32.61, and 36.85 mg/L, respectively). Therefore, this plant species was used for the synthesis and stabilization of ZnO nanoparticles based on its maximum efficacy against third-instar larvae of A. aegypti. L. leschenaultiana-encapsulated ZnO nanoparticles showed 100% mortality when tested at 10 mg/L, the LC₅₀ was extremely low,  1.57 mg/L. Zinc acetate achieved only 65.33% when tested at 60 mg/L, with a LC₅₀ of 51.62 mg/L. Additionally, ZnO nanoparticles inhibited growth of Pseudomonas aeruginosa, Proteus vulgaris, Shigella sonnei, and Vibrio parahaemolyticus and also inhibited biofilm formation on selected microbila pathogens, showing impact on EPS production and hydrophobicity. Overall, our results suggest that L. leschenaultiana-fabricated ZnO nanoparticles have a significant potential to control A. aegypti mosquitoes and Gram-negative bacterial pathogens.