Co-expression of Bacillus thuringiensis Cry4Ba and Cyt2Aa2 in Escherichia coli revealed high synergism against Aedes aegypti and Culex quinquefasciatus larvae

Cry4Ba is a delta-endotoxin produced by Bacillus thuringiensis subsp. israelensis and Cyt2Aa2 is a cytolytic delta-endotoxin produced by B. thuringiensis subsp. darmstadiensis. Cry4Ba produced in Escherichia coli was toxic to Aedes aegypti larvae (LC(50)=140 ng ml(-1)) but virtually inactive to Culex quinquefasciatus larvae. Cyt2Aa2 expressed in E. coli exhibited moderate activity against A. aegypti and C. quinquefasciatus larvae with LC(50) values of 350 and 250 ng ml(-1), respectively. Co-expression of both toxins in E. coli dramatically increased toxicity to both A. aegypti andC. quinquefasciatus larvae (LC(50)=7 and 20 ng ml(-1), respectively). This is the first report to demonstrate that Cry4Ba and Cyt2Aa2 have high synergistic activity against C. quinquefasciatus larvae.     

Screening of Brazilian Bacillus sphaericus strains for high toxicity against Culex quinquefasciatus and Aedes aegypti

Abstract:  In this work, 246 Bacillus sphaericus strains were evaluated against Aedes aegypti and Culex quinquefasciatus larvae to select the most effective ones to be used as the basis of a national product. All strains were isolated from different regions of Brazil and they are stored in a Bacillus spp. collection at Embrapa Genetic Resources and Biotechnology. The selected strains were characterized by biochemical and molecular methods. Based on selective bioassays, 87 strains were identified as toxic to one or both target species. All of these strains contain genes that encode the 42, 51 kDa proteins that constitute the binary toxin and the 100 kDa Mtx1 toxin. All toxic strains presented a very high LC₅₀ against A. aegypti , so, a product based on any of these B. sphaericus strains would not be recommended for use in programmes to control A. aegypti . S201 had highest activity against C. quinquefasciatus , presenting the lowest LC₅₀ and LC₉₀ in bioassays.     

Phytopesticidal and repellent efficacy of Litsea salicifolia (Lauraceae) against Aedes aegypti and Culex quinquefasciatus

Litsea salicifolia is one of the many plants used as phytopesticide, traditionally by various tribes of Assam. Of the five extracts of L. salicifolia tested for the bioactivity against Aedes aegypti and Culex quinquefasciatus, the aqueous extract was more effective compared to other extracts exhibiting bioactivity at 72 ppm against A. aegypti. The hexane extract (2000 ppm) exhibited 70% repellent activity for 3 hr against A. aegypti and 46% activity for 3 hr against C. quinquefasciatus. This is the first report on the insecticidal properties of L. salicifolia which can be further developed as an eco-friendly biopesticides of the future.     

Comparative delta-endotoxins of Bacillus thuringiensis against mosquito vectors (Aedes aegypti and Culex pipiens)

Pure crystals of seven Bacillus thuringiensis field isolates from the Lower Silesia region (Poland) were tested against larvae of Aedes aegypti L. and Culex pipiens L. (Culicidae, Diptera). The crystals of OpQ3 phylloplane isolate (belonging to the first biochemical type of B. thuringiensis subsp. japonensis, yoso, jinghongiensis) killed from 68 +/- 7% to 84 +/- 7% of the fourth instar larvae of A. aegypti. The crystals of two other strains (KpF3 and KpC1) of this group caused mortality between 3 +/- 2% and 70 +/- 7%. The LC50 ranged from 3.2 +/- 0.4 to 34.1 +/- 4.8 microg/ml. The effect of B. thulringiensis wratislaviensis H-47 crystals was the lowest with larval mortality from 0% to 17 +/- 3%. No significant (0%-37 +/- 6%) effect of B. thuringiensis crystals on the larvae of C. pipiens was observed. Our results show that the delta-endotoxins of B. thuringiensis act very specifically.     

Insecticidal and Repellent Activity of Siparuna guianensis Aubl. (Negramina) against Aedes aegypti and Culex quinquefasciatus

This study investigated the toxic effects of essential oils isolated from Siparuna guianensis against Aedes aegypti, Culex quinquefasciatus (eggs, larvae, pupae, and adult) and Aedes albopictus (C6/36) cells. The oviposition-deterring activity, egg viability, and repellence activity in the presence of different essential oils concentrations were determined. The essential oils showed high toxicity to all developmental stages of A. aegypti and C. quinquefasciatus. Furthermore, the oils also showed high repellent activity towards the adult stage of mosquitoes (0.025 to 0.550 μg/cm2 skin conferred 100% repellence up to 120 min) and in contact with cultured insect cells (C6/36) induced death possibly by necrosis. The results presented in this work show the potential of S. guianensis essential oils for the development of an alternative and effective method for the natural control of mosquitoes in homes and urban areas.     

Sex before or after blood feeding: Mating activities of Aedes aegypti males under conditions of different densities and female blood feeding opportunities

Blood feeding and mating are critical behaviors that regulate both mosquito population maintenance and disease transmission. However, our understanding of mosquito mating systems remains incomplete. One of the most critical issues is a lack of understanding regarding how and where males and females encounter one another. This study was performed to investigate changes in key mating behaviors of Ae. aegypti relative to female blood feeding opportunities, taking into account male density. We compared courtship latency and copulation activity between single and pooled males in a range of assays performed in the presence or absence of a blood source and after blood feeding. The time taken by grouped males to initiate courtship in the presence of a host was much shorter than that in single males. There was no significant difference in courtship latency between pooled and single males in the absence of a blood source or after blood feeding. At low male density, the presence of the host and blood meal ingestion provided better conditions for copulation. At high male density, however, copulation activity was decreased after blood feeding, but remained high regardless of the presence or absence of the host. In addition to providing insight into the mating ecology of Aedes aegypti, this study indicated that the presence of a blood source influences how males encounter and copulate with females. The observation that copulation activity decreases after blood feeding when males are numerous provides new avenues for improving mass release programs of sterile mosquitoes.     

A facile and rapid method for green synthesis of Achyranthes aspera stem extract-mediated silver nano-composites with cidal potential against Aedes aegypti L.

Aedes aegypti L. is the primary vector associated with transmission of globally concerned diseases; Zika, yellow fever, dengue and Chikungunya. Present study investigates an efficient, alternative and comparative approach for mosquito control which is safe to environment and non-target organisms. The silver nano-composites (AgNCs) were synthesized from the aqueous stem extract of Achyranthes aspera (AASE) using different concentration of aqueous silver nitrate (AgNO₃). The synthesis was tracked by UV-vis spectrophotometer and particle size analyser (DLS). The evaluation of their larvicidal potential against early fourth instars of Ae. aegypti showed significant potency, the toxicity increasing with the concentration of silver nitrate. The 24, 48 and 72 h bioassays resulted in respective LC₅₀ values of 26.693, 1.113 and 0.610 μg/mL (3 mM AASE-AgNO₃) 9.119, 0.420 and 0.407 μg/mL (4 mM AASE-AgNO₃) and that of 4.283, 0.3 and 0.248 μg/mL (5 mM AASE-AgNO₃). Keeping in view the significantly high larvicidal efficiency at lower concentration of silver nitrate, the 4 mM nano-composites were selected over 5 mM composites for further biophysical characterization carried out by X-ray Diffraction (XRD), Fourier transform infrared spectrometer (FTIR), Scanning electron microscopy (SEM), Energy dispersive X-ray (EDX) spectroscopy and Transmission electron microscopy (TEM). SEM and TEM confirmed the synthesis of spherical poly-dispersed AgNCs with average size ranging from 1–30 nm. Characterization through XRD showed the crystalline face-centered-cubic (fcc) structure of AgNCs with the highest intense peak obtained at 2θ value of 31.82°. FT-IR data suggests complex nature of AgNCs showing clearly defined peaks in different ranges. The present investigations recommend AgNCs of A. aspera stems as a low-cost and eco-friendly alternative to chemical insecticides for mosquito control.     

Insecticidal effects of Vernonanthura nebularum against two economically important pest insects

Vernonanthura nebularum (Cabrera) H. Rob. (Asteraceae), an endemic species from the north of Argentina, is a rich source of elephantopus-type sesquiterpene lactones. These compounds have proved to be promising antiparasitic agents, but there is no report about their action against pest insects. In this work we studied for the first time the antifeedant and toxic effects of V. nebularum natural products against the fall army warm Spodoptera frugiperda Smith and the oviposition deterrent activity against the fruit fly Ceratitis capitata Wiedemann. As a result, we found that extracts, fractions composed of sesquiterpene lactones and pure sesquiterpene lactones altered larval feeding behavior in the food choice test. Nutritional parameters of S. frugiperda larvae were also affected. Fraction II (300 μg/g of diet.), containing compounds 1, 2 and 3, was the most toxic substance with 80% pupal mortality and wing malformations in adults. In oviposition deterrent experiments against Ceratitis capitata, we observed a moderate effect at 30 μg/cm² of the test compound. The most active substances were the methanolic extract, dichloromethane subextract and lactone 2. According to our results, V. nebularum natural products could be used for maximizing the effectiveness and specificity in future insecticide design with specific or multiple target sites, while ensuring the economic and ecological sustainability, in addition to combat the increasing resistance rates developed by synthetic pesticides.     

Production of two entomopathogenic Aspergillus species and insecticidal activity against the mosquito Culex quinquefasciatus compared to Metarhizium anisopliae

The spore productivity and insecticidal activity of two opportunistic insect pathogenic Aspergillus species (namely: Aspergillus clavatus Desmazieres and Aspergillus flavus Link (Ascomycota: Eurotiales, Trichocomaceae)) were compared to Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin (Ascomycota: Hypocreales, Clavicipitaceae) for mosquito (Diptera: Culicidae) control. The production of aerial spores on wheat bran and white rice was investigated in solid-, semi-solid-, and liquid-state media supplemented with a nutritive solution. Wheat bran-based media increased the spore yield in solid-state from three to sevenfold: A. clavatus produced 48.4?±?5.2 and 15.7?±?1.6?×?10⁸ spores/g, A. flavus produced 22.3?±?4.1 and 3.1?±?2.5?×?10⁸ spores/g, and M. anisopliae produced 39.6?±?6.5 and 13.1?±?2.6?×?10⁸ spores/g of wheat bran or white rice, respectively. A. clavatus, A. flavus and M. anisopliae spores harvested from wheat bran-based solid-state media showed lethal concentrations (LC₅₀) of 1.1, 1.8, and 1.3?×?10⁸ spores/ml against Culex quinquefasciatus Say larvae in 72?h. Because A. clavatus and M. anisopliae displayed similar features when cultured under these conditions, our results suggest that insect pathogenic Aspergillus species may be as productive and virulent against mosquito larvae as a well-recognised entomopathogenic fungus.     

Anti-inflammatory,antioxidant and antihepatotoxic effects of Spirulina platensis against d-galactosamine induced hepatotoxicity in rats

Spirulina platensis has been advocated as safe food for human use by several investigators. In this study its beneficial dietary effect against liver injuries caused by d-galactosamine (d-GalN) was studied ensuring safety to human health using animal model. Acute hepatotoxicity was induced in Wister rats with d-GalN followed by treatment with butylated hydroxytoluene (BHT) and with Spirulina aqueous extract at various concentrations. The effect of Spirulina at different concentrations were tried and compared with BHT treatment. The animals treated with d-GalN on subsequent treatment by supplementation with Spirulina (6, 9%) in the diets, led to significant reversal in the levels of the antioxidant enzymes through hepatocytes by suppression of negative effect. Spirulina aqueous extract at 9% resulted in a significant decrease in the levels of alkaline phosphatase and infalmatory markers TNFα, IL6 and IL1β and also decreased TBARS, while it showed an increase in oxidative stress marker such as GR, GSH, GST, SOD, GPX and CAT and total protein when compared to the levels recorded with that group treated with d-GalN. Results also indicated that Spirulina aqueous extract at 9% concentration was equally effective in protecting liver damage as it was observed with BHT. Histological studies on liver treated with d-GalN, BHT and Spirulina aqueous extract showed that S. platensis is effective as diet in providing beneficial protective effect. The results obtained in the present study very clearly indicated the positive beneficial protective effect of Spirulina, when used as diet, on the safety and protection of liver from injuries caused by toxicants.     

Comparative repellency of commercial formulations of deet, permethrin and citronellal against the mosquito Aedes aegypti, using a collagen membrane technique compared with human arm tests

A collagen membrane technique, based on the membrane blood-feeding system of Cosgrove et al . (1994) , was used to compare repellents against Aedes aegypti mosquitoes. Repellency was defined in terms of inhibition of probing (ED₅₀ and ED₉₀) after 5 min exposure. A direct comparison was made with repellency from probing after 5 min on five male volunteers.
Four repellent products were compared with technical DEET as the standard. The liquid formulations tested were: Autan^® (20% deet; Bayer); Repel Plus^® (20% deet plus 0.05% permethrin; Boots); permethrin (Zeneca) and citronellal (Sigma) .
Membrane and arm tests gave similar results. Deet formulations required less active compound than citronellal for the same degree of repellency. Pure deet and Autan gave similar results, dose for dose. Permethrin was highly repellent at very low doses, but Repel Plus did not enhance the immediate repellency of deet. A technique using the same membrane system was developed to evaluate persistence of Autan, which declined to 75% after 1 h against Ae. aegypti , and to about 50% after 2–4 h.     

The population dynamics of three polyphagous owlet moths (Lepidoptera: Noctuidae) and the influence of meteorological factors and ENSO on them

The owlet moths (Lepidoptera: Noctuidae) Anicla infecta (Ochsenheimer 1816), Elaphria agrotina (Guenée 1852) and Spodoptera frugiperda (J.E. Smith 1797) occur in the entire American continent. These polyphagous moths have a preference for grasses, and have different biological habits. In this study, the populations of these three species were evaluated monthly with light traps in the Brazilian Savannah, ranging a span of four crop seasons (from July, 2013 to June, 2017). The population data were analyzed and correlated with the meteorological variables: maximum temperature, minimum temperature, relative humidity and precipitation. A total of 4719 individuals were collected in the following percentages: A. infecta (n = 459; 9.73%), E. agrotina (n = 1809; 38.33%) and S. frugiperda (n = 2451; (51.94%). The abundance of all species went down from the first crop season (2013/2014) to the third (2015/2016). In the fourth crop season (2016/2017), the populations of A. infecta and E. agrotina stabilized, but the abundance of S. frugiperda experienced further decrease. The numbers of individuals of three species declined when precipitation was much above (crop season 2014/2015) and below (crop season 2015/2016) than expected by the climatological normal. There were significant, but different degrees of correlation, between the meteorological factors and the ONI index (Oceanic Niño Index - indicator for monitoring El Niño-Southern Oscillation or “ENSO”) with respect to monthly population variations. The results are discussed in accordance with principles of the Integrated Pest Management (IPM) in mind, given the continental distribution and agricultural importance of the three owlet moth species studied.     

Plasmodium gallinaceum: effects of various compounds on immunity of susceptible Aedes aegypti and refractory Culex pipiens pipiens

Para-aminobenzoic acid (PABA) administered in the diet in 0.01, 0.1, 0.2, 0.3, 0.4, and 1.0% concentrations to Aedes aegypti was found to enhance oocyst production. The most significant increase in oocyst production was in groups receiving 0.3 and 0.1% solutions. A combination of 0.01% PABA + 0.1 M NaOH, and 0.02 M HCL, 0.1 M KOH, 0.1 M NaOH, and 0.2 M MgCl₂ likewise increased the susceptibility of A. aegypti to Plasmodium gallinaceum with 0.1 M NaOH being the most effective.Culex pipiens pipiens was found to exhibit a low degree of susceptibility to P. gallinaceum. This is the first report of oocyst development by P. gallinaceum in this species. PABA in 0.1, 0.2, 0.3, 0.4, and 1.0% concentrations was tested and all concentrations increased the susceptibility of this refractory host. This compound was most effective in a 0.4% concentration.The susceptibility of C. pipiens pipiens to P. gallinaceum was also increased by inorganic materials. NaOH and KOH in 0.1 M concentrations yielded the greatest increase. A slight increase in susceptibility was effected by 0.2 M MgCl₂ and 0.02 M HCL. Culex receiving the combination of 0.01% PABA and 0.01 M NaOH had the highest infection of those receiving inorganic compounds.Most of the oocysts produced in these experimental Culex were fairly typical of those found in A. aegypti; however, some were quite small and others showed evidence of deterioration, distortion, and shrinkage.     

Feeding behaviour of female Aedes aegypti: effects of diet temperature, bicarbonate and feeding technique on the response to ATP

Abstract. The effects of variation in diet temperature, mouthpart deployment and the addition of bicarbonate to a saline-ATP diet were investigated for their effects on amount ingested and diet destination in female Aedes aegypti (L.). Mouthpart deployment was achieved by having the insects feed through a membrane, or from a free-liquid surface with mouthparts intact, or with the fascicle separated from the labial groove.
Under the 'feeding through membrane' protocol, a 6-fold increase in the percentage of Aedes feeding on ATP-diets was recorded at 37C compared with 21C. Bicarbonate (9.5 or 19 mM) induced a 3-fold increase in numbers feeding at both 21C and 37C. Neither diet temperature nor bicarbonate content appeared to affect meal size. The pH of all diets was adjusted to 7.0. Diet was directed primarily to the midgut for all diets tested under this regimen, with the exception of the saline-ATP-19 mM NaHC0₃ at 37C which caused the diet to be directed primarily to the crop.
Under the 'feeding without membrane through feeding tube' protocol, females showed little control over diet destination. Female Aedes aegypti exhibited no strong response to ATP when the mouthparts were immersed in 21C diets in feeding tubes and few of these insects ingested large meals. The addition of bicarbonate to the ATP diets did not enhance feeding under these test conditions.     

Differences in performance of Aedes aegypti larvae raised at different densities in tires and ovitraps under field conditions in Argentina.

Alteration of fitness components was assessed in the yellow fever mosquito, Aedes aegypti, in automobile tires and vases (ovitraps) under field conditions. Larval numbers were manipulated in both kinds of containers to compare low, high, and control (natural) densities. Densities were set from a census of a wild population, then doubling and reducing to half the mean crowding, m*. Artificially altered densities were not high or low enough to produce differences among treatments. Tires generated more vigorous larval populations and females with higher fecundity than did small containers, although the mortality was more intense.     

The impact of CO2 on collection of Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say by BG-Sentinel(r) traps in Manaus,Brazil

Carbon dioxide (CO₂) is an important component for activating and attracting host-seeking mosquitoes. The BG-Sentinel^(r) trap is a well-established monitoring tool for capturing Culicidae, but CO₂ role for the trap effectiveness has not been evaluated in highly urbanised areas. The objective was to evaluate the effectiveness of BG-Sentinel traps baited with and without CO₂ for capturing urban mosquitoes. Fifteen areas were selected within the city of Manaus, Brazil, where four BG-Sentinels were operated for 24 h, two of them with CO₂ and two without CO₂. Captured Aedes aegypti females were dissected for the determination of their parity status. A significantly higher proportion of traps (from 32-79%) were positive for female Ae. aegypti when using the BG-Sentinel with CO₂ (χ² = 11.0271, p ≤ 0.001). Catches of female Culex spp were six times higher in CO₂ traps (Mann-Whitney U test = 190.5; p = 0.001). Parity rates were similar for both traps. This study showed that CO₂ has primarily an enhancing effect on the efficacy of BG-Sentinel for capturing Culex spp in Manaus. For Ae. aegypti, the positivity rate of the trap was increased, when CO₂ was added.     

Bacterial and eukaryote microbiomes of mosquito habitats in dengue-endemic southern Taiwan

Mosquitoes interact with the microbiome of the waters where they oviposit in several ways. Past work suggests adult mosquitoes can detect certain microbes that stimulate oviposition. The presence or absence of certain microbes in water containers thus can attract or repel mosquito species to different containers. I hypothesized that these relationships could be detected via metagenomics. I focused on two container breeders that coexist in Southern Taiwan: the dengue vector Aedes aegypti and the less competent vector Ae. albopictus. In addition to culturing, I performed 16S and 18S rDNA metagenomics assays, the latter of which had never been applied to mosquito waters before, to identify the microbial diversity of artificial containers with and without mosquito larvae. I found no correlation between mosquito presence to any features of the containers or to their microbiomes, which instead correlated strongly with location. Microbial diversity across containers was highly variable, even within the same location, with multiple taxa only found in single containers. This variability is reasonable, because mosquito gut microbiomes are also extremely variable. The possibility remains that microbes in natural containers differ significantly from those in artificial containers, and that these differences drive Aedes preferences for human-associated containers. Broad, single-microbe experimental work is recommended to identify possible attractant or repellent microbial taxa.     

Plant growth and yield of cucumber plants grafted on different commercial and local rootstocks grown under salinity stress

This study aimed to determine the effects of different rootstocks and soilless media on the plant growth and yield of cucumber and on the leaf ion (Na⁺, Ca⁺⁺, K⁺ and Cl^?) concentrations. Four commercial rootstocks (TZ148 F₁, RS841 F₁, Nun9075 F₁ and Avar F₁) and two local landraces (Local-1 and Local-3 belonging to Cucurbita moschata L.) were used as rootstock and grafted and non grafted plants were tested in three different salinity conditions (2.5 dS m^?1, 5.0 dS m^?1 and 7.5 dS m^?1) on three different soilless media (cocopeat, perlite and rockwool) in spring period under greenhouse conditions. Salinity found to reduce root and shoot dry weight, and yield of plants in all growing media. TZ148, Nun9075 and Local-3 are found to improve tolerance of cucumber plants to saline conditions (5.0 and 7.5 dS m^?1) when used as rootstocks. Root and shoot dry weight, yield, Ca⁺⁺ in leaves and K⁺/Na⁺ ratio in leaves were significantly decreased, but Na⁺ and Cl^? concentration in leaves were increased under salt stress. Rootstock potential of Local-3 is also found to be quite good for cucumber under saline conditions.