Assessment of biorational larvicides and botanical oils against Culex quinquefasciatus Say (Diptera: Culicidae) larvae in laboratory conditions.

Mosquitoes are known vectors that transmit deadly diseases to millions of people across the globe. The reliance on synthetic insecticides has been the sole way to combat mosquito vectors for decades. In recent years, the extensive use of conventional insecticides in mosquito suppression has led to significant pesticide resistance and serious human health hazards. In this light, investigating the potential application of biorational compounds for vector management has drawn significant attention. We, hereby, evaluated the efficacy of three microbial derivative biorational insecticides, abamectin, spinosad, and buprofezin, and two botanical oils, neem (Azadirachta indica A. Juss) and karanja oil (Pongamia pinnata Linn.) against the Culex quinquefasciatus under laboratory conditions. The fourth-instar C. quinquefasciatus larvae were exposed to different concentrations of the selected larvicides and lethality was estimated based on LC50 and LT50 with Probit analysis. All larvicides showed concentration-dependent significant effects on survival and demonstrated larvicidal activity against C. quinquefasciatus larvae. However, abamectin exerted the highest toxicity (LC50 = 10.36 ppm), exhibited statistically significant effects on C. quinquefasciatus larval mortality, followed by spinosad (LC50 = 21.32 ppm) and buprofezin (LC50 = 56.34 ppm). Abamectin caused larval mortality ranged from 30.00 to 53.33 % and 53.00-70.00 % at 06 and 07 h after treatment (HAT), respectively. In the case of botanicals, karanja oil (LC50 = 216.61 ppm) was more lethal (more than 1.5 times) and had a shorter lethal time than neem oil (LC50 = 330.93 ppm) and showed a classic pattern of relationship between concentrations and mortality over time. Overall, the present study highlighted the potential of deploying new generation biorational pesticides and botanicals in mosquito vector control programs.

Analysis of qualitative and quantitative morphological traits related to yield in country bean (Lablab purpureus L. sweet) genotypes.

Country bean is a grain legume extensively farmed for its multi-purpose uses, yet the traits related to yield are are poorly studied and yet unexplored. A study on the diversity of qualitative and quantitative morphological characteristics concerning yield among the country bean germplasms collected from Bangladesh identified considerable variation in the studied traits across the germplasms and identified a complex correlation between the qualitative and quantitative traits. Principal Component Analysis (PCA) detected five components that contributed 66.38% qualitative traits and six components contributed 74.49% quantitative traits to total variations. Eigenvalues indicated that a majority of color-related qualitative traits included cotyledon, leaf, vein, seed, flower, and petals contributed, in contrast,a majority of the seed, leaf, flower, and inflorescence-related quantitative traits contributed to the total diversity of the Lablab germplasms. Among the quantitative traits, the highest coefficient of variation (CV%) was found in average pod weight (50.98%), followed by the total number of spikes per plant (43.82%), while seed length, pod weight, length, width, thickness, number of flower/spike, spike length, and total no of spikes/plant all had more than 20.00 percent CV, suggesting suitability to use in the breeding of high yielding genotypes. The germplasms are grouped into four and three clusters based on quantitative and qualitative traits, suggesting quantitative characters offer better clustering of genotypes. Considering the above traits, our research found that the BD-10804, BD-10807, BD-11091, BD-10808, BD-10815, and BD-11089 and cultivar Goal Goda Lablab beans germplasms produced higher pod weight with corresponding higher pod length, width, and thickness suggesting to use them as high yielding genotypes for food and fodder purposes.

Potency of insect-specific scorpion toxins on mosquito control using Bacillus thuringiensis Cry4Aa.

Two insect-specific scorpion toxins, BjαIT and AahIT were produced as alkali-soluble protein inclusions in Escherichia coli. The inclusion bodies themselves exhibited no toxicity against Culex pipiens larvae. However, coadministration with Cry4Aa toxin enhanced the mosquitocidal activity by 2-3 fold. Insect-specific scorpion toxins can be good supplements for Cry toxin-based bioinsecticides.

Alanine scanning analyses of the three major loops in domain II of Bacillus thuringiensis mosquitocidal toxin Cry4Aa.

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.

Biological properties of loop-replaced mutants of Bacillus thuringiensis mosquitocidal Cry4Aa.

Cry4Aa produced by Bacillus thuringiensis subsp. israelensis (Bti) exhibits a specific toxicity to Anopheles, Aedes, and Culex larvae, which are vectors of serious diseases, and formulations of Bti are used worldwide for mosquito control. In general, domain II of the Cry toxin is believed to be important for target specificity, and three loops (loops 1, 2, and 3) in domain II have been studied extensively. In this report, to analyze the biological functions of loops 1, 2, and 3 of Cry4Aa, mutants were constructed in which one of the loops was replaced with either of the other two loops. A bioassay using Culex pipiens larvae revealed that the mosquitocidal activity was virtually lost upon replacement of loop2. The mutants in which loops 1 and/or 3 were replaced also showed decreased activity, but they still maintained some activities. This suggested that loop2, but not loops 1 and 3, was essential for the mosquitocidal activity of Cry4Aa. Proteolytic digestion revealed the involvement of loops in the stability of the Cry4Aa structure. No significant differences were observed in the amount of wild-type and mutant Cry4Aa bound to the BBMVs prepared from the C. pipiens larvae.

Design and construction of a synthetic Bacillus thuringiensis Cry4Aa gene: hyperexpression in Escherichia coli.

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is, therefore, of great interest for developing a bioinsecticide to control mosquitoes. However, the expression of Cry4Aa in Escherichia coli is relatively low, which is a major disadvantage in its development as a bioinsecticide. In this study, to establish an effective production system, a 1,914-bp modified gene (cry4Aa-S1) encoding Cry4Aa was designed and synthesized in accordance with the G + C content and codon preference of E. coli genes without altering the encoded amino acid sequence. The cry4Aa-S1 gene allowed a significant improvement in expression level, over five-fold, compared to that of the original cry4Aa gene. The product of the cry4Aa-S1 gene showed the same level of insecticidal activity against Culex pipiens larvae as that from cry4Aa. This suggested that unfavorable codon usage was one of the reasons for poor expression of cry4Aa in E. coli, and, therefore, changing the cry4Aa codons to accord with the codon usage in E. coli led to efficient production of Cry4Aa. Efficient production of Cry4Aa in E. coli can be a powerful measure to prepare a sufficient amount of Cry4Aa protein for both basic analytical and applied researches.

Flexibility and strictness in functional replacement of domain III of cry insecticidal proteins from Bacillus thuringiensis.

Cry1C, one of the lepidopteran-specific insecticidal proteins from Bacillus thuringiensis, exhibits potent cytotoxicity against Sf9, an insect cell line. Cry1Aa and Cry4A, which are lepidopteran- and dipteran-specific insecticidal proteins, respectively, show no cytotoxicity against Sf9. When domain III of Cry1C was replaced with that of Cry1Aa or Cry4A, the hybrid Cry1C protein retained the cytotoxicity. These results suggest that domain III of Cry1C is not crucial in determining the cytocidal specificity of Cry1C against Sf9.