Larvicides play a vital role in controlling mosquitoes in their breeding sites. The present study was carried out to establish the larvicidal activities of mycosynthesized silver nanoparticles (AgNPs) against vectors: Aedes aegypti and Anopheles stephensi responsible for diseases of public health importance. The AgNPs synthesized by filamentous fungus Cochliobolus lunatus, characterized by UV–Vis spectrophotometry, Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. The characterization studies confirmed the spherical shape and size (3–21 nm) of silver nanoparticles. The efficacy of mycosynthesized AgNPs at all the tested concentrations (10, 5, 2.5, 1.25, 0.625, and 0.3125 ppm) against second, third, and fourth instar larvae of A. aegypti (LC50 1.29, 1.48, and 1.58; LC90 3.08, 3.33, and 3.41 ppm) and against A. stephensi (LC50 1.17, 1.30, and 1.41; LC90 2.99, 3.13, and 3.29 ppm) were observed, respectively. The mortality rates were positively correlated with the concentration of AgNPs. Significant (P < 0.05) changes in the larval mortality was also recorded between the period of exposure against fourth instar larvae of A. aegypti and A. stephensi. The possible larvicidal activity may be due to penetration of nanoparticles through membrane. Toxicity studies carried out against non-target fish species Poecilia reticulata, the most common organism in the habitats of A. aegypti and A. stephensi showed no toxicity at LC50 and LC90 doses of the AgNPs. This is the first report on mosquito larvicidal activity of mycosynthesized nanoparticles. Thus, the use of fungus C. lunatus to synthesize silver nanoparticles is a rapid, eco-friendly, and a single-step approach and the AgNps formed can be potential mosquito larvicidal agents.

Larvicidal potential of silver nanoparticles synthesized using fungus Cochliobolus lunatus against Aedes aegypti (Linnaeus, 1762) and Anopheles stephensi Liston (Diptera; Culicidae)

Laboratory bioassays on insecticidal activity of essential oils (EOs) extracted from six Mediterranean plants (Achillea millefolium, Lavandula angustifolia, Helichrysum italicum, Foeniculum vulgare, Myrtus communis, and Rosmarinus officinalis) were carried out against the larvae of the Culicidae mosquito Aedes albopictus. The chemical composition of the six EOs was also investigated. Results from applications showed that all tested oils had insecticidal activity, with differences in mortality rates as a function of both oil and dosage. At the highest dosage (300 ppm), EOs from H. italicum, A. millefolium, and F. vulgare caused higher mortality than the other three oils, with mortality rates ranging from 98.3% to 100%. M. communis EO induced only 36.7% larval mortality at the highest dosage (300 ppm), a similar value to those recorded at the same dosage by using R. officinalis and L. angustifolia (51.7% and 55%, respectively). Identified compounds ranged from 91% to 99%. The analyzed EOs had higher content of monoterpenoids (80-99%) than sesquiterpenes (1-15%), and they can be categorized into three groups on the basis of their composition. Few EOs showed the hydrocarbon sesquiterpenes, and these volatile compounds were generally predominant in comparison with the oxygenated forms, which were detected in lower quantities only in H. italicum (1.80%) and in M. communis (1%).

Essential oil composition and larvicidal activity of six Mediterranean aromatic plants against the mosquito Aedes albopictus (Diptera: Culicidae)

Cyclotides are plant proteins whose defining structural features are a head-to-tail cyclized backbone and three interlocking disulfide bonds, which in combination are known as a cyclic cystine knot. This unique structural motif confers cyclotides with exceptional resistance to proteolysis. Their endogenous function is thought to be as plant defense agents, associated with their insecticidal and larval growth-inhibitory properties. However, in addition, an array of pharmaceutically relevant biological activities has been ascribed to cyclotides, including anti-HIV, anthelmintic, uterotonic, and antimicrobial effects. So far, >150 cyclotides have been elucidated from members of the Rubiaceae, Violaceae, and Cucurbitaceae plant families, but their wider distribution among other plant families remains unclear. Clitoria ternatea (Butterfly pea) is a member of plant family Fabaceae and through its usage in traditional medicine to aid childbirth bears similarity to Oldenlandia affinis, from which many cyclotides have been isolated. Using a combination of nanospray and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analyses, we examined seed extracts of C. ternatea and discovered cyclotides in the Fabaceae, the third-largest family of flowering plants. We characterized 12 novel cyclotides, thus expanding knowledge of cyclotide distribution and evolution within the plant kingdom. The discovery of cyclotides containing novel sequence motifs near the in planta cyclization site has provided new insights into cyclotide biosynthesis. In particular, MS analyses of the novel cyclotides from C. ternatea suggest that Asn to Asp variants at the cyclization site are more common than previously recognized. Moreover, this study provides impetus for the examination of other economically and agriculturally significant species within Fabaceae, now the largest plant family from which cyclotides have been described.

Discovery of Cyclotides in the Fabaceae Plant Family Provides New Insights into the Cyclization, Evolution, and Distribution of Circular Proteins

Plant extracts for developing mosquito larvicides: From laboratory to the field, with insights on the modes of action.

Chrysosporium tropicum is a pathogenic fungus. It is known to be an effective mosquito control agent. In the present study, we have synthesized the silver and gold nanoparticles using C. tropicum. These nanoparticles have been characterized through Microscan reader, X-ray diffractometer, transmission electron microscopy, and further confirmed by scanning electron microscopy. The characterization study confirmed the spherical shape and size (2–15 and 20–50 nm) of gold and silver nanoparticles. These silver and gold nanoparticles have been tested as a larvicide against the Aedes aegypti larvae. The larvicidal efficacy was noted when performed against all instars of A. aegypti at six different log concentrations, and significant results could be observed. The gold nanoparticles used as an efficacy enhancer have shown mortality at three times higher concentration than the silver nanoparticles. The larval mortality was observed after different time of exposures. The mortality values were obtained using the probit analysis. The larvae of A. aegypti were found to be highly susceptible for the silver nanoparticles. The second instar larvae have shown 100% mortality against the silver nanoparticles after 1 h, whereas the first, third, and fourth instars have shown efficacy (LC50 = 3.47, 4, and 2; LC90 = 12.30, 8.91, and 4; LC99 = 13.18, 13.18, and 7.58, respectively) after 1 h. The results could suggest that the use of fungus C. tropicum, silver, and gold nanoparticles is a rapid, environmentally safer, and greener approach for mosquito control. This could lead us to a new possibility in vector control strategy.

Efficacy of fungus mediated silver and gold nanoparticles against Aedes aegypti larvae

Screening of natural products for mosquito larvicidal activity against three major mosquito vectors Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi resulted in the identification of three potential plant extracts viz., Saraca indica/asoca, Nyctanthes arbor-tristis, and Clitoria ternatea for mosquito larval control. In the case of S. indica/asoca, the petroleum ether extract of the leaves and the chloroform extract of the bark were effective against the larvae of C. quinquefasciatus with respective LC50 values 228.9 and 291.5 ppm. The LC50 values of chloroform extract of N. arbor-tristis leaves were 303.2, 518.2, and 420.2 ppm against A. aegypti, A. stephensi, and C. quinquefasciatus, respectively. The methanol and chloroform extracts of flowers of N. arbor-tristis showed larvicidal activity against larvae of A. stephensi with the respective LC50 values of 244.4 and 747.7 ppm. Among the methanol extracts of C. ternatea leaves, roots, flowers, and seeds, the seed extract was effective against the larvae of all the three species with LC50 values 65.2, 154.5, and 54.4 ppm, respectively, for A. stephensi, A. aegypti, and C. quinquefasciatus. Among the three plant species studied for mosquito larvicidal activity, C. ternatea was showing the most promising mosquito larvicidal activity. The phytochemical analysis of the promising methanolic extract of the seed extract was positive for carbohydrates, saponins, terpenoids, tannins, and proteins. In conclusion, bioassay-guided fractionation of effective extracts may result in identification of a useful molecule for the control of mosquito vectors.

S. M. Sivakumar

Papers

Larvicidal activity of Saraca indica, Nyctanthes arbor-tristis, and Clitoria ternatea extracts against three mosquito vector species