Synthesis of new series of quinoline derivatives with insecticidal effects on larval vectors of malaria and dengue diseases.

Mosquito borne diseases are on the rise because of their fast spread worldwide and the lack of effective treatments. Here we are focusing on the development of a novel anti-malarial and virucidal agent with biocidal effects also on its vectors. We have synthesized a new quinoline (4,7-dichloroquinoline) derivative which showed significant larvicidal and pupicidal properties against a malarial and a dengue vector and a lethal toxicity ranging from 4.408 µM/mL (first instar larvae) to 7.958 µM/mL (pupal populations) for Anopheles stephensi and 5.016 µM/mL (larva 1) to 10.669 µM/mL (pupae) for Aedes aegypti. In-vitro antiplasmodial efficacy of 4,7-dichloroquinoline revealed a significant growth inhibition of both sensitive strains of Plasmodium falciparum with IC50 values of 6.7 nM (CQ-s) and 8.5 nM (CQ-r). Chloroquine IC50 values, as control, were 23 nM (CQ-s), and 27.5 nM (CQ-r). In vivo antiplasmodial studies with P. falciparum infected mice showed an effect of 4,7-dichloroquinoline compared to chloroquine. The quinoline compound showed significant activity against the viral pathogen serotype 2 (DENV-2). In vitro conditions and the purified quinoline exhibited insignificant toxicity on the host system up to 100 µM/mL. Overall, 4,7-dichloroquinoline could provide a good anti-vectorial and anti-malarial agent.

Efficacy and side effects of bio-fabricated sardine fish scale silver nanoparticles against malarial vector Anopheles stephensi.

Mosquitoes are a great menace for humankind since they transmit pathogenic organisms causing Malaria, Dengue, Chikungunya, Elephantiasis and Japanese encephalitis. There is an urgent need to discover new and novel biological tools to mitigate mosquito-borne diseases. To develop bioinsecticides through newly developed nanotechnology is another option in the present research scenario. In this study we synthesize and characterize sardine fish scales with silver nitrate by adopting various instrumental techniques such as UV- and FTIR-spectroscopy, energy-dispersive X-ray (EDAX), X-ray diffraction analyses (XRD) and scanning electron microscopy (SEM). Toxicity bioassays were conducted with young developmental stages of mosquito vectors. Significant mortality appeared after different life stages of mosquito vectors (young larval and pupal instars were exposed to the nanomaterials). LC50 values were 13.261 ppm for young first instar larvae and 32.182 ppm for pupae. Feeding and predatory potential of G. affinis, before and after exposure to nanoparticles against mosquito larval (I & II) instars of the mosquitoes showed promising results in laboratory experiments. Feeding potential of mosquito fish without nanoparticle treatment was 79.7% and 70.55% for the first and second instar larval populations respectively. At the nanoparticle-exposed situation the predatory efficiency of mosquitofish was 94.15% and 84.3%, respectively. Antioxidant enzymes like (SOD), (CAT), and (LPO) were estimated in the gill region of sardine fish in control and experimental waters. A significant reduction of egg hatchability was evident after nanoparticle application. It became evident from this study that the nano-fabricated materials provide suitable tools to control the malaria vector Anopheles stephensi in the aquatic phase of its life cycle. This finding suggests an effective novel approach to mosquito control.

Zinc oxide nanoparticles using plant Lawsonia inermis and their mosquitocidal, antimicrobial, anticancer applications showing moderate side effects.

Microbes or parasites spread vector-borne diseases by mosquitoes without being affected themselves. Insecticides used in vector control produce a substantial problem for human health. This study synthesized zinc oxide nanoparticles (ZnO NPs) using Lawsonia inermis L. and were characterized by UV-vis, FT-IR, SEM with EDX, and XRD analysis. Green synthesized ZnO NPs were highly toxic against Anopheles stephensi, whose lethal concentrations values ranged from 5.494 ppm (I instar), 6.801 ppm (II instar), 9.336 ppm (III instar), 10.736 ppm (IV instar), and 12.710 ppm (pupae) in contrast to L. inermis treatment. The predation efficiency of the teleost fish Gambusia affinis and the copepod Mesocyclops aspericornis against A. stephensi was not affected by exposure at sublethal doses of ZnO NPs. The predatory potency for G. affinis was 45 (I) and 25.83% (IV), copepod M. aspericornis was 40.66 (I) and 10.8% (IV) while in an ZnO NPs contaminated environment, the predation by the fish G. affinis was boosted to 71.33 and 34.25%, and predation of the copepod M. aspericornis was 60.35 and 16.75%, respectively. ZnO NPs inhibited the growth of several microbial pathogens including the bacteria (Escherichia coli and Bacillus subtilis) and the fungi (Alternaria alternate and Aspergillus flavus), respectively. ZnO NPs decreased the cell viability of Hep-G2 with IC50 value of 21.63 µg/mL (R2 = 0.942; P < 0.001) while the concentration increased from 1.88 to 30 µg/mL. These outcomes support the use of L. inermis mediated ZnO NPs for mosquito control and drug development.

Iron and iron oxide nanoparticles are highly toxic to Culex quinquefasciatus with little non-target effects on larvivorous fishes.

The control of filariasis vectors has been enhanced in several areas, but there are main challenges, including increasing resistance to insecticides and lack of cheap and eco-friendly products. The toxicity of iron (Fe0) and iron oxide (Fe2O3) nanoparticles has been scarcely investigated yet. We studied the larvicidal and pupicidal activity of Fe0 and Fe2O3 nanoparticles against Culex quinquefasciatus. Fe0 and Fe2O3 nanoparticles produced by green (using a Ficus natalensis aqueous extract) and chemical nanosynthesis, respectively, were analyzed by UV-Vis spectrophotometry, FT-IR spectroscopy, XRD analysis, SEM, and EDX assays. In larvicidal and pupicidal experiments on Cx. quinquefasciatus, LC50 of Fe0 nanoparticles ranged from 20.9 (I instar larvae) to 43.7 ppm (pupae) and from 4.5 (I) to 22.1 ppm (pupae) for Fe2O3 nanoparticles synthesized chemically. Furthermore, the predation efficiency of the guppy fish, Poecilia reticulata, after a single treatment with sub-lethal doses of Fe0 and Fe2O3 nanoparticles was magnified. Overall, this work provides new insights about the toxicity of Fe0 and Fe2O3 nanoparticles against mosquito vectors; we suggested that green and chemical fabricated nano-iron may be considered to develop novel and effective pesticides.

Magnetic nanoparticles are highly toxic to chloroquine-resistant Plasmodium falciparum, dengue virus (DEN-2), and their mosquito vectors.

A main challenge in parasitology is the development of reliable tools to prevent or treat mosquito-borne diseases. We investigated the toxicity of magnetic nanoparticles (MNP) produced by Magnetospirillum gryphiswaldense (strain MSR-1) on chloroquine-resistant (CQ-r) and sensitive (CQ-s) Plasmodium falciparum, dengue virus (DEN-2), and two of their main vectors, Anopheles stephensi and Aedes aegypti, respectively. MNP were studied by Fourier-transform infrared spectroscopy and transmission electron microscopy. They were toxic to larvae and pupae of An. stephensi, LC50 ranged from 2.563 ppm (1st instar larva) to 6.430 ppm (pupa), and Ae. aegypti, LC50 ranged from 3.231 ppm (1st instar larva) to 7.545 ppm (pupa). MNP IC50 on P. falciparum were 83.32 µg ml-1 (CQ-s) and 87.47 µg ml-1 (CQ-r). However, the in vivo efficacy of MNP on Plasmodium berghei was low if compared to CQ-based treatments. Moderate cytotoxicity was detected on Vero cells post-treatment with MNP doses lower than 4 µg ml-1. MNP evaluated at 2-8 µg ml-1 inhibited DEN-2 replication inhibiting the expression of the envelope (E) protein. In conclusion, our findings represent the first report about the use of MNP in medical and veterinary entomology, proposing them as suitable materials to develop reliable tools to combat mosquito-borne diseases.

Fabrication of nano-mosquitocides using chitosan from crab shells: Impact on non-target organisms in the aquatic environment.

Mosquitoes are arthropods of huge medical and veterinary relevance, since they vector pathogens and parasites of public health importance, including malaria, dengue and Zika virus. Currently, nanotechnology is considered a potential eco-friendly approach in mosquito control research. We proposed a novel method of biofabrication of silver nanoparticles (AgNP) using chitosan (Ch) from crab shells. Ch-AgNP nanocomposite was characterized by UV-vis spectroscopy, FTIR, SEM, EDX and XRD. Ch-AgNP were tested against larvae and pupae of the malaria vector Anopheles stephensi obtaining LC50 ranging from 3.18 ppm (I) to 6.54 ppm (pupae). The antibacterial properties of Ch-AgNP were proved against Bacillus subtilis, Klebsiella pneumoniae and Salmonella typhi, while no growth inhibition was reported in assays conducted on Proteus vulgaris. Concerning non-target effects, in standard laboratory considtions the predation efficiency of Danio rerio zebrafishes was 68.8% and 61.6% against I and II instar larvae of A. stephensi, respectively. In a Ch-AgNP-contaminated environment, fish predation was boosted to 89.5% and 77.3%, respectively. Quantitative analysis of antioxidant enzymes SOD, CAT and LPO from hepatopancreas of fresh water crabs Paratelphusa hydrodromous exposed for 16 days to a Ch-AgNP-contaminated aquatic environment were conducted. Notably, deleterious effects of Ch-AgNP contaminating aquatic enviroment on the non-target crab P. hydrodromous were observed, particularly when doses higher than 8-10ppm are tested. Overall, this research highlights the potential of Ch-AGNP for the development of newer control tools against young instar populations of malaria mosquitoes, also highlighting some risks concerned the employ of nanoparticles in aquatic environments.

Eco-friendly drugs from the marine environment: spongeweed-synthesized silver nanoparticles are highly effective on Plasmodium falciparum and its vector Anopheles stephensi, with little non-target effects on predatory copepods.

Mosquitoes act as vectors of devastating pathogens and parasites, representing a key threat for millions of humans and animals worldwide. The control of mosquito-borne diseases is facing a number of crucial challenges, including the emergence of artemisinin and chloroquine resistance in Plasmodium parasites, as well as the presence of mosquito vectors resistant to synthetic and microbial pesticides. Therefore, eco-friendly tools are urgently required. Here, a synergic approach relying to nanotechnologies and biological control strategies is proposed. The marine environment is an outstanding reservoir of bioactive natural products, which have many applications against pests, parasites, and pathogens. We proposed a novel method of seaweed-mediated synthesis of silver nanoparticles (AgNP) using the spongeweed Codium tomentosum, acting as a reducing and capping agent. AgNP were characterized by UV-Vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). In mosquitocidal assays, the 50 % lethal concentration (LC50) of C. tomentosum extract against Anopheles stephensi ranged from 255.1 (larva I) to 487.1 ppm (pupa). LC50 of C. tomentosum-synthesized AgNP ranged from 18.1 (larva I) to 40.7 ppm (pupa). In laboratory, the predation efficiency of Mesocyclops aspericornis copepods against A. stephensi larvae was 81, 65, 17, and 9 % (I, II, III, and IV instar, respectively). In AgNP contaminated environment, predation was not affected; 83, 66, 19, and 11 % (I, II, III, and IV). The anti-plasmodial activity of C. tomentosum extract and spongeweed-synthesized AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. Fifty percent inhibitory concentration (IC50) of C. tomentosum were 51.34 µg/ml (CQ-s) and 65.17 µg/ml (CQ-r); C. tomentosum-synthesized AgNP achieved IC50 of 72.45 µg/ml (CQ-s) and 76.08 µg/ml (CQ-r). Furthermore, low doses of the AgNP inhibited the growth of Bacillus subtilis, Klebsiella pneumoniae, and Salmonella typhi, using the agar disk diffusion and minimum inhibitory concentration protocol. Overall, C. tomentosum metabolites and spongeweed-synthesized AgNP may be potential candidates to develop novel and effective tools in the fight against Plasmodium parasites and their mosquito vectors. The employ of ultra-low doses of nanomosquitocides in synergy with cyclopoid crustaceans seems a promising green route for effective mosquito control programs.

In vivo and in vitro effectiveness of Azadirachta indica-synthesized silver nanocrystals against Plasmodium berghei and Plasmodium falciparum, and their potential against malaria mosquitoes.

Malaria transmission is a serious emergence in urban and semiurban areas worldwide, becoming a major international public health concern. Malaria is transmitted through the bites of Anopheles mosquitoes. The extensive employ of synthetic pesticides leads to negative effects on human health and the environment. Recently, plant-synthesized nanoparticles have been proposed as highly effective mosquitocides. In this research, we synthesized silver nanoparticles (AgNP) using the Azadirachta indica seed kernel extract as reducing and stabilizing agent. AgNP were characterized by UV-vis spectrophotometry, SEM, EDX, XRD and FTIR spectroscopy. The A. indica seed kernel extract was toxic against Anopheles stephensi larvae and pupae, LC50 were 232.8ppm (larva I), 260.6ppm (II), 290.3ppm (III), 323.4ppm (IV), and 348.4ppm (pupa). AgNP LC50 were 3.9ppm (I), 4.9ppm (II), 5.6ppm (III), 6.5ppm (IV), and 8.2ppm (pupa). The antiplasmodial activity of A. indica seed kernel extract and AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC50 of A. indica seed kernel extract were 63.18µg/ml (CQ-s) and 69.24µg/ml (CQ-r). A. indica seed kernel-synthesized AgNP achieved IC50, of 82.41µg/ml (CQ-s) and 86.12µg/ml (CQ-r). However, in vivo anti-plasmodial experiments conducted on Plasmodium berghei infecting albino mice showed moderate activity of the A. indica extract and AgNP. Overall, this study showed that the A. indica-mediated fabrication of AgNP is of interest for a wide array of purposes, ranging from IPM of mosquito vectors to the development of novel and cheap antimalarial drugs.

Earthworm-mediated synthesis of silver nanoparticles: A potent tool against hepatocellular carcinoma, Plasmodium falciparum parasites and malaria mosquitoes.

The development of parasites and pathogens resistant to synthetic drugs highlighted the needing of novel, eco-friendly and effective control approaches. Recently, metal nanoparticles have been proposed as highly effective tools towards cancer cells and Plasmodium parasites. In this study, we synthesized silver nanoparticles (EW-AgNP) using Eudrilus eugeniae earthworms as reducing and stabilizing agents. EW-AgNP showed plasmon resonance reduction in UV-vis spectrophotometry, the functional groups involved in the reduction were studied by FTIR spectroscopy, while particle size and shape was analyzed by FESEM. The effect of EW-AgNP on in vitro HepG2 cell proliferation was measured using MTT assays. Apoptosis assessed by flow cytometry showed diminished endurance of HepG2 cells and cytotoxicity in a dose-dependent manner. EW-AgNP were toxic to Anopheles stephensi larvae and pupae, LC(50) were 4.8 ppm (I), 5.8 ppm (II), 6.9 ppm (III), 8.5 ppm (IV), and 15.5 ppm (pupae). The antiplasmodial activity of EW-AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. EW-AgNP IC(50) were 49.3 µg/ml (CQ-s) and 55.5 µg/ml (CQ-r), while chloroquine IC(50) were 81.5 µg/ml (CQ-s) and 86.5 µg/ml (CQ-r). EW-AgNP showed a valuable antibiotic potential against important pathogenic bacteria and fungi. Concerning non-target effects of EW-AgNP against mosquito natural enemies, the predation efficiency of the mosquitofish Gambusia affinis towards the II and II instar larvae of A. stephensi was 68.50% (II) and 47.00% (III), respectively. In EW-AgNP-contaminated environments, predation was boosted to 89.25% (II) and 70.75% (III), respectively. Overall, this research highlighted the EW-AgNP potential against hepatocellular carcinoma, Plasmodium parasites and mosquito vectors, with little detrimental effects on mosquito natural enemies.

Multipurpose effectiveness of Couroupita guianensis-synthesized gold nanoparticles: high antiplasmodial potential, field efficacy against malaria vectors and synergy with Aplocheilus lineatus predators.

Mosquito-borne diseases represent a deadly threat for millions of people worldwide. According to recent estimates, about 3.2 billion people, almost half of the world's population, are at risk of malaria. Malaria control is particularly challenging due to a growing number of chloroquine-resistant Plasmodium and pesticide-resistant Anopheles vectors. Newer and safer control tools are required. In this research, gold nanoparticles (AuNPs) were biosynthesized using a cheap flower extract of Couroupita guianensis as reducing and stabilizing agent. The biofabrication of AuNP was confirmed by UV-vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD), zeta potential, and particle size analysis. AuNP showed different shapes including spheres, ovals, and triangles. AuNPs were crystalline in nature with face-centered cubic geometry; mean size was 29.2-43.8 nm. In laboratory conditions, AuNPs were toxic against Anopheles stephensi larvae, pupae, and adults. LC50 was 17.36 ppm (larva I), 19.79 ppm (larva II), 21.69 ppm (larva III), 24.57 ppm (larva IV), 28.78 ppm (pupa), and 11.23 ppm (adult). In the field, a single treatment with C. guianensis flower extract and AuNP (10 × LC50) led to complete larval mortality after 72 h. In standard laboratory conditions, the predation efficiency of golden wonder killifish, Aplocheilus lineatus, against A. stephensi IV instar larvae was 56.38 %, while in an aquatic environment treated with sub-lethal doses of the flower extract or AuNP, predation efficiency was boosted to 83.98 and 98.04 %, respectively. Lastly, the antiplasmodial activity of C. guianensis flower extract and AuNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC50 of C. guianensis flower extract was 43.21 µg/ml (CQ-s) and 51.16 µg/ml (CQ-r). AuNP IC50 was 69.47 µg/ml (CQ-s) and 76.33 µg/ml (CQ-r). Overall, our results showed the multipurpose effectiveness of C. guianensis-synthesized AuNPs, since they may be proposed as newer and safer tools in the fight against CQ-r strains of P. falciparum and for field control of malaria vectors, in synergy with wonder killifish predators.

Fern-synthesized nanoparticles in the fight against malaria: LC/MS analysis of Pteridium aquilinum leaf extract and biosynthesis of silver nanoparticles with high mosquitocidal and antiplasmodial activity.

Malaria remains a major public health problem due to the emergence and spread of Plasmodium falciparum strains resistant to chloroquine. There is an urgent need to investigate new and effective sources of antimalarial drugs. This research proposed a novel method of fern-mediated synthesis of silver nanoparticles (AgNP) using a cheap plant extract of Pteridium aquilinum, acting as a reducing and capping agent. AgNP were characterized by UV-vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). Phytochemical analysis of P. aquilinum leaf extract revealed the presence of phenols, alkaloids, tannins, flavonoids, proteins, carbohydrates, saponins, glycosides, steroids, and triterpenoids. LC/MS analysis identified at least 19 compounds, namely pterosin, hydroquinone, hydroxy-acetophenone, hydroxy-cinnamic acid, 5, 7-dihydroxy-4-methyl coumarin, trans-cinnamic acid, apiole, quercetin 3-glucoside, hydroxy-L-proline, hypaphorine, khellol glucoside, umbelliferose, violaxanthin, ergotamine tartrate, palmatine chloride, deacylgymnemic acid, methyl laurate, and palmitoyl acetate. In DPPH scavenging assays, the IC50 value of the P. aquilinum leaf extract was 10.04 µg/ml, while IC50 of BHT and rutin were 7.93 and 6.35 µg/ml. In mosquitocidal assays, LC50 of P. aquilinum leaf extract against Anopheles stephensi larvae and pupae were 220.44 ppm (larva I), 254.12 ppm (II), 302.32 ppm (III), 395.12 ppm (IV), and 502.20 ppm (pupa). LC50 of P. aquilinum-synthesized AgNP were 7.48 ppm (I), 10.68 ppm (II), 13.77 ppm (III), 18.45 ppm (IV), and 31.51 ppm (pupa). In the field, the application of P. aquilinum extract and AgNP (10 × LC50) led to 100 % larval reduction after 72 h. Both the P. aquilinum extract and AgNP reduced longevity and fecundity of An. stephensi adults. Smoke toxicity experiments conducted against An. stephensi adults showed that P. aquilinum leaf-, stem-, and root-based coils evoked mortality rates comparable to the permethrin-based positive control (57, 50, 41, and 49 %, respectively). Furthermore, the antiplasmodial activity of P. aquilinum leaf extract and green-synthesized AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of P. falciparum. IC50 of P. aquilinum were 62.04 µg/ml (CQ-s) and 71.16 µg/ml (CQ-r); P. aquilinum-synthesized AgNP achieved IC50 of 78.12 µg/ml (CQ-s) and 88.34 µg/ml (CQ-r). Overall, our results highlighted that fern-synthesized AgNP could be candidated as a new tool against chloroquine-resistant P. falciparum and different developmental instars of its primary vector An. stephensi. Further research on nanosynthesis routed by the LC/MS-identified constituents is ongoing.

Characterization and mosquitocidal potential of neem cake-synthesized silver nanoparticles: genotoxicity and impact on predation efficiency of mosquito natural enemies.

Mosquitoes (Diptera: Culicidae) serve as important vectors for a wide number of parasites and pathogens of huge medical and veterinary importance. Aedes aegypti is a primary dengue vector in tropical and subtropical urban areas. There is an urgent need to develop eco-friendly mosquitocides. In this study, silver nanoparticles (AgNP) were biosynthesized using neem cake, a by-product of the neem oil extraction from the seed kernels of Azadirachta indica. AgNP were characterized using a variety of biophysical methods, including UV-vis spectrophotometry, FTIR, SEM, EDX, and XRD analyses. Furthermore, the neem cake extract and the biosynthesized AgNP were tested for acute toxicity against larvae and pupae of the dengue vector Ae. aegypti. LC50 values achieved by the neem cake extract ranged from 106.53 (larva I) to 235.36 ppm (pupa), while AgNP LC50 ranged from 3.969 (larva I) to 8.308 ppm (pupa). In standard laboratory conditions, the predation efficiency of a Carassius auratus per day was 7.9 (larva II) and 5.5 individuals (larva III). Post-treatment with sub-lethal doses of AgNP, the predation efficiency was boosted to 9.2 (larva II) and 8.1 individuals (larva III). The genotoxic effect of AgNP was studied on C. auratus using the comet assay and micronucleus frequency test. DNA damage was evaluated on peripheral erythrocytes sampled at different time intervals from the treatment; experiments showed no significant damages at doses below 12 ppm. Overall, this research pointed out that neem cake-fabricated AgNP are easy to produce, stable over time, and can be employed at low dosages to reduce populations of dengue vectors, with moderate detrimental effects on non-target mosquito natural enemies.

DNA barcoding and molecular evolution of mosquito vectors of medical and veterinary importance.

Mosquitoes (Diptera: Culicidae) are a key threat for millions of people worldwide, since they act as vectors for devastating pathogens and parasites. The standard method of utilisation of morphological characters becomes challenging due to various factors such as phenotypical variations. We explored the complementary approach of CO1 gene-based identification, analysing ten species of mosquito vectors belonging to three genera, Aedes, Culex and Anopheles from India. Analysed nucleotide sequences were found without pseudo genes and indels; they match with high similarity in nucleotide Basic Local Alignment Search Tool (BLASTn) search. The partial CO1 sequence of Anopheles niligricus was the first time record submitted to National Center for Biotechnology Information (NCBI). Mean intra- and interspecies divergence was found to be 1.30 and 3.83 %, respectively. The congeneric divergence was three times higher than the conspecifics. Deep intraspecific divergence was noted in three of the species, and the reason could be explained more accurately in the future by improving the sample size across different locations. The transitional and transversional substitutions were tested individually. Ts and Tv substitutions in all the 1st, 2nd and 3rd codons were estimated to be (0.44, 99.51), (40.35, 59.66) and (59.16, 40.84), respectively. Saturation of the sequences was resolved, since both the Ts and Tv exhibited a linear relationship suggesting that the sequences were not saturated. NJ and ML tree analysis showed that the individuals of the same species clustered together based on the CO1 sequence similarity, regardless of their collection site and geographic location. Overall, this study adds basic knowledge to molecular evolution of mosquito vectors of medical and veterinary importance and may be useful to improve biotechnological tools employed in Culicidae control programmes.

Hydrothermal synthesis of titanium dioxide nanoparticles: mosquitocidal potential and anticancer activity on human breast cancer cells (MCF-7).

Mosquito vectors (Diptera: Culicidae) are responsible for transmission of serious diseases worldwide. Mosquito control is being enhanced in many areas, but there are significant challenges, including increasing resistance to insecticides and lack of alternative, cost-effective, and eco-friendly products. To deal with these crucial issues, recent emphasis has been placed on plant materials with mosquitocidal properties. Furthermore, cancers figure among the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. It is expected that annual cancer cases will rise from 14 million in 2012 to 22 million within the next two decades. Nanotechnology is a promising field of research and is expected to give major innovation impulses in a variety of industrial sectors. In this study, we synthesized titanium dioxide (TiO2) nanoparticles using the hydrothermal method. Nanoparticles were subjected to different analysis including UV-Vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), zeta potential, and energy-dispersive spectrometric (EDX). The synthesized TiO2 nanoparticles exhibited dose-dependent cytotoxicity against human breast cancer cells (MCF-7) and normal breast epithelial cells (HBL-100). After 24-h incubation, the inhibitory concentrations (IC50) were found to be 60 and 80 µg/mL on MCF-7 and normal HBL-100 cells, respectively. Induction of apoptosis was evidenced by Acridine Orange (AO)/ethidium bromide (EtBr) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. In larvicidal and pupicidal experiments conducted against the primary dengue mosquito Aedes aegypti, LC50 values of nanoparticles were 4.02 ppm (larva I), 4.962 ppm (larva II), 5.671 ppm (larva III), 6.485 ppm (larva IV), and 7.527 ppm (pupa). Overall, our results suggested that TiO2 nanoparticles may be considered as a safe tool to build newer and safer mosquitocides and chemotherapeutic agents with little systemic toxicity.

Carbon and silver nanoparticles in the fight against the filariasis vector Culex quinquefasciatus: genotoxicity and impact on behavioral traits of non-target aquatic organisms.

Mosquito-borne diseases represent a deadly threat for millions of people worldwide. The Culex genus, with special reference to Culex quinquefasciatus, comprises the most common vectors of filariasis across urban and semi-urban areas of Asia. In recent years, important efforts have been conducted to propose green-synthesized nanoparticles as a valuable alternative to synthetic insecticides. However, the mosquitocidal potential of carbon nanoparticles has been scarcely investigated. In this study, the larvicidal and pupicidal activity of carbon nanoparticle (CNP) and silver nanoparticle (AgNP) was tested against Cx. quinquefasciatus. UV-Vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy, and Raman analysis confirmed the rapid and cheap synthesis of carbon and silver nanoparticles. In laboratory assays, LC50 (lethal concentration that kills 50 % of the exposed organisms) values ranged from 8.752 ppm (first-instar larvae) to 18.676 ppm (pupae) for silver nanoparticles and from 6.373 ppm (first-instar larvae) to 14.849 ppm (pupae) for carbon nanoparticles. The predation efficiency of the water bug Lethocerus indicus after a single treatment with low doses of silver and carbon nanoparticles was not reduced. Moderate evidence of genotoxic effects induced by exposure to carbon nanoparticles was found on non-target goldfish, Carassius auratus. Lastly, the plant extract used for silver nanosynthesis was tested for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity. Overall, our results pointed out that AgNP and CNP can be a candidate for effective tools to reduce larval and pupal populations of filariasis vectors, with reduced genotoxicity and impact on behavioral traits of other aquatic organisms sharing the same ecological niche of Cx. quinquefasciatus.

Fighting arboviral diseases: low toxicity on mammalian cells, dengue growth inhibition (in vitro), and mosquitocidal activity of Centroceras clavulatum-synthesized silver nanoparticles.

Dengue is a mosquito-borne viral disease that has rapidly spread in all regions of the world in recent years. Female mosquitoes, mainly Aedes aegypti, transmit dengue. Approximately 3,900 million people, in 128 countries, are at risk of dengue infection. Recently, a focus has been provided on the potential of green-synthesized nanoparticles as inhibitors of the production of dengue viral envelope (E) protein in Vero cells and downregulators of the expression of dengue viral E gene. Algae are an outstanding reservoir of novel compounds, which may help in the fight against mosquito-borne diseases. In this research, silver nanoparticles (AgNP) were rapidly synthesized using a cheap extract of the alga Centroceras clavulatum. AgNP were characterized by UV­vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). In mosquitocidal assays, LC50 values of C. clavulatum extract against A. aegypti larvae and pupae were 269.361 ppm (larva I), 309.698 ppm (larva II), 348.325 ppm (larva III), 387.637 ppm (larva IV), and 446.262 ppm (pupa). C. clavulatum extract also exhibited moderate antioxidant activity, both in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging assays. LC50 values of C. clavulatum-synthesized AgNP were 21.460 ppm (larva I), 23.579 ppm (larva II), 25.912 ppm (larva III), 29.155 ppm (larva IV), and 33.877 ppm (pupa). Furthermore, C. clavulatum-synthesized AgNP inhibited dengue (serotype dengue virus type-2 (DEN-2)) viral replication in Vero cells. Notably, 50 µg/ml of green-synthesized AgNP showed no cytotoxicity on Vero cells while reduced DEN-2 viral growth of more than 80%; 12.5 µg/ml inhibited viral growth of more than 50%. Cellular internalization assays highlighted that untreated infected cells showed high intensity of fluorescence emission, which denotes high level of viral internalization. Conversely, AgNP-treated infected cells showed reduced levels of fluorescence, failing to show significant viral load. Overall, our study showed that alga-mediated synthesis of metal nanoparticles may be considered to develop newer, safer, and cheap tools in the fight against the dengue virus, serotype DEN-2, and its vector A. aegypti, with little cytotoxicity on mammalian cells.

Green-synthesised nanoparticles from Melia azedarach seeds and the cyclopoid crustacean Cyclops vernalis: an eco-friendly route to control the malaria vector Anopheles stephensi?

The impact of green-synthesised mosquitocidal nanoparticles on non-target aquatic predators is poorly studied. In this research, we proposed a single-step method to synthesise silver nanoparticles (Ag NP) using the seed extract of Melia azedarach. Ag NP were characterised using a variety of biophysical methods, including UV-vis spectrophotometry, scanning electron microscopy, energy-dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy. In laboratory assays on Anopheles stephensi, Ag NP showed LC50 ranging from 2.897 (I instar larvae) to 14.548 ppm (pupae). In the field, the application of Ag NP (10 × LC50) lead to complete elimination of larval populations after 72 h. The application of Ag NP in the aquatic environment did not show negative adverse effects on predatory efficiency of the mosquito natural enemy Cyclops vernalis. Overall, this study highlights the concrete possibility to employ M. azedarach-synthesised Ag NP on young instars of malaria vectors.

Rapid biosynthesis of silver nanoparticles using Crotalaria verrucosa leaves against the dengue vector Aedes aegypti: what happens around? An analysis of dragonfly predatory behaviour after exposure at ultra-low doses.

Aedes aegypti is a primary vector of dengue, a mosquito-borne viral disease infecting 50-100 million people every year. Here, we biosynthesised mosquitocidal silver nanoparticles (AgNP) using the aqueous leaf extract of Crotalaria verrucosa. The green synthesis of AgNP was studied by UV-vis spectroscopy, SEM, EDX and FTIR. C. verrucosa-synthesised AgNPs were toxic against A. aegypti larvae and pupae. LC50 of AgNP ranged from 3.496 ppm (I instar larvae) to 17.700 ppm (pupae). Furthermore, we evaluated the predatory efficiency of dragonfly nymphs, Brachydiplax sobrina, against II and III instar larvae of A. aegypti in an aquatic environment contaminated with ultra-low doses of AgNP. Under standard laboratory conditions, predation after 24 h was 87.5% (II) and 54.7% (III). In an AgNP-contaminated environment, predation was 91 and 75.5%, respectively. Overall, C. verrucosa-synthesised AgNP could be employed at ultra-low doses to reduce larval population of dengue vectors enhancing predation rates of dragonfly nymphs.

Datura metel-synthesized silver nanoparticles magnify predation of dragonfly nymphs against the malaria vector Anopheles stephensi.

Malaria is a life-threatening disease caused by parasites transmitted to people and animals through the bites of infected mosquitoes. The employ of synthetic insecticides to control Anopheles populations leads to high operational costs, non-target effects, and induced resistance. Recently, plant-borne compounds have been proposed for efficient and rapid extracellular synthesis of mosquitocidal nanoparticles. However, their impact against predators of mosquito larvae has been poorly studied. In this study, we synthesized silver nanoparticles (AgNPs) using the Datura metel leaf extract as reducing and stabilizing agent. The biosynthesis of AgNPs was confirmed analyzing the excitation of surface plasmon resonance using ultraviolet-visible (UV-vis) spectroscopy. Scanning electron microscopy (SEM) showed the clustered and irregular shapes of AgNPs, with a mean size of 40-60 nm. The presence of silver was determined by energy-dispersive X-ray (EDX) spectroscopy. Fourier transform infrared (FTIR) spectroscopy analysis investigated the identity of secondary metabolites, which may be acting as AgNP capping agents. In laboratory, LC50 of D. metel extract against Anopheles stephensi ranged from 34.693 ppm (I instar larvae) to 81.500 ppm (pupae). LC50 of AgNP ranged from 2.969 ppm (I instar larvae) to 6.755 ppm (pupae). Under standard laboratory conditions, the predation efficiency of Anax immaculifrons nymphs after 24 h was 75.5 % (II instar larvae) and 53.5 % (III instar larvae). In AgNP-contaminated environment, predation rates were boosted to 95.5 and 78 %, respectively. Our results documented that D. metel-synthesized AgNP might be employed at rather low doses to reduce larval populations of malaria vectors, without detrimental effects on behavioral traits of young instars of the dragonfly Anax immaculifrons.