Culex Quinquefasciatus Larvae Research Articles

WDP formulations using a novel mosquitocidal bacteria, Bacillus thuringiensis subsp. israelensis/tochigiensis (VCRC B-474) – Development and storage stability

A novel mosquito active strain, Bacillus thuringiensis (VCRC B474) sharing the antigens of 2 serotypes, namely israelensis &tochigiensis was characterized by scanning electron microscopy and SDS-PAGE. The spherical and ovoid crystals present in this strain was composed of major polypeptides the size of 28, 65, and 130 kDa respectively. The sporulated cell mass was formulated into water dispersible powder (WDP) formulations with different carrier materials and checked for activity against Culex quinquefasciatus larvae at monthly intervals for up to a year. The formulation containing chalk was the most effective with LC50 values ranging between 0.274–0.523 μg/ml compared to the formulations containing bentonite (0.335-0.775) μg/ml and talc (0.348-0.808 μg/ml). The decline in the activity of these formulations with storage period was as follows: 3 months −14%, 22%, 20% respectively, 6 months - 25%, 35%, 37% respectively, 9 months − 39%, 50%, 47% respectively and 12 months −52%, 43%, 40% respectively. This study demonstrated that wet biomass of bacterial isolates could be simply mixed with carrier materials, dried and used for mosquito larval control without significant loss of activity for up to 6 months at room temperature. Further, this strain of Bacillus thuringiensis var. israelensis/tochigiensis (H14/19) can be a prospective candidate for use in mosquito control programs.

Isolation and growth inhibition potential of entomopathogenic nematodes against three public health important mosquito vectors

The prevalence of mosquito vector borne diseases and the resistance of mosquitoes to conventional pesticides have been of important public concern to the mosquito endemic countries. Present study was conducted to identify the native bio-larvicidal potential of the entomopathogenic nematodes; Steinernema siamkayai (KPR-4) Heterohabditis indica (KPR-8), Steinernema glaseri and Steinernema abbasi. The isolated nematodes were subsequently cultured and evaluated their larvicidal potential against the larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Among the tested four different nematode species, the S. abassi exerted the highest mortality against A. aegypti (97.33%), the H. indica (KPR-8) against A. stephensi (97.33%) and the S. siamkayai (KPR-4) against C. quinquefasciatus (98.67%). The maximal mosquito-larvicidal property of EPNs was found with the LC50 and LC90 values (IJs/larvae): S. abbasi = 12.47 & 54.35 on A. aegypti; H. indica KPR-8 = 19.88 & 66.81 on A. stephensi and S. siamkayai KPR-4 = 16.69 & 58.97 on C. quinquefasciatus, respectively. The presently generated data on the molecular and larvicidal characteristics of the entomopathogenic nematodes form an important baseline data that upon further research would lead to the development of eco-friendly mosquito-control agent.

An aromatic cluster in Lysinibacillus sphaericus BinB involved in toxicity and proper in-membrane folding

The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. Based on structural alignments with other toxins, an aromatic cluster in the C-terminal domain of BinB (termed here BC) has been proposed to be important for toxicity. We tested this experimentally using BinB mutants bearing single mutations in this aromatic cluster. Consistent with the hypothesis, two of these mutations, F311A and F315A, were not toxic to Culex quinquefasciatus larvae and were unable to permeabilize liposomes or elicit ion channel activity, in contrast to wild-type BinB. Despite these effects, none of these mutations altered significantly the interaction between the activated forms of the two subunits in solution. These results indicate that these aromatic residues on the C-terminal domain of BinB are critical for toxin insertion in membranes. The latter can be by direct contact of these residues with the membrane surface, or by facilitating the formation a membrane-inserting oligomer.

Enhancement of insect susceptibility and larvicidal efficacy of Cry4Ba toxin by calcofluor

BackgroundMosquitoes transmit many vector-borne infectious diseases including malaria, dengue, chikungunya, yellow fever, filariasis, and Japanese encephalitis. The insecticidal δ-endotoxins Cry4, Cry11, and Cyt produced from Bacillus thuringiensis have been used for bio-control of mosquito larvae. Cry δ-endotoxins are synthesised as inactive protoxins in the form of crystalline inclusions in which they are processed to active toxins in larval midgut lumen. Previously, we demonstrated that the activated Cry4Ba toxin has to alter the permeability of the peritrophic membrane (PM), allowing toxin passage across PM to reach specific receptors on microvilli of larval midgut epithelial cells, where the toxin undergoes conformational changes, followed by membrane insertion and pore formation, resulting in larval death. A peritrophic membrane (PM)-binding calcofluor has been proposed to inhibit chitin formation and enhance baculovirus infection of lepidopteran Trichoplusia ni.MethodsIn this study, Aedes aegypti larvae were fed with the calcofluor and Cry4Ba toxin to investigate the effect of this agent on the toxicity of the Cry4Ba toxin.ResultsCalcofluor displayed an enhancing effect when co-fed with the Cry4Ba wild-type toxin. The agent could restore the killing activity of the partially active Cry4Ba mutant E417A/Y455A toward Ae. aegypti larvae. PM destruction was observed after larval challenge with calcofluor together with the toxin. Interestingly, calcofluor increased Cry4Ba toxin susceptibility toward semi-susceptible Culex quinquefasciatus larvae. However, calcofluor alone or in combination with the toxin showed no mortality effect on non-susceptible fresh-water fleas, Moina macrocopa.ConclusionsOur results suggest that PM may contribute to the resistance of the mosquito larvae to Cry4Ba toxin. The PM-permeability alternating calcofluor might be a promising candidate for enhancing insect susceptibility, which will consequently improve Cry4Ba efficacy in field settings in the future.

Highly specific host-pathogen interactions influence Metarhizium brunneum blastospore virulence against Culex quinquefasciatus larvae

ABSTRACT Entomopathogenic fungi are potential biological control agents of mosquitoes. Our group observed that not all mosquitoes were equally susceptible to fungal infection and observed significant differences in virulence of different spore types. Conidiospores and blastospores were tested against Culex quinquefasciatus larvae. Blastospores are normally considered more virulent than conidia as they form germ tubes and penetrate the host integument more rapidly than conidia. However, when tested against Cx. quinquefasciatus, blastospores were less virulent than conidia. This host-fungus interaction was studied by optical, electron and atomic force microscopy (AFM). Furthermore, host immune responses and specific gene expression were investigated. Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores did not readily adhere to Culex larval integument and the main route of infection was through the gut. Adhesion forces between blastospores and Culex cuticle were significantly lower than for other insects. Larvae challenged with blastospores showed enhanced immune responses, with increased levels of phenoloxidase, glutathione-S-transferase, esterase, superoxide dismutase and lipid peroxidase activity. Interestingly, M. brunneum pathogenicity/stress-related genes were all down-regulated in blastospores exposed to Culex. Conversely, when conidia were exposed to Culex, the pathogenicity genes involved in adhesion or cuticle degradation were up-regulated. Delayed host mortality following blastospore infection of Culex was probably due to lower adhesion rates of blastospores to the cuticle and enhanced host immune responses deployed to counter infection. The results here show that subtle differences in host-pathogen interactions can be responsible for significant changes in virulence when comparing mosquito species, having important consequences for biological control strategies and the understanding of pathogenicity processes.

Chicory (Cichorium intybus) and wormwood (Artemisia absinthium) extracts exhibit strong larvicidal activity against mosquito vectors of malaria, dengue fever, and filariasis

Vector-borne diseases transmitted by mosquitoes cause globally important diseases such as malaria, dengue fever, and filariasis. The incidence of these diseases can be reduced through mosquito control programs but these control programs currently rely on synthetic insecticides that can impact the environment, and has selected widespread mosquito resistance. Environment friendly and biodegradable natural insecticides discovered in plants offer an alternative approach to mosquito control. Here, we investigated extracts from root or aerial parts of Chicory (Cichorium intybus) and wormwood (Artemisia absinthium) against the early 4th instar larvae of Anopheles stephensi (malaria vector), Aedes aegypti (dengue fever vector), and Culex quinquefasciatus (filariasis vector). The root and aerial parts extracts of A. absinthium and C. intybus at 200, 100, 50, 25 and 12.5 ppm caused significant mortality of the tested mosquito species. Root extracts exhibited higher larvicidal activity that aerial part extracts. The highest larvicidal activity was recorded in methanol extract of roots of C. intybus with LC50 = 66.16, 18.88 and LC¬90 = 197.56, 107.16 ppm for An. stephensi; LC50 = 78.51, 40.15 and LC90 = 277.31, 231.28 ppm for Ae. aegypti and LC50 = 103.99, 64.56 and LC¬90 = 314.04, 247.54 ppm for Cx. quinquefasciatus. These results reveal potent mosquito larvicidal activity against vectors of malaria, dengue fever, and filariasis is present in extracts of chicory and wormwood.

Toxicity of Fusarium oxysporum-VKFO-01 Derived Silver Nanoparticles as Potential Inseciticide Against Three Mosquito Vector Species (Diptera: Culicidae)

Mosquitoes are important public health pest transmitting major diseases such as dengue, malaria, zika and chikungunya. In the current study, isolation and identification of an entomopathogenic fungus was confirmed using 18s rDNA sequencing analysis. Silver nanoparticles were synthesized using Fusarium oxysporum-VKFO-01 culture filtrate with silver nitrate and green synthesized AgNPs were tested on larvae of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Fusarium oxysporum-VKFO-01 culture filtrate derived synthesized AgNPs were characterized using, UV–Vis spectroscopy, FT-IR, EDaX, XRD, SEM, TEM and AFM analysis. Larvicidal bioassay results show that strong mortality produced by F. oxysporum-VKFO-01 derived AgNPs. LC50 and LC90 values in An. stephensi, 69.985–401.639 μg/ml; Ae. aegypti, 42.942–272.842 μg/ml and Cx. quinquefasciatus, 97.557–706.962 μg/ml respectively. The present study shows that F. oxysporum-VKFO-01 derived green AgNPs are very effective against An. stephensi, Ae. aegypti and Cx. quinquefasciatus mosquito larvae. Phylogenetic construction of 18s rDNA sequence was done for finding the taxonomic identification and the evolutionary steps of isolated Fusarium sp. Our evaluation highlighted those F. oxysporum-VKFO-01 culture filtrate mediated synthesized silver nanoparticles as a very effective green pesticide for control of mosquitoes.

Antimicrobial and larvicidal activity of eco-friendly silver nanoparticles synthesized from endophytic fungi Phomopsis liquidambaris

Abstract In the quest for developing a biological-based nanomaterial for therapeutic applications, here we report a novel benign method of silver nanoparticles synthesized using the endophytic fungi Phomopsis liquidambaris strain SA1 (SAAgNPs). A viable method was developed to generate silver nanoparticles by reacting with silver nitrate and an extracellular filtrate of P.liquidambaris as a reducing and stabilizing agent. After formation of brown color, synthesized SAAgNPs was measured for its Surface Plasmon Resonance (SPR) which produces an intense absorption spectrum at 430 nm. Fourier Transform Infrared Spectroscopy (FTIR) revealed the functional group moieties involved as a capping and reducing agent in SAAgNPs synthesis. Stable, spherical and polydispersed nanoparticles with an average size of 18.7 nm were affirmed by Transmission Electron Microscope (TEM). X-ray Diffraction study showed the diffraction peaks corresponds to (111), (200), (220) and (311) planes resembling cubic crystalline in nature. Energy dispersive analysis (EDAX) identifies the silver as a major compositional element. Moreover, SAAgNPs performed better growth inhibitory activity against the tested pathogens. Further, SAAgNPs arrest the growth of IInd and IVth instar larvae of Aedes aegypti and Culex quinquefasciatus in a dose-dependent method. Finally, SAAgNPs exhibited very low hemolytic activity; suggesting the biocompatibility and feasibility of SAAgNPs as a therapeutic agent. Thus, the study bestowed the isolation of endophytic fungi Phomopsis liquidambaris and its mediated synthesis of silver nanoparticles. Further, SAAgNPs proved its efficacy as an effective antimicrobial and mosquitocidal agent.

Nanosilver crystals capped with Bauhinia acuminata phytochemicals as new antimicrobials and mosquito larvicides

To develop novel nanoformulated insecticides and antimicrobials, herein we produced Ag nanoparticles (AgNPs) using the Bauhinia acuminata leaf extract. This unexpensive aqueous extract acted as a capping and reducing agent for the formation of AgNPs. We characterized B. acuminata-synthesized AgNPs by UV–vis and FTIR spectroscopy, XRD and TEM analyses. UV–vis spectroscopy analysis of B. acuminata-synthesized AgNPs showed a peak at 441.5 nm. FTIR shed light on functional groups from the phytoconstituents involved in nanosynthesis. XRD of B. acuminata-synthesized AgNPs suggested a face-centered cubic structure, with a highly crystalline nature. TEM of B. acuminata-synthesized AgNPs revealed mean size of 25 nm, with round shape. AgNPs tested at 60 μg/mL inhibited the growth of 5 bacteria and 3 fungal pathogens. In the insecticidal assays on important mosquito species, LC50 of the aqueous extract of B. acuminata leaves on the larvae of Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus were 204.07, 226.02, and 249.24 μg/mL, respectively. The B. acuminata-synthesized AgNPs exhibited higher larvicidal efficacy, with LC50 values of 24.59, 27.19, and 30.19 μg/mL, respectively. Therefore, herein we developed a single-step, reliable, inexpensive, and environmentally non-toxic synthesis process to obtain AgNPs with high bioactivity against pathogens and vectors. Given the effective antimicrobial and larvicidal activity, nanoparticles fabricated using plant extracts and extremely low concentrations of trace elements, such as silver, can be exploited for multipurpose activities. Our results pointed out that B. acuminata-synthesized AgNPs have a promising potential in antimicrobial food packaging, as well as a foliar spray to control plant pathogens in the field, and to synergize the efficacy of fungicidal and larvicidal formulations.

LARVICIDAL ACTIVITY OF LANTANA INDICA AND VITEX NEGUNDO ON CULEX QUINQUEFASCIATUS

Objectives: The activity of two different leaf extracts of Lantana indica and Vitex negundo is tested against the 3rd and 4th instar Culex quinquefasciatus larvae to evaluate the potency of the extracts as a larvicide and to find an ecologically sustainable alternative to chemical insecticides. A bioinformatics screening approach was performed to evaluate the in vivo results.Methods: The obtained larvae’s from nearby water sources were tested with N, N-diethyl-meta-toluamide (DEET) as the positive control which is the commercial chemical mosquito repellent and the solvents used for the respective plant extracts act as the negative control. Petroleum ether (PE), ethyl acetate (EA) and an aqueous (AQ) extract were prepared for both L. indica and V. negundo extracts, and its larvicidal activity was tested. A docking based approach was used to study the inhibitory effect of known active compounds from L. indica and V. negundo against acetylcholine esterase (AChE) and sterol binding protein as targets.Results: On comparing the results between three plants extract for its larvicidal activity, the EA extract of V. negundo and L. indica is found to be potent with a low LC50 value. Further, the docking studies between active compounds of L. indica and V. negundo with AChE and Sterol binding protein as targets showed that the compound tangeritin-1 had a good docking score compared to DEET and could be a natural alternative for larvicidal activity in the mosquito.Conclusion: Individual activity of tangeritin-1 could be further studied with mosquito mortality studies and molecular simulations and develop tangeritin-1 as a potential larvicidal compound for commercial use.

Interspecific variation, habitat complexity and ovipositional responses modulate the efficacy of cyclopoid copepods in disease vector control

The use of predatory biological control agents can form an effective component in the management of vectors of parasitic diseases and arboviruses. However, we require predictive methods to assess the efficacies of potential biocontrol agents under relevant environmental contexts. Here, we applied functional responses (FRs) and reproductive effort as a proxy of numerical responses (NRs) to compare the Relative Control Potential (RCP) of three cyclopoid copepods, Macrocyclops albidus, M. fuscus and Megacyclops viridis towards larvae of the mosquito Culex quinquefasciatus. The effects of habitat complexity on such predatory impacts were examined, as well as ovipositional responses of C. quinquefasciatus to copepod cues in pairwise choice tests. All three copepod species demonstrated a population destabilising Type II FR. M. albidus demonstrated the shortest handling time and highest maximum feeding rate, whilst M. fuscus exhibited the highest attack rate. The integration of reproductive effort estimations in the new RCP metric identifies M. albidus as a very promising biocontrol agent. Habitat complexity did not impact the FR form or maximum feeding rate of M. albidus, indicating that potentially population destabilising effects are robust to habitat variations; however, attack rates of M. albidus were reduced in the presence of such complexity. C. quinquefasciatus avoided ovipositing where M. albidus was physically present, however it did not avoid chemical cues alone. C. quinquefasciatus continued to avoid M. albidus during oviposition when both the treatment and control water were dyed; however, when an undyed, predator-free control was paired with dyed, predator-treated water, positive selectivity towards the treatment water was stimulated. We thus demonstrate the marked predatory potential of cyclopoid copepods, utilising our new RCP metric, and advocate their feasibility in biological control programmes targeting container-style habitats. We also show that behavioural responses of target organisms and environmental context should be considered to maximise agent efficacy.

Toxicity of different fatty acids and methyl esters on Culex quinquefasciatus larvae

The Culex quinquefasciatus mosquito is a vector of several diseases, and its control has been performed with synthetic insecticides, which may have human and environmental side effects. Thus, the use of new and safe molecules are important, and this study evaluated the toxicity of active substances against this mosquito. The oleic, linoleic, linolenic, palmitic and stearic acids and their respective methyl esters were tested against fourth instar C. quinquefasciatus larvae. Oleic, linoleic and linolenic acids had LC50 values of 8.58, 10.04 and 19.78 mg/L, respectively. Histological analysis showed that these three compounds caused cell instability with an increase in the number of vesicles in the fat body and in the midgut cells. Based on these results, glucose, triglyceride, and protein levels were evaluated after 1 h of acid exposure. These compounds decreased in insects treated with linoleic acid. Linolenic acid also caused a significant increase in acetylcholinesterase activity. These results show that oleic, linoleic, and linoleic acids have a lower LC50 for C. quinquefasciatus, affecting its metabolism and the morphology of midgut and fat body.

Screening of phytochemical and pharmacological activities of Syzygium caryophyllatum (L.) Alston

BackgroundThe systematic screening of plant species with the purpose of discovering new bioactive compounds is prerequisite for any bioprospecting study. Therefore the present study was carried out to assess the phytochemical content and the evaluation of in vitro antioxidant, antibacterial, larvicidal and antidiabetic activities of the methanol extracts of S.caryophyllatum bark, leaves, fruit pulp and seeds.MethodsThe quantitative estimation of total phenol, flavonoid and tannin content of the extracts of S. caryophyllatum bark, leaf, fruit pulp and seeds were analyzed by using standard methods. The present study also conducted to screen antioxidant (DPPH, ABTS assays), antimicrobial, antidiabetic (in vitro α-amylase and α-glucosidase inhibitory assays) and larvicidal (against fourth instars larvae of Aedes aegypti and Culex quinquefasciatus) activities of methanol extract of S. caryophyllatum.ResultsThe results of quantitative phytochemical analysis revealed the presence of maximum amount of phytoconstituents such as phenol, flavonoids and tannins in the leaf, bark and seed extract when compared to fruit pulp extracts. Free radical scavenging activity indicated that methanol bark, leaves, fruit and seeds extracts has significant free radical scavenging ability on DPPH with percentage inhibition of 88.15%, 81.31%, 75.24% and 83.36% respectively. The in vitro α-amylase and α-glucosidase inhibitory studies of the methanol crude extracts of four different plant parts of S. caryophyllatum (Bark, leaf fruit pulp and seed) showed good inhibitory activities in concentration dependent manner. The maximum percentage inhibitory activity of 78.03% was showed at concentration of 500 μg/ml seed extracts followed by bark (78.03%), leaf (69.4%) and fruit pulp (56.9%) at the same concentration. While the percentage inhibitory activity of four extracts also showed potent inhibition of α-glucosidase; maximum inhibition exhibited at the concentration of 100 μg/ml by bark extract (80.9%) compared with other extracts, leaf (78.2%), seed (77.59%) and fruit pulp (63.35%). The leaf essential oil and four extracts showed significant mortality against fourth instars larvae of Ae. aegypti and Cx. quinquefasciatus respectively.ConclusionThus the present study suggests that S.caryophyllatum plant parts can be used as natural antioxidant source to prevent diseases associated with free radicals. Also, this plant can be a good source for further purification studies for isolation and characterization of compounds related to these antioxidants, antidiabetic and antibacterial activities.

Larvicidal efficacy of 4-Dodecene derived from marine Streptomyces sp. VITVSK1 on Blood-Sucking Parasites

The present study was aimed to assess the larvicidal activity of the lead compound 4-dodecene isolated from Streptomyces sp. VITVSK1 against cattle ticks, Haemaphysalis bispinosa and Rhipicephalus (Boophilus) microplus; Anopheles subpictus and Culex quinquefasciatus larvae. The compound was methanol extracted and purified by silica gel column chromatography and preparative thin layer chromatography using a solvent system of chloroform: methanol (9:1 v/v). The active fractions were concentrated and analyzed by mass spectroscopy (MS) and based on the similarity index with reference compounds available in the mass spectra library, National Institute of Standards and Technology (NIST) the structure of the pure compound was elucidated. The structure of the purified compound was further confirmed with the help of 1H-NMR and 13C-NMR spectra. In this study, the crude extract and the lead compound 4-dodecene extracted from Streptomyces sp. VITVSK1 were tested against the larvae of the selected parasites at varying concentrations. Based on the spectral studies the compound was identified as 4-dodecene and the molecular formula of the aliphatic compound was established as C14H20O2. The methanolic extract (1000 ppm) showed antiparasitic effects after 24 h of exposure and the mortality of the parasites was observed in the larvae of A. subpictus (LC50=116.1 ppm; r2=0.864); R. microplus (LC50=331.79 ppm; r2=0.948); C. quinquefasciatus (LC50=168.59 ppm; r2=0.425) and H. bispinosa (LC50=284.02 ppm; r2= 0.885). The maximum efficacy was observed with the extracted compound 4-dodecene and the LC50 and r2 values were calculated against the larvae of A. subpictus (82.65 ppm; 0.688), R. microplus (173.48 ppm; 0.857), C. quinquefasciatus (78.32 ppm; 0.769) and against H. bispinosa (126.59 ppm; 0.840) respectively. The active compound 4-dodecene from Streptomyces sp. VITVSK1 exhibited profound larvicidal activity against blood-sucking parasites, A. subpictus, R. microplus, C. quinquefasciatus and H. bispinosa. The results of this study suggest that the active compound 4-dodecene can be probed further to be developed as a larvicidal agent.

High Levels of Resistance in A Culex quinquefasciatus Population to the Insecticide Permethrin in Filariasis Endemic Areas in Central Java

Resistance among Culex mosquitoes to various insecticides has been reported in many countries. However, there have been no studies of the resistance status of Culex in Indonesia. There is a need for such studies to develop a database for use in vector control management. This study aimed to investigate the insecticide resistance status of C. quenquefasciatus , which is the primary vector of filariasis, to aid the planning of a vector control management program. In the present study, Culex quinquefasciatus larvae were collected from five districts/municipalities where filariasis is endemic in Central Java. The larvae were reared to adult stage, and insecticide susceptibility testing was then conducted according to standard bioassay procedures of the World Health Organization (WHO). The results of the bioassays showed that C. quinquefasciatus had a high level of resistance against 0.75% permethrin, with mortality rates ranging from 4.8 to 21.6%. The lowest resistance was found among mosquitoes collected from Grobogan district. This may be explained by the district’s remote geographical location. The high level of resistance found in the present study may be caused by exposure to local insecticides, which have been applied for many years as part of a dengue vector control program. These insecticides may also have contaminated the breeding sites of C. quinquefasciatus mosquitoes. Better vector control management is needed to help prevent the development and spread of resistance. Such management should include routine insecticide surveillance and insecticide alternation.

Receptor protein of Lysinibacillus sphaericus mosquito-larvicidal toxin displays amylomaltase activity

The activated binary toxin (BinAB) from Lysinibacillus sphaericus binds to surface receptor protein (Cqm1) on the midgut cell membrane and kills Culex quinquefasciatus larvae on internalization. Cqm1 is attached to cells via a glycosyl-phosphatidylinositol (GPI) anchor. It has been classified as a member of glycoside hydrolase family 13 of the CAZy database. Here, we report characterization of the ordered domain (residues 23–560) of Cqm1. Gene expressing Cqm1 of BinAB susceptible mosquito was chemically synthesized and the protein was purified using E. coli expression system. Values for the Michaelis-Menten kinetics parameters towards 4-nitrophenyl α-D-glucopyranoside (α-pNPG) substrate were estimated to be 0.44 mM (Km) and 1.9 s−1 (kcat). Thin layer chromatography experiments established Cqm1 as α-glucosidase competent to cleave α-1,4-glycosidic bonds of maltose and maltotriose with high glycosyltransferase activity to form glucose-oligomers. The observed hydrolysis and synthesis of glucose-oligomers is consistent with open and accessible active-site in the structural model. The protein also hydrolyses glycogen and sucrose. These activities suggest that Cqm1 may be involved in carbohydrate metabolism in mosquitoes. Further, toxic BinA component does not inhibit α-glucosidase activity of Cqm1, while BinB reduced the activity by nearly 50%. The surface plasmon resonance study reveals strong binding of BinB with Cqm1 (Kd, 9.8 nM). BinA interaction with Cqm1 however, is 1000-fold weaker. Notably the estimated Kd values match well with dissociation constants reported earlier with larvae brush border membrane fractions. The Cqm1 protein forms a stable dimer that is consistent with its apical localization in lipid rafts. Its melting temperature (Tm) as observed by thermofluor-shift assay is 51.5 °C and Ca2+ provides structural stability to the protein.

Efficacy of the Volatile Oil from Water Celery (Helosciadium nodiflorum, Apiaceae) against the Filariasis Vector Culex quinquefasciatus, the Housefly Musca domestica, and the African Cotton Leafworm Spodoptera littoralis.

Water celery (Helosciadium nodiflorum, Apiaceae) is a common and invasive hydrophyte occurring in rivers and slow waters of Europe. In the past, it was consumed as a vegetable and spices, but currently it has mostly been abandoned. As a result, this plant produces an unexploited biomass occluding rivers and slow waters. With the aim to valorise water celery as an important biomass source for the development of botanical insecticides, in this work, we investigated the inhibitory effects of the essential oil hydrodistilled from its aerial parts against larvae of the filariasis vector Culex quinquefasciatus, adults of the housefly Musca domestica, and larvae of the African cotton leafworm Spodoptera littoralis. The essential oil, which was characterized by the insecticidal limonene (39.8%) and myristicin (35.3%), exerted important toxic effects on 3rd instar larvae of C. quinquefasciatus and S. littoralis and on adults of M. domestica, with LD50 of 50.4 μl l-1 , 116.7 μg larva-1 , and 45.1 (male) and 70.1 (female) μg adult-1 , respectively. The oil did not show any noticeable inhibitory activity on the acetylcholinesterase enzyme. Our findings add scientific evidences highlighting water celery as an important biomass source to be used for the control of insect pests and vectors of high economic importance.

Chemical composition and insecticidal activity of the essential oil from Helichrysum faradifani endemic to Madagascar

Helichrysum faradifani (Asteraceae) is a perennial shrub growing in rocky and sandy places of Madagascar. The plant is used in the Malagasy traditional medicine as a wound-healing agent, disinfectant and for the treatment of syphilis, diarrhea, cough and headache. In the present work, we analysed the chemical composition of the essential oil distilled from the aerial parts of H. faradifani by GC-MS and evaluated its insecticidal activity against 2nd, 3rd and 4th instar larvae of the lymphatic filariasis vector Culex quinquefasciatus by acute toxicity assays. The most sensitive were 2nd instar (LC50 = 85.7 μL L−1) larvae. For the 3rd and 4th instar larvae, the estimated LC50 were 156.8 and 134.1 μL L−1, respectively. Monoterpene hydrocarbons (51.6%) were the major fraction of the essential oil, with the bicyclic α-fenchene (35.6%) as the predominant component. Sesquiterpene hydrocarbons (34.0%) were the second major group characterising the oil, with γ-curcumene (17.7%) as the most abundant component.

Larvicidal, Histopathological Efficacy of Penicillium daleae against Larvae of Culex quinquefasciatus and Aedes aegypti Plus Biotoxicity on Artemia nauplii a Non-target Aquatic Organism.

Mosquitoes can transmit the terrible diseases to human beings. Soil-borne fungal products act as potential source for low-cost chemicals, used for developing eco-friendly control agents against mosquito-vector borne diseases. The prime aim of study was to check the larvicidal potential of fungus mycelia (by ethyl acetate solvent) extract from Penicillium daleae (KX387370) against Culex quinquefasciatus and Aedes aegypti and to test the toxicity of brine shrimp Artemia nauplii, by observing the physiological activity. The ethyl acetate extract of P. daleae mycelia (after 15 days) from Potato dextrose broth (PDB) medium revealed better result with least LC50 and LC90 values of I-IV instars larvae of Cx. quinquefasciatus (LC50 = 127.441, 129.087, 108.683, and 93.521; LC90 = 152.758, 158.169, 139.091, and 125.918 μg/ml) and Ae. aegypti (LC50 = 105.077, 83.943, 97.158, and 76.513; LC90 = 128.035, 106.869, 125.640, and 104.606 μg/ml) respectively. At higher concentration (1000 μg/ml) of extracts, mortality begins at 18 h of exposure and attained 100% mortality after 48 h exposure. Overall, the activity was depends on the dose and time of exposure to the extracts. The stereomicroscopic and histopathological analysis of Ae. aegypti and Cx. quinquefasciatus larvae treated with mycelium ethyl acetate extract showed complete disintegration of abdominal region, particularly the midgut and caeca, loss of cuticular parts and caudal hairs. Morphological characterization of the fungi was performed and taxonomically identified through 5.8s rDNA technique. The phylogenetic analysis of rDNA sequence was carried out to find out the taxonomic and the evolutionary sketch of isolate in relation to earlier described genus Penicillium. Behavior and swimming speed alteration was analyzed together with mortality. The results of the experiment indicates that swimming behavior recorder (SBR) is a appropriate tool to detect individual swimming speed of the A. nauplii organisms, since the values have been obtained in accordance with control monitored results showed the 2.75 mm s-1 and after 24 h treated found to be 0.72 mm s-1, respectively. The extract-exposed to A. nauplii showed changes in body structures, i.e., intestine enlargement, eye formation, outer shell malformations and loss of antennae. In the present study, we aimed to investigate the toxicity of the ethyl acetate extract of P. daleae on A. nauplii larvae by performing the mortality, behavior and alterations in swimming responses. This is the first time report on the larvicidal efficacy of P. daleae ethyl acetate extract against Cx. quinquefasciatus and Ae. aegypti larvae.

Characterization of a novel mosquitocidal toxin of Cry50Ba and its potential synergism with other mosquitocidal toxins

A putative toxin gene of cry50Ba was successfully expressed in E. coli cells and confirmed that the purified Cry50Ba toxin had very high toxic activity against Culex quinquefasciatus larvae. Furthermore, the potential synergism of Cry50Ba toxin with Cry2Aa, Cry4Aa and Cry11Aa at a ratio of 1:1 was investigated. Although no significant synergism with other toxins was observed, the Cry50Ba as a novel toxin could be used to delay rapid onset of resistance in mosquito.