To characterize the production and larvicidal activity of Xenorhabdus stockiae KUT6 Petroleum ether extracts from Luria Broth and induced Quorum sensing medium containing N-3- oxododecanoyl Homoserine Lactone inducer against dengue vector Aedes aegypti. The Galleria mellonella larvae were reared for the isolation of Steinernema saimkayi symbiont Xenorhabdus stockiae KUT6 from Cucumber field soil sample in NBTA. Then for the extraction of compounds the KUT6 strains were cultured in Luria Broth and Quorum Sensing optimized media using N-3-oxododecanoyl homoserine lactone inducer. The larvicidal activity of Xenorhabdus stockiae KUT6 of petroleum ether extracts were bioassayed against 4th instar Aedes aegypti dengue vector. The maximum rate of mortality were recorded of the samples A-24h, B-48h, C-72h, A1-24h, B1-48h, C1-72h at different concentrations 50 µg/ml, 100 µg/ml and 150 µg/ml respectively for 24h to 72h of exposure treatment. The morphological characteristics of Xenorhabdus stockiae KUT6 in NBTA were red core colonies with blue background surrounded by zone of inhibition. After 24h exposure maximum rate of 100% mortality of Aedes aegypti 4th instar larvae was attained when treated with sample C1-72h 50 µg/ml of the petroleum ether extracts of quorum sensed medium whereas the sample C 72h petroleum ether extracts of KUT6 cultured in Luria broth recorded 100% mortality at 150 µg on 24h exposure indicates enhancement in the product yield. The study assures the use of Xenorhabdus stockiae KUT6 petroleum ether extracts as biocontrol agent could be beneficial for the control of dengue vectors.
Simulium (Simulium) contractum Takaoka from Sulawesi, Indonesia was known only as the pupa. Its female, male and mature larva are described for the first time. The tentative assignment of this species in the Simulium dumogaense species-group is confirmed by the adult characters including the female and male genitalia. The female and male of this species are similar to those of Simulium (Simulium) tumpaense Takaoka and Roberts but are distinguished by the yellowish femora.
Goat Warble Fly Infestation (GWFI) is also known as subcutaneous myiasis caused by Przhevalskiana silenus (Diptera: Oestridae). It is widely distributed in tropical and sub-tropical areas of the world. In goats, WFI is usually detected through conventional procedure which underestimated the infestation. The current study was designed to determine the serodiagonsis of GWFI (through IDEXX Hypodermosis serum antibody test) and also aimed to investigate its seroepizootiological profile in Pothwar region, Pakistan from 2013-14. The results showed that average seropositivity (ELISA kit) of GWFI was 18.5% whereas, it was 11% by using conventional procedure (Palpation method) depicting a significant difference (p<0.05). Higher seropositivity (30.8%) was observed in Jhelum district as compared to e Attock district (6%). The L1 larvae were found in September, while nodules start appearing in October to December and last until the end of February. The month wise peaks of optical density (OD) was higher in December which gradually decrease along with the end of winter season. The prevalence of GWFI revealed no significant difference among three host breeds (Jattal, Beetal and Tedy). According to the results, high infestation rate (28%) was observed in young animals of age group < 1 year as compared to old animals (> 2 years). Topographically, hilly areas (33%) provide favourable climatic conditions for the propagating of larval stages. Sex difference showed no significant difference. The seroprevalence varied significantly with respect to age, month, districts and topographical location. The current study proved that serologic diagnosis (commercial ELISA kit) as more sensitive and accurate for timely diagnosis of GWFI than traditional method. The information on the epizootiology of P. silenus in goats of Pothwar region would help in devising effective control strategies.
This research was undertaken to evaluate the nematicidal activity of various concentrations of aqueous tinctures of 80 plant species towards L1-2 of S. papillosus. For the experiment with larvae of S. papillosus, there were used 0.19%, 0.75% and 3.00% aqueous tinctures of plants. Out of 80 tested species, nematicidal activity against L1-2 of S. papillosus was displayed by 20 plants. The greatest activity (LC50 = 0.060-0.069%) towards larvae of S. papillosus was exerted by Teucrium polium, Achillea millefolium, Genista tinctoria and Ulmus laevis. Less expressed nematicidal activity (LC50 = 0.070-0.079%) was recorded for Thalictrum minus, Stachys recta, Falcaria vulgaris, Lavatera thuringiaca. Even lower effect (LC50 = 0.080-0.089%) was shown by aqueous tinctures of Mentha × piperita, Achillea millefolium, Salvia nutans, Eryngium campestre and Cerasus fruticosa. The following plants could be arranged in declining order of effectiveness of nematicidal activity (LC50 = 0.090-0.165%) Malus sylvestris, Tragopogon orientalis, Erigeron annuus, Grindelia squarrosa, Urtica dioica, Daucus carota, Medicago sativa, Carduus acanthoides, Ulmus minor and Hieracium umbellatum. A far weaker effect on the nematodes was displayed by Bromopsis inermis and Tragopogon podolicus. Aqueous tinctures of 60 other studied species of plants exhibited low nematicidal activity in 3.00% aqueous tincture, while in 0.19% and 0.75% aqueous tinctures, no nematicidal activity was seen. The results of the research suggest that in the conditions of natural ecosystems, some species of plants of the Apiaceae, Asteraceae, Fabaceae, Lamiaceae, Malvaceae, Rosaceae, Ulmaceae and Urticaceae families could reduce vitality of free-living L1-2 larvae of S. papillosus.
Cx. quinquefasciatus is a common nuisance mosquito widely distributed in tropical and subtropical areas. This mosquito is also a vector of urban filariasis. Control with chemicals has been hampered by the development of resistance against chemical insecticides and rising problems of environmental contamination associated with them. Therefore, it is important to adopt more integrated mosquito management approaches that include sustainable, non chemical solutions. The mermithid nematode Romanomermis iyengari is one of several natural control alternatives to synthetic pesticides for mosquito suppression. This study evaluated the effectiveness of the nematode R. iyengari for control of Cx. quinquefasciatus. The nematode R. iyengari was mass-produced, and pre-parasitics (J2) were used for laboratory and field experiments. In laboratory experiments, two concentrations of pre-parasitics (5 and 10 J2 per larva) were tested against L1, L2 and L3 instars larvae of Cx. quinquefasciatus. Infected larvae were observed daily to determine their mortality rate and the number of postparasitic nematodes emerging from dead larvae. In field experiments, 1000, 2000 and 3000 J2/m2 were sprayed in separate natural Cx. quinquefasciatus breeding sites. After treatment, the larval mosquito density in the breeding sites was assessed every 5 days. Laboratory results showed that all tested Cx. quinquefasciatus instars larvae were susceptible to nematode infection. The mortality rates observed for each larval stage indicated that the concentration of 10 J2 kills larvae faster, and that the L1 larvae died earlier than older larvae. The average number of post-parasitic nematodes emerging per larva increases with increasing nematode concentration; also more post-parasitic nematodes emerged from the L2 larvae. Field data showed that, in breeding site treated with 3000 J2 per square meter, larval mosquito reduction reached 97% after nematode application. The dosage of 1000 J2 per square meter did not reduce the larval density. The insect parasitic nematode R. iyengari could be easily used as component of integrated mosquitoes control program in lymphatic filariasis endemic countries.
A T. spiralis serine protease 1.2 (TsSP1.2) was identified in the muscle larvae (ML) and intestinal larvae surface/excretory-secretory (ES) proteins by immunoproteomics. The aim of this study was to determine the TsSP1.2 function in the process of T. spiralis intrusion, growth and reproduction by using RNA interference (RNAi). RNAi was used to silence the expression of TsSP1.2 mRNA and protein in the nematode. On 2 days after the ML were electroporated with 2 µM of TsSP1.2-specific siRNA 534, TsSP1.2 mRNA and protein expression declined in 56.44 and 84.48%, respectively, compared with untreated ML. Although TsSP1.2 silencing did not impair worm viability, larval intrusion of intestinal epithelium cells (IEC) was suppressed by 57.18% (P < 0.01) and the suppression was siRNA-dose dependent (r = 0.976). Infection of mice with siRNA 534 transfected ML produced a 57.16% reduction of enteral adult burden and 71.46% reduction of muscle larva burden (P < 0.05). Moreover, silencing of TsSP1.2 gene in ML resulted in worm development impediment and reduction of female fertility. The results showed that silencing of TsSP1.2 by RNAi inhibited larval intrusion and development, and reduced female fecundity. TsSP1.2 plays a crucial role for worm invasion and development in T. spiralis life cycle, and is a potential vaccine/drug target against Trichinella infection.
Rhipicephalus (Boophilus) microplus serves as an important ectoparasite of livestock and a vector of several pathogens resulting in diseases, subsequently affecting the agricultural field as well as the economy. The extensive use of synthetic acaricides is known to cause resistance over time and therefore a much safer, effective and environmentally friendly alternative to overcome tick infestation should be implemented. Larval immersion tests (LIT) were done to evaluate the effects of Citrus hystrix (Family: Rutaceae) and Cymbopogon citratus (Family: Poaceae) essential oils (EOs) for their individual and combined (1:1) acaricidal activity against the cattle tick. Results showed that LC50 and LC90 values in 24 and 48 hours for Cit. hystrix EO were 11.98% and 24.84%, and 10.95% and 21.71% respectively. LC50 and LC90 values for Cym. citratus EO were 1.21% and 6.28%, and 1.05% and 6.12% respectively. The mixture of EOs from two plants in 1:1 ratio (Cit. hystrix 50%: Cym. citratus 50%) was found to exhibit antagonistic effect (synergistic factor < 1). The 24 hours and 48 hours LC50 and LC90 values for combined EOs were 1.52% and 2.84%, and 1.50% and 2.76% respectively. Individual and combined essential oils were subjected to qualitative analysis using gas chromatography-mass spectrometry (GC-MS) to screen the chemical components present in EOs. Our results showed that the combination of Cit. hystrix and Cym. citratus at 1:1 ratio resulted in an antagonistic effect and the use of Cym. citratus alone is more toxic to R. (B.) microplus, making it a better alternative to chemical based acaricide.
In addition to the scarcity of forensic entomology baseline data on oviposition of necrophagous insects and completion of their life cycles in the Borneo region, similar data derived from caves remain unreported. Since entomological baseline data can differ from one biogeoclimatic region to another, the lack of such data would limit the practical values of applying entomological evidence in estimating minimum postmortem interval (mPMI). Therefore, this present research that investigated oviposition and completion of life cycles of necrophagous flies infesting rabbit carcasses decomposing in Mount Kapur Cave and its surrounding forest habitat in Kuching, Sarawak merits forensic consideration. In general, 13 taxa of necrophagous flies were identified viz. Hypopygiopsis violacea, Hypopygiopsis fumipennis, Hemipyrellia ligurriens, Hemipyrellia tagaliana, Chrysomya megacephala, Chrysomya villeneuvi, Chrysomya rufifacies, Chrysomya chani, Chrysomya pinguis, Chrysomya nigripes, Ophyra spinigera and Ophyra chalcogaster, as well as unidentified Sarcophagidae. In addition, Hyp. violacea and Hyp. fumipennis were the two earlier necrophagous flies that oviposited in all rabbit carcasses decomposing in both habitats. While all these necrophagous flies were observed infesting carcasses in Mount Kapur Cave, Hem. ligurriens and Hem. tagaliana were not found infesting carcasses in the surrounding forest habitat. Complete life cycles for six and five different necrophagous fly species were successfully observed in Mount Kapur Cave and its surrounding forest habitat, respectively. Significant delay in oviposition, as well as longer durations for completing the life cycles in several necrophagous fly species were observed in Mount Kapur Cave when compared with those of surrounding forest habitat (p < 0.05). These findings deserve consideration as the first ever forensic empirical baseline data on oviposition and completion of life cycles for necrophagous flies in Sarawak as well as in a cave habitat, in view of its practical values for estimating mPMI for forensic practical caseworks.
The sand fly Phlebotomus papatasi is an important disease-bearing vector. Five entomopathogenic nematodes (EPNs) - Steinernema carpocapsae DD136, Steinernema sp. (SII), S. carpocapsae all, S. abbasi, and Heterorhabditis bacteriophora HP88 - were applied as biocontrol agents against the late third instar larvae of P. papatasi. In addition, the effect of toxin complexes (TCs) of Xenorhabdus nematophila and Photorhabdus luminescens laumondii bacteria was evaluated. Results revealed that S. carpocapsae DD136 was the most virulent species followed by Steinernema sp. (SII) and S. carpocapsae all where LC50 were 472, 565, 962 IJs/ml, respectively. Also, the crude TCs were slightly more active and toxic than their fractionated protein. Histopathological examination of infected larvae with H. bacteriophora HP88 showed negative effect on their midgut cells. In conclusion, EPNs with their symbiotic bacteria are more effective as biocontrol agents than the crude or fractionated TCs against sand fly larvae.
Although myiasis caused by the blow fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), has been reported in Thailand, all of the cases were human. This study described three cutaneous myiasis cases caused by C. bezziana in dogs in Chiang Mai province, northern Thailand. The removal of fly larvae together with specific treatment to cure all cases in this study was represented.
The immature stages of necrophagous insects such as Diptera and Coleoptera play a vital ecological role in carrion decomposition. These invertebrates reduce the necromass significantly through consumption and recycle nutrients into organic forms which are readily being used by autotrophs or served as an abiotic storage in the soil ecosystem. Fly and beetle larvae are frequently encountered decomposers on ephemeral resource patches; however, lepidopterans associated with carrion decomposition is seldom reported. Here, we report colonization of Monopis sp. (Tineidae) and an unknown species of Psychidae on a rat carcass, and a Lithosiini caterpillar (Arctiidae) on a rabbit carcass in Peninsular Malaysia for the first time. The feeding behaviour and their potential forensic implications are discussed.
In previous studies, a Trichinella spiralis serine protease (TsSP) was identified in excretion/secretion (ES) products from intestinal infective L1 larvae (IIL1) using immunoproteomics. The complete cDNA sequence of TsSP gene was 1372 bp, which encoded 429 amino acids with 47.55 kDa. The TsSP was transcribed and expressed at all T. spiralis life cycle phases, as well as mainly located at the cuticle and stichosome of the parasitic nematode. Recombinant TsSP bind to intestinal epithelial cells (IEC) and promoted larva invasion, however, its exact function in invasion, development and reproduction are still unknown. The aim of this study was to confirm the biological function of TsSP during T. spiralis invasion and growth using RNA interference (RNAi) technology. The results showed that on 1 day after electroporation using 2.5 µM siRNA156, TsSP mRNA and protein expression of muscle larvae (ML) was suppressed by 48.35 and 59.98%, respectively. Meanwhile, silencing of TsSP gene by RNAi resulted in a 61.38% decrease of serine protease activity of ML ES proteins, and a significant reduction of the in vitro and in vivo invasive capacity of IIL1 to intrude into the IEC monolayer and intestinal mucosa. When mice were infected with siRNA 156-transfected larvae, adult worm and muscle larva burdens were decreased by 58.85 and 60.48%, respectively. Moreover, intestinal worm growth and female fecundity were evidently inhibited after TsSP gene was knockdown, it was demonstrated that intestinal adults became smaller and the in vitro newborn larval yield of females obviously declined compared with the control siRNA group. The results indicated that knockdown of TsSP gene by RNAi significantly reduced the TsSP expression and enzymatic activity, impaired larvae intrusion and growth, and lowered the female reproductive capacity, further verified that TsSP might participate in diverse processes of T. spiralis life cycle, it will be a new prospective candidate molecular target of anti-Trichinella vaccines.
Susceptibility status of Aedes albopictus (Skuse) sampled from residential areas in Interior, Sandakan and Tawau divisions of Sabah, Malaysia, was evaluated based on the WHOrecommended doses of organochlorine and organophosphate larvicides. To determine susceptibility status, larval bioassays were carried out and post 24-hour mortalities based on WHO resistance classifications were adopted. The results demonstrated that Ae. albopictus larvae were resistant toward 5 out of the 8 larvicides tested. Larvae from all populations were resistant against bromophos, fenitrothion, malathion, temephos and dichlorodiphenyltrichloroethane (DDT), with mortalities ranging from 0.00 to 89.33%. Dieldrin, on the other hand, could induce 100.00% mortalities in all populations, followed by fenthion and chlorpyrifos, with mortalities ranging from 97.33 to 100.00% and 81.33 to 100.00% respectively. Despite most populations exhibiting similitude in their resistance status, larvae from Sandakan exhibited the highest resistance level whereas the lowest level was observed in Keningau. In view of the inadequacy of some larvicides in controlling Ae. albopictus in this study, integrated management such as insecticide rotation or combination of interventions is warranted.
Plants contain bioactive compounds and are constantly explored as safer alternatives to conventional insecticides. Despite numerous studies on many plants, information on the insecticidal potential of underutilised plants like tiger nut, Cyperus esculentus L., are scant, although their pharmacological potentials are well known. Hence, this study investigated the larvicidal potential of crude aqueous extracts of two C. esculentus varieties (black and yellow) on the mosquitoes Aedes aegypti (L.) and Culex quinquefasciatus (Say). Mosquito larvae were exposed to C. esculentus crude extracts using the larval bioassay technique of the World Health Organization. Differential larvicidal responses were observed in the test mosquitoes and extracts of Black Dried Tiger nuts (BDT) were more larvicidal than Yellow Dried Tiger nuts (YDT). Acute larval toxicity of the extracts was more pronounced on Cx. quinquefasciatus than Ae. aegypti. The results indicate the potential of C. esculentus (particularly BDT) as a source of mosquito bioinsecticide and merits further studies as a safer alternative to conventional insecticide-based vector control.
Melia azedarach L. (Meliaceae) is a botanical species with focal point of global research for its biological properties. The Melia azedarach tree is distinguished by its rapid growth, its adaptation to different temperate zones, as well as its insecticidal properties. All this made us think of exploiting it in biological control against different stages of mosquitoes. To this end, we aim, through the present work, to evaluate the effectiveness of Melia azedarach extracts against Culex pipiens mosquito. More specifically, our study focuses on determining the chemical composition of Melia almond oil, as well as the larvicidal, ovicidal and repellent activities on Culex pipiens L. mosquito as well as the activities of acetylcholinesterase (AChE) and glutathione-S-transferase (GST). Almond oil was extracted by a Soxhlet and subjected to gas chromatography-mass spectrometry (GC/MS). The yield was found to be 35.17%. The chemical composition revealed the presence of various phytoconstituents. A total of 7 compounds were identified, the main ones being 9,11-Octadecadienoic acid, methyl ester, (E,E)- (79.32%), 9-octadecenoic acid (Z)-, methyl ester (13.24%), hexadecanoic acid and methyl ester (3.69%). The larvicidal bioassays were performed according to the protocol recommended by the World Health Organization with concentrations varying from 20 to 80 mg/L depending on the exposure time (24, 48 and 72 hours). The almond oil exhibited remarkable larvicidal activity against fourth instar larvae and the lethal concentrations were determined (LC25= 23.70 mg/L, LC50=35.49 mg/L, LC90=79.61 mg/L). The results also showed that the oil caused an ovicidal activity with a significant effect on egg hatch. The recorded hatching percentages were respectively 88.79% and 72.40% for the LC25 and LC50, and this compared to the control series. Moreover, this oil exhibited significant repellency against adult mosquitoes. Furthermore, the enzymatic measurements performed on LC50 and LC90 treated larvae revealed a neurotoxic activity and a stimulation of the detoxification system as evidenced, respectively, by an inhibition of AChE and induction in GST activity. Overall, our data proved that Melia azedarach almond oil could be considered as a potent biorational alternative to synthetic insecticides for mosquito control.
Haemonchus contortus (Hc) is a hematophagous parasite affecting the health and productivity of flocks. The administration of chemical anthelmintic drugs (AH) is the common method of deworming; however, generates resistance in the parasites to AH and it is a public health risk due to drug residues in milk, meat and sub-products. Natural compounds from plants are explored to diminish this parasitosis, improving their health and productivity, without the negative effects of AH. Ipomoea genus is a group of climbing plants belonging to the Convulvulaceae family possessing perennial leaves and tuberous roots. Medicinal properties has been attributed to this plant including nutritional agents, emetics, diuretics, diaphoretics, purgatives and pesticides. The objective of this study was assessing the in vitro nematocidal activity of a hydroalcoholic extract (HA-E) obtained from Ipomoea pauciflora (Cazahuate) flowers against Hc infective larvae (L3) and to identify its phytochemical profile (PhC-P). The assay was carried out using microtiter plates (MTP). Four HA-E concentrations were assessed and Ivermectin and distilled water were used as positive and negative control groups, respectively. Approximately 100 Hc L3 were deposited in each well (n=12) and incubated at 25-35°C for 7 days. Data were analyzed using ANOVA and a General Linear Model (GLM) followed by Tukey test (P<0.05). The treatments showing a concentration-dependent effect (CDE) were analyzed to identify their 50% and 90% lethal concentrations (CL50, 90) via a Probit Analysis. The highest mortality was observed at 50 mg/mL (82.64 ± 0.71%) and the lowest at 6.25 mg/mL (56.46 ± 2.49%), showing a CDE with increasing mortality from 6.25 to 50 mg/mL. The PhC-P revealed the presence of alkaloids, coumarins, flavonoids, tannins and triterpenes/ sterols. A HA-E from flowers of I. pauciflora will be considered to assess its potential use in the control of haemonchosis in small ruminants.
Simulium (Gomphostilbia) okinawense Takaoka and S. (G.) tokarense Takaoka, both from the Nansei Islands, Japan, were morphologically reexamined and genetically analysed by using the COI gene sequences. The female, male, pupa and mature larva of the two species are redescribed. Morphological reexamination shows that both species are more similar to species in the S. asakoae species-group than to those in the S. ceylonicum species-group, by having a medium-long female sensory vesicle, yellow tuft hairs (S. (G.) okinawense) or yellow tuft hairs mixed with a few to several dark hairs (S. (G.) tokarense) at the base of the radial vein in the female and male, and medium-long larval postgenal cleft. However, the body of the male ventral plate (viewed ventrally) is parallel-sided (S. (G.) okinawense) or parallelsided or slightly narrowed (S. (G.) tokarense) and not emarginated basally, differing from those of most species in the S. asakoae species-group. Our genetic analysis shows that S. (G.) tokarense is in the S. asakoae species-group, and S. (G.) okinawense formed a separate sister clade with other members of the S. asakoae species-group with high bootstrap support. From the results of morphological and genetic analysis combined, S. (G.) okinawense and S. (G.) tokarense are transferred from the S. ceylonicum species-group to the S. asakoae species-group.
The estimation of the postmortem interval (PMI) is an essential information in death investigations. It is necessary to know the developmental data of the most important necrophagous insect species in every geographical area. Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) is one of the most common species associated with human body decomposition, especially in the tropics, so a precise knowledge of its life cycle is crucial. However, despite its ubiquity, developmental data in a range of tropical temperatures is scarce. For this reason, the aim of this study is to provide the developmental data of the blowfly, C. megacephala, in a range of tropical temperatures and to apply these data in forensic entomology. Four temperatures were examined (i.e., 27.0°C 29.5°C, 32.0°C and 34.5°C) and the time of developments from egg to adult were recorded. To build the growth curves, five larvae per day were measured with a digital caliper. Accumulated degree-days were calculated and the isomorphen diagram for this species was plotted. As we predicted, the results showed that the life cycle of this species was affected by the increasing temperature. The implications of these new data for determining the minimum PMI in forensic entomology were discussed.
Simulium takahasii (Rubtsov), which was originally described from Japan, and recorded from Korea and China, is the first among the 19 species of the subgenus Wilhelmia Enderlein recorded from East Asia. It is striking in mating, blood-feeding and ovipositing in captivity and in experimentally transmitting Dirofilaria immitis (Leidy) and Brugia pahangi (Buckley & Edeson), and it is a severe biter of cattle and horses, rarely of humans. Nevertheless, updated information about its morphological characteristics was lacking, making comparisons with related species described from China difficult, since species of the subgenus Wilhelmia are almost indistinguishable from one another, in particular, in their female terminalia, male genitalia and most of larval features. In this study, as many morphological characteristics as possible of S. takahasii based on specimens from Japan are redescribed. New information about many features of this species including the length of the female sensory vesicle against the third palpal segment, number of male upper-eye (large) facets, arrangement of the eight pupal gill filaments, presence or absence of tiny dark setae on the dorsum of the larval abdomen and the number of rows and hooklets of the larval posterior circlet will be useful in evaluating the species status of several Wilhelmia species in China including the species regarded as S. takahasii.
The development of new alternatives strategies to synthetic insecticides aimed at reducing pest populations by developing pesticides based on plant extracts without negative effects in non target organisms and environment. The present study was undertaken in order to assess the insecticidal activity of the crude methanolic extract of the Algerian Asteraceae Cotula cinerea, against the larval and the pupal stage of Culex pipiens (Diptera: Culicidae). It is also to determine the chemical composition of the used extract, and to understand the mechanism of toxic action of the tested extract. Based on the preliminary tests, five concentrations of the crude methanolic extract of C. cinerea (0.62, 1.25, 2.50, 3.75, and 5 mg/mL) were tested for their insecticidal activity according to the protocol recommended by the World Health Organization. The chemical profile of the extract was also obtained by high performance liquid chromatography (HPLC). Histopathological effects and inhibition of acetylcholinesterase activity in treated mosquitoes with LC90 were examined to elucidate the mechanism of the toxic effect of the tested extract (48 h post treatment). Eight compounds have been identified by HPLC. That includes four flavonoids (rutin, quercetin, myrcetin and cathechin), three phenolic acids (benzoic acid, vanillic acid, p-coumaric acid) and one alkaloid (berberine). C. cinerea methanolic extract showed good larvicidal and pupicidal activities with LC50 and LC90 values of 1.10 and 4.37 mg/mL respectively against pupae, 24h post treatment and 1.26, 2.35 mg/mL respectively against the fourth instar larvae. Data of enzymatic assay performed on LC50 and LC90 pupae and larvae revealed prominent neurotoxic effects. C. cinerea extract reduced the activity of acetylcholinesterase (AChE) enzyme in a concentration dependent manner. Obtained inhibition percentages, 48 h after treatment, were 35.11 ± 7.44 and 51.83 ± 4.04% for pupal stage and 30.98 ± 2.97 % and 48.77 ± 4.72% for the fourth instar larvae for LC50 and LC90 values respectively. Treated larvae and pupae showed also histopathological damages in the pupal cuticle and larval midgut. The results of this study showed that C. cinerea crude methanolic extract could be considered as an eco-friendly alternative for mosquito control.