Sublethal effects of oregano essential oil and its major compound carvacrol on biological parameters of Aedes albopictus (Diptera: Culicidae).

Mosquito management programs rely basically on the use of conventional synthetic larvicides. However, frequent applications and misuse of some synthetic insecticides have led to problems related to mosquito resistance development, harmful effects on human health and unacceptable environmental effects on non-target organisms. Recently, a growing number of phytochemicals has been tested as more eco-friendly larvicides against various mosquito species, exerting high efficacy with multiple modes of action. In the laboratory, we investigated for the first time the sublethal effects of oregano oil and its major compound carvacrol, against Aedes albopictus (Asian tiger mosquito), a mosquito of great medical importance. We determined the effects of short term (24h) exposure of 3rd- 4th larvae to LC50 concentrations on survival and development of survived larvae until adulthood, as well as on fecundity, fertility, longevity and wing length of emerged adults. Only half of 24h survived larvae from oregano oil and carvacrol treatment finally reached adulthood. Abnormal shapes of dead larvae and pupae, and failed adult emergence were also observed, indicating a potential growth inhibitory activity of the tested materials. No particular effects from exposure to larvicidal LC50 concentrations were recorded on life cycle parameters of successfully emerged adults. These findings suggest the tested oregano oil and carvacrol as sufficiently effective larvicides against Ae. albopictus at lower than the acutely toxic concentrations, promoting a more eco-friendly and less costly profile for these biopesticides.

Evaluating the Sublethal Effects of Origanum vulgare Essential Oil and Carvacrol on the Biological Characteristics of Culex pipiens biotype molestus (Diptera: Culicidae).

Culex pipiens is a mosquito species complex spread worldwide that poses a serious threat to human health as the primary vector of West Nile virus. Its control is mainly based on larvicidal applications with synthetic insecticides on mosquito breeding sites. However, the excessive use of synthetic larvicides may provoke mosquito resistance issues and negative side effects to the aquatic environment and human health. Plant-derived essential oils, including those from the Lamiaceae family, can be eco-friendly alternative larvicidal agents causing acute larval toxicity and/or growth inhibitory effects on the developmental stages of mosquitoes through different modes of action. In the current laboratory study, we evaluated the sublethal effects of carvacrol-rich oregano essential oil and pure carvacrol on Cx. pipiens biotype molestus, the autogenous member of the Cx. pipiens species complex, after the exposure of 3rd-4th instar larvae to LC50 concentrations. The short-term (24 h) larvicidal treatment with the sublethal concentrations of both tested materials exhibited an acute lethal effect on the exposed larvae as well as significant delayed mortality for surviving larvae and pupae. Larvicidal treatment with carvacrol reduced the longevity of the emerged males. In addition, the morphological abnormalities that were observed at the larval and pupal stage along with failed adult emergence indicate the potential growth inhibitory properties of the tested bioinsecticides. Our findings suggest that carvacrol and carvacrol-rich oregano oil are effective plant-based larvicides at doses lower than the acute lethal ones, thus promoting an environmentally friendly and more affordable perspective for their use against the WNV vector Cx. pipiens biotype molestus.

Laboratory study on larval competition between two related mosquito species: Aedes (Stegomyia) albopictus and Aedes (Stegomyia) cretinus.

Aedes albopictus (Asian tiger mosquito) and Aedes cretinus are closely related mosquito species with similar morphological and bio-ecological characteristics. These species have been detected in specific areas of Athens, Greece, with Ae. albopictus developing significantly higher population densities than the native mosquito Ae. cretinus. In a laboratory factorial experimental design, we investigated the potential of interspecific and intraspecific competition between larvae of these species under various food and population density conditions. Duration of larval development, survival rate from the first larval instar until adulthood and the wing length of females were measured. When these two species developed on their own, larvae developed faster and the females were larger at high food provision, indicating intraspecific competition. When the two species developed in the same environment and food provision was low, Ae. albopictus outcompeted Ae. cretinus. In particular, the larval developmental time when these species competed with each other was 1.3 to 2.4 days shorter for Ae. albopictus and 0.9 to 1.4 days longer for Ae. cretinus, compared with single species development. Interspecific competition resulted to larger Ae. albopictus females at limited food availability and low density of individuals. Our findings indicate that Ae. albopictus is a superior competitor to Ae. cretinus, primarily at limited larval food resources, and this may account for the expansion of Ae. albopictus and the limited presence of Ae. cretinus in areas of Athens, Greece, where these related species co-exist.

Behavioural response of Culex pipiens biotype molestus to oviposition pheromone.

Insect behaviour relies on an olfactory sensory system that controls a range of activities, from food choice and mating to oviposition, where pheromones play a central role. In Culex mosquitoes, egg-laying is accompanied by the release of mosquito oviposition pheromone (MOP), which has been shown to affect the oviposition behaviour of conspecifics. Here, we investigated for the first time the effect of MOP on the oviposition rate of Culex pipiens biotype molestus, examining separately males and females, before and after mating and oviposition. Our results demonstrate that MOP is more likely to act as an oviposition stimulant rather than an attractant, since more gravid females laid eggs in its presence, while the number of male or female mosquitoes (virgin or mated) captured on pheromone-treated pots was similar to those treated with control water.

Efficacy Evaluation of Oregano Essential Oil Mixed with Bacillus thuringiensis israelensis and Diflubenzuron against Culex pipiens and Aedes albopictus in Road Drains of Italy.

Mosquito management programs in the urban environment of Italian cities mainly rely on larval control with conventional insecticides, primarily targeting the road drains that constitute the principal mosquito breeding sites encountered in public. The repeated utilization of synthetic insecticides may have adverse effects on non-targets and lead to resistance development issues, while the performance of biopesticides encounters limitations in field use. Botanical insecticides as single larval control agents or in binary mixtures with conventional insecticides have been extensively studied in the laboratory as an effective and eco-friendly alternative mosquito control method with promising results. The study herein concerns the investigation, for the first time under realistic conditions in the field, of the joint action of a carvacrol-rich oregano Essential Oil (EO) with two conventional insecticides, namely, the insect growth regulator diflubenzuron and the bio-insecticide Bacillus thuringiensis israelensis (B.t.i.), in road drains in Crevalcore city, Italy, against Culex pipiens and Aedes albopictus. According to the obtained results, the application of both plain EO and its mixtures with diflubenzuron and B.t.i. exerted very high efficacy in terms of immature mosquito population reduction over a two-week period. Three weeks after treatment, the performance of the oil and its mixtures diminished but remained high, while the addition of diflubenzuron potentiated the persistent action of the oil against Cx. pipiens. These findings are indicative of the potential of mixing carvacrol-rich EO with diflubenzuron and B.t.i. as an efficient eco-friendly alternative to mono-insecticide applications in road drains against Cx. pipiens and Ae. albopictus larvae.

Physicochemical Characteristics of Four Limonene-Based Nanoemulsions and Their Larvicidal Properties against Two Mosquito Species, Aedes albopictus and Culex pipiens molestus.

Negative impacts on the environment from the continuous use of synthetic insecticides against mosquitoes has driven research towards more ecofriendly products. Phytochemicals, classified as low-risk substances, have been recognized as potential larvicides of mosquitoes; however, problems related to water solubility and stability are limiting factors for their use in mosquito control programs in the field. In this context, many researchers have focused on formulating essential oils in nanoemulsions, exploiting innovative nanotechnology. In the current study, we prepared 4 (R)-(+)-limonene oil-in-water nanoemulsions using low and high energy methods, and we evaluated their physicochemical characteristics (e.g., viscosity, stability, mean droplet diameter, polydispersity index) and their bioactivity against larvae of two mosquito species of great medical importance, namely, Cx. pipiens molestus and Ae. albopictus. According to the dose-response bioassays with the limonene-based nanoemulsions and pure limonene (dissolved in organic solvent), the tested nanoformulations improved the activity of limonene against Ae. albopictus larvae, while the performance of limonene was either the same or better than limonene against Cx. pipiens molestus, depending on the applied system. Overall, we achieved the production of limonene-based delivery nanosystems, with sufficient lethal properties against mosquito larvae to consider them promising larvicidal formulations applicable to mosquito breeding sites.

Citrus essential oils and four enantiomeric pinenes against Culex pipiens (Diptera: Culicidae).

The aim of this study was to evaluate the toxicity of pinenes (enantiomers of alpha- and beta-) and essential oils from Greek plants of the Rutaceae family against the mosquito larvae of Culex pipiens (Diptera: Culicidae). Essential oils were isolated by hydrodistillation from fruit peel of orange (Citrus sinensis L.), lemon (Citrus limon L.), and bitter orange (Citrus aurantium L.). The chemical composition was determined by gas chromatography/mass spectrometry (GC/MS) analysis. Citrus essential oils contained in high proportion limonene and in lower quantities p-menthane molecules and pinenes. The insecticidal action of these essential oils and enantiomers of their pinenes on mosquito larvae was evaluated. Plant essential oils exhibited strong toxicity against larvae with the LC(50) values ranging from 30.1 (lemon) to 51.5 mg/L (orange) depending on Citrus species and their composition. Finally, the LC(50) value of pinenes ranging from 36.53 to 66.52 mg/L indicated an enantioselective toxicity only for the beta-pinene enantiomer.