A rapid method for screening mosquito repellents on Anopheles pseudopunctipennis and Aedes aegypti.

One of the main vectors for malaria in Latin America is Anopheles pseudopunctipennis (Theobald), whereas Aedes aegypti (L.) is the primary vector of dengue, yellow fever, Zika and chikungunya viruses. The use of repellents is recommended as a personal protection method against these mosquitoes. However there are very few studies evaluating the effect of repellents on An. pseudopunctipennis. The use of a Petri dish to study repellence has been applied by several authors on flies, cockroaches, kissing bugs and mosquitoes, being a valuable technique for species that are difficult to breed under laboratory conditions, such as An. pseudopunctipennis. In the present study, we evaluated the repellence of the essential oil of the Eucalyptus nitens (Shining gum), its main component (1,8-cineole) and the commercial repellent DEET on An. pseudopunctipennis and Ae. aegypti adult females using the plaque repellency method coupled to EthoVision XT10.1 video-tracking software. Repellent effect and locomotor activity were studied through a repellence index (RI) together with an axis constructed from the behavioural variables obtained using the tracking software. DEET repellent effect was observed at 0.01 mg/mL for Ae. aegypti and 0.01 and 0.1 mg/mL for An. pseudopunctipennis. In addition, the essential oil showed significant repellence at 1 and 10 mg/mL for Ae. aegypti, and 1, 5, 10 and 25 mg/mL for An. pseudopunctipennis. Neither of these species were repelled at any concentration of 1,8-cineole. This is the first study that evaluates these compounds on An. pseudopunctipennis females and quantifies their effects on the activity of both species.

Oviposition Behavior in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Response to the Presence of Heterospecific and Conspecific Larvae.

In mosquitoes, location of suitable sites for oviposition requires a set of visual, tactile, and olfactory cues that influences females before laying their eggs. The ability of gravid females to distinguish among potential oviposition sites that will or will not support the growth, development, and survival of their progeny is critical. Aedes aegypti (L.) and Aedes albopictus (Skuse) share ecological niches, being highly competitive in larval stage. We studied the oviposition behavior of both species in the presence of larvae of one or the other species (heterospecific or conspecific larvae). The number of eggs laid by gravid females on oviposition sites (water with different or the same species of Aedes larvae) were compared. The presence and density of heterospecific or conspecific larvae had a positive or negative effect on the ovipositional responses, measured as an oviposition activity index. For both species, the oviposition was not affected by heterospecific larvae with densities between 10 and 100 larvae in water, but a strong attractant behavior was observed for a density of 500 larvae in water. For Ae. albopictus in the presence of larvae of the same species (conspecific oviposition), we observed an attractant effect for larvae density of 10 but not for 100 or 500 larvae in water. Instead, for Ae. aegypti, we observed attraction only for 100 larvae, not for 10 or 500 larvae. Results presented here provide an additional insight about oviposition behavior responses of gravid females in the presence of conspecific and heterospecific larvae in breeding sites.

The use of Aedes aegypti larvae attractants to enhance the effectiveness of larvicides.

Aedes aegypti (L.) is an important dengue, chikungunya, and yellow fever vector. Immature stages of this species inhabit human-made containers placed in residential landscapes, and the application of larvicides inside containers that cannot be eliminated is still considered a priority in control programs. Larvicidal efficacy is influenced by several factors, including the formulation used, the water quality, and the susceptibility of larvae, among others. If an attractant can be incorporated into a slow-release larvicide formulation, it will be feasible to direct the larvae into the source of insecticide and thereby improving its efficacy. We studied the influence of 1-octen-3ol and 3-methylphenol on the rate of Ae. aegypti larvae mortality using the larvicides Bacillus thuringiensis var. israelensis (Bti), temephos, and spinosad. These chemicals were combined with the larvicides mixed with agar during the bioassays. Mortality was registered every 10 min, and a lethal time 50 (LT50) was calculated. The inclusion of the Ae. aegypti larvae attractants with the larvicides into a solid agar matrix improved their efficiency obtaining a strong and marked reduction in the LT50 compared with the use of larvicides alone.

Behavioral Response of Aedes aegypti (Diptera: Culicidae) Larvae to Synthetic and Natural Attractants and Repellents.

Aedes aegypti (L.) (Diptera: Culicidae) is the key vector of three important arboviral diseases: dengue, yellow fever, and chikungunya. Immature stages of this species inhabit human-made containers placed in residential landscapes. In this study, we evaluated a few compounds in a sensitive behavioral assay with Ae. aegypti larvae. The orientation of larvae to different compounds was surveyed using a performance index (PI). The PI represents the response to each odorant, where a value of +1 is indicative of full attraction and -1 represents complete repulsion. The widely used insect repellent N, N-diethyl-m-toluamide elicited a significantly negative PI, as did acetophenone and indole. A yeast extract, a known food source, elicited a significantly positive PI, as did 2-methylphenol, 1-octen-3-ol, 3-methylphenol, and fish food. On the other hand, no response was observed for the essential oil of Eucalyptus grandis x Eucalyptus camaldulensis at the concentration evaluated. Pretreatment of larvae with N-ethylmaleimide and ablation of the antennae resulted in a suppression of behavioral responses. The overall mobility of ablated larvae was indistinguishable from unablated controls, and absence of any visible locomotor dysfunction was observed. This work is a contribution to the study of the chemical ecology of disease vectors with the aim of developing more efficient tools for surveillance and control.Natural and synthetic compounds attractive to Ae. aegypti larvae should be incorporated into integrated pest management programs through the use of baited traps or by improving the efficacy of larvicides commonly used in control campaigns.

Validation of models to estimate the fumigant and larvicidal activity of Eucalyptus essential oils against Aedes aegypti (Diptera: Culicidae).

The aim of this work is to validate the pre-existing models that relate the larvicidal and adulticidal activities of the Eucalyptus essential oils on Aedes aegypti. Previous works at our laboratory described that the larvicidal activity of Eucalyptus essential oils can be estimated from the relative concentration of two main components (p-cymene and 1,8-cineole) and that the adulticidal effectiveness can be explained, to a great extent, by the presence of large amounts of the component 1,8-cineole in it. In general, the results show that the higher adulticidal effect of essential oils the lower their larvicidal activity. Fresh leaves was harvested and distilled. Once the essential oil was obtained, the chemical composition was analysed, evaluating the biological activity of 15 species of the genus Eucalyptus (Eucalyptus badjensis Beuzev and Welch, Eucalyptus badjensis × nitens, Eucalyptus benthamii var Benthamii Maiden and Cambage, Eucalyptus benthamii var dorrigoensis Maiden and Cambage, Eucalyptus botryoides Smith, Eucalyptus dalrympleana Maiden, Eucalyptus fastigata Deane and Maiden, Eucalyptus nobilis L.A.S. Johnson and K.D.Hill, Eucalyptus polybractea R. Baker, Eucalyptus radiata ssp radiata Sieber ex Spreng, Eucalyptus resinifera Smith, Eucalyptus robertsonii Blakely, Eucalyptus robusta Smith, Eucalyptus rubida Deane and Maiden, Eucalyptus smithii R. Baker). Essential oils of these plant species were used for the validation of equations from preexistent models, in which observed and estimated values of the biological activity were compared. The regression analysis showed a strong validation of the models, re-stating the trends previously observed. The models were expressed as follows: A, fumigant activity [KT(50(min)) = 10.65-0.076 × 1,8-cineole (%)](p < 0.01; F, 397; R (2), 0.79); B, larval mortality (%)((40 ppm)) = 103.85 + 0.482 × p-cymene (%) - 0.363 × α-pinene (%) - 1.07 × 1,8-cineole (%) (p < 0.01; F, 300; R (2), 0.90). These results confirmed the importance of the mayor components in the biological activity of Eucalyptus essential oils on A. aegypti. However, it is worth mentioning that two or three species differ in the data estimated by the models, and these biological activity results coincide with the presence of minor differential components in the essential oils. According to what was previously mentioned, it can be inferred that the model is able to estimate very closely the biological activity of essential oils of Eucalyptus on A. aegypti.

Insecticidal activity of essential oils from eleven Eucalyptus spp. and two hybrids: lethal and sublethal effects of their major components on Blattella germanica.

The aim of the current study was to evaluate the fumigant activity of the essential oils from 11 species of the genus Eucalyptus and two of their hybrids on first instar of Blattella germanica L. The fumigant activity and repellence of the four major monoterpene components of these essential oils also were tested. Fumigant activity was evaluated by exposing nymphs to the vapors emitted by 50 microl of essential oil or monoterpene in a closed container. The lowest knockdown time 50% (KT50) values, expressed in minutes, were elicited by the essential oils of the Eucalyptus grandis X Eucalyptus tereticornis (57.9) hybrid, Eucalyptus sideroxylon A. Cunn (62.0), E. grandis X Eucalyptus camaldulensis (63.8) hybrid, Eucalyptus viminalis Labill (64.1), Eucalyptus dunnii Maiden (64.5), and Eucalyptus grandis (Hill) ex Maiden (68.7). The KT50 values for the remaining essential oils ranged between 74.5 (E. saligna Smith) and 161.4 min (E. tereticornis Smith). The essential oil from the hybrid E. grandis X E. tereticornis was 3.7 times less toxic than dichlorvos (positive control). The KT50 values of monoterpenes were 38.8 for alpha-pinene, 55.3 for 1,8-cineole, 175.6 for p-cymene, and 178.3 for gamma-terpinene. Alpha-pinene was 2.5 times less toxic than dichlorvos. There was a strong positive correlation between the fumigant activity of essential oils and their corresponding 1,8-cineole and alpha-pinene concentration. Repellency was quantified using a video tracking system. Two concentrations of monoterpenes were studied (7 and 70 microg/cm2). All compounds produced a light repellent effect but only when applied at 70 microg/cm2. In all cases, the repellent effect was less than that produced by the broad-spectrum insect repellent N,N-diethyl-3-methylbenzamide (positive control).

Chemical composition and fumigant toxicity of the essential oils from 16 species of Eucalyptus against Haematobia irritans (Diptera: Muscidae) adults.

Oils extracted from various species of Eucalyptus (Eucalyptus badjensis Beuzev & Welch, Eucalyptus badjensis x Eucalyptus nitens, Eucalyptus benthamii variety dorrigoensis Maiden & Cambage, Eucalyptus botryoides Smith, Eucalyptus dalrympleana Maiden, Eucalyptus fastigata Deane & Maiden, Eucalyptus nobilis L.A.S. Johnson & K. D. Hill, Eucalyptus polybractea R. Baker, Eucalyptus radiata ssp. radiata Sieber ex Spreng, Eucalyptus resinifera Smith, Eucalyptus robertsonii Blakely, Eucalyptus rubida Deane & Maiden, Eucalyptus smithii R. Baker, Eucalyptus elata Dehnh, Eucalyptus fraxinoides Deane & Maiden, E. obliqua L'Hér) were obtained by hydrodistillation. The chemical composition of essential oils was determined by gas chromatography coupled to mass spectrometry. Essential oils were mainly composed of 1,8-cineole, alpha-pinene, alpha-terpineol, 4-terpineol, and p-cymene. Vapors from these essential oils and their major components were found to be toxic to Haematobia irritans (L.) (Diptera: Muscidae) adults. An aliquot of each oil was placed in a cylindrical test chamber, and the number of knocked down flies was recorded as a function of time. Knockdown time 50% was then calculated. Results showed that essential oil of E. polybractea had the highest knockdown activity of 3.44 min. A correlation was observed between the content of 1,8-cineole in the Eucalyptus essential oils and the corresponding toxic effect.

Effects of Temephos, Permethrin, and Eucalyptus nitens Essential Oil on Survival and Swimming Behavior of Aedes aegypti and Anopheles pseudopunctipennis (Diptera: Culicidae) Larvae.

An essential strategy to deal with mosquito-borne diseases is the control of larvae in their development sites. The mosquitoes Anopheles pseudopunctipennis (Theobald) (Diptera: Culicidae), a malaria vector, and Aedes aegypti (L.) (Diptera: Culicidae), vector of dengue, Zika, yellow fever, and chikungunya viruses, breed in very different habitats. Insecticide treatments of mosquito larvae focus mainly on their lethal effects. However, insecticide degradation or the poor dosage of larvicides will invariably lead to the sublethal exposure of a target (and nontarget) species, the nonlethal effects of these compounds may have important effects on vital insect activities, and therefore their evaluation is necessary. In this study, we assessed the survival and swimming behavior of larvae of Ae. aegypti and An. pseudopunctipennis exposed to increasing concentrations of three larvicides. We found that Ae. aegypti, was more sensitive to the larvicides than An. pseudopunctipennis, we also observed an overall decrease in the movement of those larvae of both species, which survive the treatments. This decrease might have ecological relevance in their natural habitats, increasing the chance to be predated and decreasing their ability to obtain food. Finally, this information will be valuable to assist authorities to make decisions in the implementation of further control programs.

Synergism between cis-permethrin and benzoyl phenyl urea insect growth regulators against Aedes aegypti larvae.

Emulsifiable concentrate formulations of 2 insect growth regulator (IGR) benzoyl ureas (novaluron and diflubenzuron) and the pyrethroid cis-permethrin were individually bioassayed against late 3rd- or early 4th-stage larvae of susceptible Aedes aegypti. The emergence inhibition 50% were 0.038, 0.048, and 0.179 ppb for novaluron, diflubenzuron, and cis-permethrin, respectively. We also tested 1:1 (v:v) mixtures of cis-permethrin and benzoyl ureas. Emergence inhibition 50% for novaluron plus cis-permethrin and diflubenzuron plus cis-permethrin were 0.030 and 0.037, respectively, with combination indexes of 0.49 and 0.56, indicating a synergistic interaction between these insecticides. The percentage of larval mortality produced by 0.5 ppb of the mixtures of cis-permethrin and each IGR was compared to the addition of larval mortality produced in separated experiments by 0.25 ppb of each component, after 5, 9 and 13 days of continuous exposure. In all bioassays, the novaluron plus cis-permethrin mixture produced a significantly higher larvicidal effect (P < 0.05) compared with the individual contribution of each compound. The diflubenzuron plus cis-permethrin mixture showed significantly higher larval mortality (P < 0.05) compared with the contributive effect of each insecticide after 9 and 13 days of exposure, but there was no significant difference after 5 days of exposure.

A Video-Tracking Analysis-Based Behavioral Assay for Larvae of Anopheles pseudopunctipennis and Aedes aegypti (Diptera: Culicidae).

Aedes aegypti (L.) is the primary vector of dengue, yellow fever, Zika, and chikungunya viruses, whereas Anopheles pseudopunctipennis (Theobald) is the principal vector for malaria in Latin America. The larval stage of these mosquitoes occurs in very different development habitats, and the study of their respective behaviors could give us valuable information to improve larval control. The aim of this study was to set up a bioassay to study basic larval behaviors using a video-tracking software. Larvae of An. pseudopunctipennis came from two localities in Salta Province, Argentina, while Ae. aegypti larvae were of the Rockefeller laboratory strain. Behaviors of individual fourth-instar larvae were documented in an experimental petri dish arena using EthoVision XT10.1 video-tracking software. The overall level of movement of larval An. pseudopunctipennis was lower than that for Ae. aegypti, and, while moving, larval An. pseudopunctipennis spent significantly more time swimming near the wall of the arena (thigmotaxis). This is the first study that analyzes the behavior of An. pseudopunctipennis larvae. The experimental system described here may be useful for future studies on the effect of physiological, toxicological, and chemosensory stimuli on larval behaviors.

Repellent and Larvicidal Activity of the Essential Oil From Eucalyptus nitens Against Aedes aegypti and Aedes albopictus (Diptera: Culicidae).

Dengue, chikungunya, and yellow fever are important vector-borne diseases transmitted by female mosquitoes when they feed on humans. The use of repellents based on natural products is an alternative for personal protection against these diseases. Application of chemicals with larvicidal activity is another strategy for controlling the mosquito population. The repellent and larvicidal activities of the essential oil from Eucalyptus nitens were tested against Aedes aegypti and Aedes albopictus, the main vectors of these arboviruses. The essential oil was extracted by hydrodistillation and then analyzed by gas chromatography-mass spectrometry. The main components of Eucalyptus nitens essential oil were found to be terpenes such as 1,8-cineole and p-cymene, followed by ß-triketones and alkyl esters. The repellent activity of the essential oil against both species was significantly higher when compared with the main component, 1,8-cineole, alone. These results indicate that the repellent effect of E. nitens is not due only to the main component, 1,8-cineole, but also that other compounds may be responsible. Aedes aegypti was found to be more tolerant to the essential oil larvicidal effects than Ae. albopictus (Ae. aegypti LC50 = 52.83 ppm, Ae. albopictus LC 50 = 28.19 ppm). The repellent and larvicidal activity could be associated to the presence of cyclic ß-triketones such as flavesone, leptospermone, and isoleptospermone.