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.

First Report of Adult Aedes aegypti (Diptera: Culicidae) Resistance to Pyrethroids in Argentina.

Severe human arboviral diseases can be transmitted by the mosquito Aedes aegypti (Linnaeus), including dengue, chikungunya, Zika, and yellow fever. Adult control using spatial sprays with adulticides is recommended only when dengue outbreaks occur. In Argentina, mainly pyrethroids, like cis-permethrin, have been used as an adulticide, especially since 2008. The evolution and spread of resistance to insecticides is a major concern for vector control. This study reports for the first time pyrethroid resistance in Ae. aegypti adults from Argentina, in the city of Salvador Mazza (Salta). WHO discriminating doses of 0.75% were used for permethrin, 0.05% for deltamethrin, and 5% for malathion. Also the discriminating dose for cis-permethrin (0.6%) was calculated and evaluated for the first time. We found a resistance ratio 50 (RR50) of 10.3 (9.7-10.4) for cis-permethrin, which is considered as high resistance. Our results also indicated resistance to deltamethrin (22.6% mortality) and permethrin (53.6% mortality), and a total susceptibility to malathion (100% mortality). Results from this study highlight the importance of the correct use of insecticides within an Integrated Vector Management (IVM) approach and of early detection of resistance to enable Ae. aegypti control in Argentina. More studies are needed to determine the spread of mosquito resistance to pyrethroids.

Transcriptomic analysis and molecular docking reveal genes involved in the response of Aedes aegypti larvae to an essential oil extracted from Eucalyptus.

BACKGROUND: Aedes aegypti (L.) is an urban mosquito, vector of several arboviruses that cause severe diseases in hundreds of million people each year. The resistance to synthetic insecticides developed by Ae. aegypti populations worldwide has contributed to failures in vector control campaigns, increasing the impact of arbovirus diseases. In this context, plant-derived essential oils with larvicidal activity could be an attractive alternative for vector control. However, the mode of action and the detoxificant response of mosquitoes to plant derived compounds have not been established, impairing the optimization of their use. METHODS AND FINDINGS: Here we compare gene expression in Ae. aegypti larvae after 14 hrs of exposure to Eucalyptus camaldulensis essential oil with a control group exposed to vehicle (acetone) for the same lapse, by using RNA-Seq. We found differentially expressed genes encoding for cuticle proteins, fatty-acid synthesis, membrane transporters and detoxificant related gene families (i.e. heat shock proteins, cytochromes P450, glutathione transferases, UDP-glycosyltransferases and ABC transporters). Finally, our RNA-Seq and molecular docking results provide evidence pointing to a central involvement of chemosensory proteins in the detoxificant response in mosquitoes. CONCLUSIONS AND SIGNIFICANCE: Our work contributes to the understanding of the physiological response of Ae. aegypti larvae to an intoxication with a natural toxic distilled from Eucalyptus leafs. The results suggest an involvement of most of the gene families associated to detoxification of xenobiotics in insects. Noteworthy, this work provides important information regarding the implication of chemosensory proteins in the detoxification of a natural larvicide. Understanding the mode of detoxification of Eucalyptus distilled compounds could contribute to their implementation as a tool in mosquito control.

Lufenuron can be transferred by gravid Aedes aegypti females to breeding sites and can affect their fertility, fecundity and blood intake capacity.

BACKGROUND: Aedes aegypti (L.) is the main vector of dengue, yellow fever, Zika and chikungunya viruses. A new method for controlling this mosquito has been developed based on the possibility that wild adult mosquitoes exposed to artificial resting sites contaminated with a larvicide, can disseminate it to larval breeding sites, is named "auto-dissemination". The present study was undertaken to evaluate if a chitin synthesis inhibitor like lufenuron can be disseminated to larval breeding sites and prevent adult emergence and also if forced contact of Ae. aegypti females with treated surfaces can affect its fertility, fecundity, and blood intake capacity. METHODS: Larval susceptibility to lufenuron was measured through EI50 and EI90. On the other hand, gravid females were exposed by tarsal contact to lufenuron-treated papers, we used the WHO susceptibility test kit tube to line the papers, and 1, 3 or 5 females for the transference. We also evaluated if the exposure of female mosquitoes to lufenuron-treated papers (0.4 and 1 mg a.i./cm2) has an effect on their fertility, fecundity or in the ability to feed on blood. In each assay 12-15 female mosquitoes were exposed to lufenuron for 1 h, 24 h before blood meal (BBM) or 24 h after a blood meal (ABM). RESULTS: Lufenuron proved to be very active against Ae. aegypti larvae with an EI50 of 0.164 ppb and EI90 of 0.81 ppb. We also found that lufenuron can be transferred by females from treated surfaces to clean containers causing the inhibition of emergence of the larvae (between 30 and 50%). This effect was dependent on the concentration applied on the paper and the number of females added to each cage. CONCLUSIONS: This study introduces an innovation by first exploring the possibility that an insect growth regulator (IGR) belonging to the group of benzoylphenyl ureas, such as lufenuron, can be transferred by gravid females to breeding sites and that at the same time can have an effect on fertility, fecundity and blood intake capacity of adult mosquitoes.

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.

N-substituted methyl maleamates as larvicidal compounds against Aedes aegypti (Diptera: Culicidae).

Severe human arboviral diseases can be transmitted by the mosquito Aedes aegypti (L.), including dengue, chikungunya, zika, and yellow fever. The use of larvicides in containers that can result as potential breeding places and cannot be eliminated is the main alternative in control programs. However, their continuous and widespread use caused an increase in insecticide-resistant populations of this mosquito. The aim of this study was to evaluate the effect of three N-substituted methyl maleamates as larvicides on Ae. aegypti, the N-propyl methyl maleamate (PMM), N-butyl methyl maleamate (BMM), and N-hexyl methyl maleamate (HMM). These compounds could have a different mode of action from those larvicides known so far. We evaluated the larva mortality after 1 and 24 h of exposure and we found that mortality was fast and occurs within the first 60 min. HMM was slightly more effective with LC50 values of 0.7 and 0.3 ppm for 1 and 24 h of exposure and LC95 of 11 and 3 ppm. Our results demonstrate that N-substituted methyl maleamates have insecticidal properties for the control of Ae. aegypti larvae. These compounds could become useful alternatives to traditional larvicides after studying their insecticidal mechanism as well as their toxicity towards non target organisms.

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.

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.

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.

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.

Estudo do efeito agudo da fadiga na força de membros inferiores durante saltos com contramovimento / Estudio del efecto de la fatiga aguda en la fuerza del miembros inferiores durante saltos con contramovimiento / Acute fatigue effects on ground reaction force of lower limbs during countermovement jumps

Parameters associated with the performance of countermovement jumps were identified from vertical ground reaction force recordings during fatigue and resting conditions. Fourteen variables were defined, dividing the vertical ground reaction force into negative and positive external working times and times in which the vertical ground reaction force values were lower and higher than the participant's body weight. We attempted to explain parameter variations by considering the relationship between the set of contractile and elastic components of the lower limbs. We determined that jumping performance is based on impulsion optimization and not on instantaneous ground reaction force value: the time in which the ground reaction force was lower than the body weight, and negative external work time was lower under fatigue. The results suggest that, during fatigue, there is less contribution from elastic energy and from overall active state. However, the participation of contractile elements could partially compensate for the worsening of jumping performance.

Parâmetros associados com o desempenho do salto contramovimento foram identificados a partir de registros da força vertical em condições de fadiga e não fadiga. Quatorze variáveis foram definidas dividindo a força de reação vertical do solo em valores negativos e positivos de trabalho externo e o tempo em que os valores de força vertical foi menor e maior do que o peso corporal do indivíduo. As variações dos parâmetros foram avaliadas considerando a relação entre conjunto de elementos contráteis e elásticos nos membros inferiores. Determinou-se que o desempenho é baseado na otimização do impulso e não em um valor instantâneo da força; o tempo em que a força de reação do solo foi menor do que o peso corporal e o tempo de trabalho externo negativo são mais baixos sob fadiga. Os resultados sugerem que durante a fadiga há uma menor contribuição da energia elástica e do estado ativo de forma global, mas a participação de elementos contráteis poderia compensar parcialmente a queda de desempenho.

Se identificaron parámetros asociados con el desempeño de saltos con contramovimiento a partir de registros de fuerza vertical en condiciones de fatiga y no fatiga. Catorce variables fueron definidas dividiendo la componente vertical de la curva de fuerza de reacción del suelo en valores negativos y positivos de trabajo externo y tiempo en que los valores de fuerza resultaron menores o mayores que el valor del peso del individuo. Las variaciones de los parámetros fueron evaluadas considerando la relación entre el conjunto de elementos contráctiles y elásticos en los miembros inferiores. Se determinó que el desempeño está basado en la optimización del impulso y no en un valor instantáneo de fuerza; el tiempo en que la fuerza de reacción del suelo fue menor que el peso corporal y el tiempo de trabajo externo negativo resultaron más bajos en fatiga. Los resultados sugieren que durante la fatiga hay una menor contribución de la energía elástica y del estado activo de forma global, pero la participación de elementos contráctiles podría compensar parcialmente la caída en el desempeño.