Challenging Popular Belief, Mosquito Larvae Breathe Underwater.

Immature mosquitoes are thought to breathe only atmospheric air through their siphons despite reports of prolonged submerged survival. We studied the survival of last-instar larvae of Aedes aegypti fully submerged at different temperatures and measured the oxygen consumption from air and water-dissolved larvae and pupae of this species under different conditions. Larvae survived much longer than expected, reaching 50% mortality only after 58, 10, and 5 days at 15°, 25°, and 35 °C, respectively. Larval to pupa molt was only observed in larvae with access to air, whereas individuals kept submerged never molted. Although most of the oxygen was obtained from the air, larvae obtained 12.72% of their oxygen from the water, while pupae took only 5.32%. In both media, temperature affected the respiration rate of the larvae, with relatively close Q10 values (1.56 and 1.83 for water and air, respectively). A similar pattern of O2 consumption was observed in Ae. albopictus, whose larvae obtained 12.14% of their oxygen from the water. The detailed quantification of oxygen consumption by mosquito larvae showed that water-dissolved oxygen is not negligible and physiologically relevant, challenging the idea that mosquito larvae only breathe atmospheric oxygen.

Machine-learning model led design to experimentally test species thermal limits: The case of kissing bugs (Triatominae).

Species Distribution Modelling (SDM) determines habitat suitability of a species across geographic areas using macro-climatic variables; however, micro-habitats can buffer or exacerbate the influence of macro-climatic variables, requiring links between physiology and species persistence. Experimental approaches linking species physiology to micro-climate are complex, time consuming and expensive. E.g., what combination of exposure time and temperature is important for a species thermal tolerance is difficult to judge a priori. We tackled this problem using an active learning approach that utilized machine learning methods to guide thermal tolerance experimental design for three kissing-bug species: Triatoma infestans, Rhodnius prolixus, and Panstrongylus megistus (Hemiptera: Reduviidae: Triatominae), vectors of the parasite causing Chagas disease. As with other pathogen vectors, triatomines are well known to utilize micro-habitats and the associated shift in microclimate to enhance survival. Using a limited literature-collected dataset, our approach showed that temperature followed by exposure time were the strongest predictors of mortality; species played a minor role, and life stage was the least important. Further, we identified complex but biologically plausible nonlinear interactions between temperature and exposure time in shaping mortality, together setting the potential thermal limits of triatomines. The results from this data led to the design of new experiments with laboratory results that produced novel insights of the effects of temperature and exposure for the triatomines. These results, in turn, can be used to better model micro-climatic envelope for the species. Here we demonstrate the power of an active learning approach to explore experimental space to design laboratory studies testing species thermal limits. Our analytical pipeline can be easily adapted to other systems and we provide code to allow practitioners to perform similar analyses. Not only does our approach have the potential to save time and money: it can also increase our understanding of the links between species physiology and climate, a topic of increasing ecological importance.

Surfactantless Emulsions Containing Eugenol for Imidacloprid Solubilization: Physicochemical Characterization and Toxicity against Insecticide-Resistant Cimex lectularius.

Synthetic insecticides have been used for a long time as one of the most effective tools for insect pest control. However, the re-emergence of insect pests and their fast development of resistance, as has occurred for pyrethroid-resistant bed bugs Cimex lectularius L., make it necessary to develop new and safe strategies for effective pest control. This has fostered the research on new eco-sustainable formulations based on essential oils, which allows reducing the impact associated with the intensive use of synthetic insecticides on the environment and their effects on human health. This research explores the stability of water/eugenol/ethanol surfactantless emulsions loaded with imidacloprid (0.003 wt%), and their toxicity against a resistant bed bug strain. The results have shown that these emulsions enable the solubilization of a poorly water-soluble drug, such as the imidacloprid, without any significant modification of their stability. Furthermore, the application of the obtained formulations against the pyrethroid-resistant bed bug results in mortality in the 50-85% range upon topical and spray applications, with the increase of the eugenol content enhancing the effectiveness of the formulations. It may be expected that the ternary water/eugenol/ethanol mixtures could be further developed in the preparation of ready to use formulations, enabling the dispersion of insecticides for pest control.

High effectiveness of an adulticide-larvicide formulation for field control of sandflies (Diptera: Psychodidae) in the city of Clorinda, Argentina.

In Argentina, Leishmania infantum (syn. L. chagasi) is the etiologic agent of human visceral leishmaniosis (HVL), and Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) is the main vector. The objective of this study was to evaluate the effectiveness and residual effect of two commercial insecticide formulations, one with permethrin and pyriproxyfen as active ingredients (Dragon Max®) and the other with only permethrin (Flop®) for the control of sandflies. Both formulations were applied in chicken coops and other surroundings structures of the peridomicile of urban houses in Clorinda, Formosa (Argentina). Entomological monitoring was carried out weekly for 44 weeks after the intervention. The results showed great effectiveness and residual effect up to 21 weeks post-intervention for Dragon Max®. This result could be explained by the excellent larvicidal activity of the Insect Growth Regulator (IGR) pyriproxyfen against the immature forms of phlebotomines and by the delay on the restoration of the natural threshold of the vector population in treated sites.

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.

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.