The yellow fever mosquito, Aedes aegypti, particularly in Neotropical regions, is the principal vector of dengue, yellow fever, Zika and Chikungunya viruses. Pyrethroids remain one of the most used insecticides to control Aedes mosquitoes, despite the development of pyrethroid resistance in many mosquito populations worldwide. Here, we report a Brazilian strain of A. aegypti with high levels (approximately 100–60,000 fold) of resistance to both type I and type II pyrethroids. We detected two mutations (V410L and F1534C) in the sodium channel from this resistant strain. This study is the first report of the V410L mutation in mosquitoes. Alone or in combination with the F1534C mutation, the V410L mutation drastically reduced the sensitivity of mosquito sodium channels expressed in Xenopus oocytes to both type I and type II pyrethroids. The V410L mutation presents a serious challenge for the control of A. aegypti and will compromise the use of pyrethroids for the control of A. aegypti in Brazil; therefore, early monitoring of the frequency of the V410L mutation will be a key resistance management strategy to preserve the effectiveness of pyrethroid insecticides.
The in vitro rearing of worker honey bees (Apis mellifera L.) has become an increasingly important method in honey bee research in general, and in pesticide risk assessment specifically. Authorities from the European Organization for Economic Co-operation and Development and the United States Environmental Protection Agency are requesting data on pesticide impacts on immature bee survivorship prior to registering new crop protection products. Those using the current in vitro rearing protocols have had variable success with immature bee survival and protocol repeatability. Here, we present an improved method for the in vitro rearing of worker honey bees from larvae to adult emergence. We have achieved consistently high survival (>95%) in our control and solvent-control rearing trials. Changes in the proportion of diet components, royal jelly source, maintenance of the developing bee, and rearing environment are the main contributors for our high rearing success and are discussed herein. Our in vitro rearing protocol can be implemented as the standard protocol to determine the impact of pesticides on immature bees because of the protocol's high control survivability, ease in end point determination, and high overall repeatability. Protocolo para la cría in vitro de obreras de Apis melliferaLa cría in vitro de las abejas obreras (Apis mellifera L.) se ha convertido en un método cada vez más importante en la investigació n de la abeja de la miel en general, y específicamente, en la evaluació n del riesgo de los plaguicidas. Las autoridades de la Organizació n Europea para la Cooperació n y el Desarrollo, y la Agencia de Protecció n Ambiental de los Estados Unidos están solicitando datos sobre los efectos de los plaguicidas en la supervivencia de abejas inmaduras antes de registrar nuevos productos para la protecció n de los cultivos. Aquellos que utilizan actualmente los protocolos de la cría in vitro han tenido un éxito variable en la supervivencia de abejas inmaduras y la repetitividad del protocolo. Aquí presentamos un método mejorado para la cría in vitro de las abejas obreras a partir de larvas para la eclosió n de los adultos. Hemos logrado consistentemente una alta supervivencia (>95%) en nuestro control y en los ensayos de cría con el disolvente del control. Los cambios en la proporció n de los componentes de la dieta, la fuente de jalea real, el mantenimiento de las abejas en desarrollo, y el ambiente de cría son los principales constituyentes para nuestro alto éxito de cría y son discutidos en este trabajo. Nuestro protocolo de cría in vitro puede ser implementado como el protocolo estándar para determinar el impacto de los plaguicidas en las abejas inmaduras debido a la alta capacidad de supervivencia del control, la facilidad en la determinació n del punto final, y la alta capacidad de repetitividad.
Declines in pollinator colonies represent a worldwide concern. The widespread use of agricultural pesticides is recognized as a potential cause of these declines. Previous studies have examined the effects of neonicotinoid insecticides such as imidacloprid on pollinator colonies, but these investigations have mainly focused on adult honey bees. Native stingless bees (Hymenoptera: Apidae: Meliponinae) are key pollinators in neotropical areas and are threatened with extinction due to deforestation and pesticide use. Few studies have directly investigated the effects of pesticides on these pollinators. Furthermore, the existing impact studies did not address the issue of larval ingestion of contaminated pollen and nectar, which could potentially have dire consequences for the colony. Here, we assessed the effects of imidacloprid ingestion by stingless bee larvae on their survival, development, neuromorphology and adult walking behavior. Increasing doses of imidacloprid were added to the diet provided to individual worker larvae of the stingless bee Melipona quadrifasciata anthidioides throughout their development. Survival rates above 50% were only observed at insecticide doses lower than 0.0056 µg active ingredient (a.i.)/bee. No sublethal effect on body mass or developmental time was observed in the surviving insects, but the pesticide treatment negatively affected the development of mushroom bodies in the brain and impaired the walking behavior of newly emerged adult workers. Therefore, stingless bee larvae are particularly susceptible to imidacloprid, as it caused both high mortality and sublethal effects that impaired brain development and compromised mobility at the young adult stage. These findings demonstrate the lethal effects of imidacloprid on native stingless bees and provide evidence of novel serious sublethal effects that may compromise colony survival. The ecological and economic importance of neotropical stingless bees as pollinators, their susceptibility to insecticides and the vulnerability of their larvae to insecticide exposure emphasize the importance of studying these species.
The risks imposed by novel insecticides, mainly bioinsecticides, are largely unknown despite their increased use and their perceived environmental safety, which is based on their natural origin. Furthermore, unlike honeybees, native pollinator species have received little attention. In the present study, the lethal and sublethal effects of the neonicotinoid imidacloprid and the bioinsecticide spinosad were assessed in the stingless bee species Meliponaquadrifasciata, an important native pollinator in the Neotropical region. The adult stingless bee workers exhibited high oral insecticide susceptibility, with LD50s of 23.54 and 12.07 ng a.i./bee for imidacloprid and spinosad, respectively. Imidacloprid also impaired worker respiration and overall group activity and flight, while spinosad significantly impaired only worker flight despite exhibiting higher oral toxicity to adult workers than imidacloprid. These findings indicate the hazardous nature not only of imidacloprid but also the bioinsecticide spinosad to adult workers of the native pollinator M. quadrifasciata. Therefore, bioinsecticides should not be exempted from risk assessment analysis due to their lethal and sublethal components.
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