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.

In the female insect, the spermatheca is an ectodermal organ responsible for receiving, maintaining, and releasing sperm to fertilize eggs. The number and morphology of spermathecae vary according to species. Within the spermathecal lumen, substances in the semen and secretions from the spermathecal gland nourish the sperm. Thus, the spermatheca provides an appropriate environment that ensures the long-term viability of sperm. Maintaining sperm viability for long periods within the spermatheca is crucial for insect reproductive success; however, the details of this process remain poorly understood. This review examines several aspects of and gaps in the current understanding of spermatheca biology, including morphology, function, reservoir filling, development, and biochemistry. Despite the importance of the spermatheca in insects, there is little information on the gland secretions and their role in the maintenance and protection of male gametes. Furthermore, in this review, we highlight the current information on spermathecal gland secretions and the likely roles they play in the maintenance and protection of sperm.

Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 6C but had different LD 50 values, ranging from 10 4 to 10 7 c.f.u. per larva.The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.

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.

The midgut epithelium of bees is formed by the digestive cells, responsible for enzyme secretion and nutrient absorption and for small regenerative cells that are placed in nests scattered among the digestive cells. During metamorphosis, the larval midgut epithelium degenerates and a new adult midgut epithelium is built during larval differentiation of regenerative cells. The present work focuses on the midgut epithelial modifications during the post-embryonic development of the stingless bee Melipona quadrifasciata anthidioides worker and the occurrence of regenerative cell proliferation during midgut metamorphosis in order to test the hypothesis that adult midgut epithelium of worker bees results from regenerative cell proliferation during the pupal stage. Regenerative cell proliferation was detected during larval lifespan. Larval aging is followed by an increase in the number and the size of the nests of regenerative cells. Larval epithelium degeneration begins 2 days after the start of defecation process and in this period the nests of regenerative cells are in contact by means of cytoplasmic extension which have many septate desmosomes and gap junctions. The BrdU immunoreactive regenerative cells were found in the prepupae 12 h after BrdU injection, suggesting that regenerative cell population increase during this larval period. Regenerative cell proliferation results in the increase of the regenerative cell population and not in the formation of new digestive cells because the proliferation of regenerative cells would not be enough to reestablish the nests of regenerative cells and at the same time form new adult digestive cells. In this sense the hypothesis that digestive adult cells originate from regenerative cell proliferation during midgut metamorphosis in M. quadrifasciata anthidioides was rejected.

The toxicological stress induced by pesticides, particularly neonicotinoid insecticides, and its consequences in bees has been the focus of much recent attention, particularly for honey bees. However, the emphasis on honey bees and neonicotinoids has led to neglect of the relevance of stingless bees, the prevailing pollinators of natural and agricultural tropical ecosystems, and of other agrochemicals, including other pesticides and even leaf fertilizers. Consequently, studies focusing on agrochemical effects on stingless bees are sparse, usually limited to histopathological studies, and lack a holistic assessment of the effects of these compounds on physiology and behavior. Such effects have consequences for individual and colony fitness and are likely to affect both the stingless bee populations and the associated community, thereby producing a hierarchy of consequences thus far overlooked. Herein, we review the current literature on stingless bee-agrochemical interactions and discuss the underlying mechanisms involved in reported stress symptoms, as well as the potential consequences based on the peculiarities of these pollinators.