Gustavo Ferreira Martins

Imidacloprid-Induced Impairment of Mushroom Bodies and Behavior of the Native Stingless Bee Melipona quadrifasciata anthidioides

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.

Biochemical and Morphological Aspects of Salivary Glands of the Predator Brontocoris tabidus (Heteroptera: Pentatomidae)

The aim of this work was to study the feeding habits of the predator Brontocoris tabidus (Heteroptera: Pentatomidae) analyzing the salivary gland complex (SGC). The SGC was dissected and subjected to histological analyses and biochemical assays. Results showed that a pair of bilobed principal salivary glands and one pair of tubular accessory salivary glands form the SGC and different parts of salivary gland synthesizes similar substances. Lipases, a-amylase and trypsin-like enzymes were detected at both lobes of the principal salivary gland. These data together with observations related to the predators diet suggested that it could be considered an obligate zoophytophagous.

Spinosad in the native stingless bee Melipona quadrifasciata: Regrettable non-target toxicity of a bioinsecticide

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.

Aedes aegypti midgut remodeling during metamorphosis

The Aedes aegypti midgut is restructured during metamorphosis; its epithelium is renewed by replacing the digestive and endocrine cells through stem or regenerative cell differentiation. Shortly after pupation (white pupae) begins, the larval digestive cells are histolized and show signs of degeneration, such as autophagic vacuoles and disintegrating microvilli. Simultaneously, differentiating cells derived from larval stem cells form an electron-dense layer that is visible 24h after pupation begins. Forty-eight hours after pupation onset, the differentiating cells yield an electron-lucent cytoplasm rich in microvilli and organelles. Dividing stem cells were observed in the fourth instar larvae and during the first 24h of pupation, which suggests that stem cells proliferate at the end of the larval period and during pupation. This study discusses various aspects of the changes during midgut remodeling for pupating A. aegypti.

A comparative study of the ovaries in some Brazilian bees (Hymenoptera; Apoidea)

A morfologia do ovario em 33 especies de abelhas apresentando diferentes graus de sociabilidade foi estudada. Todas as especies apresentaram ovario do tipo meroistico politrofico formado por um germario anterior e um vitelario basal. No germario estao localizadas as celulas germinativas e o vitelario apresenta foliculos arranjados linearmente. Cada foliculo e constituido pela câmara nutridora e pela câmara ovocitica, ambas revestidas por uma camada unica de celulas foliculares. O numero de ovariolos/ovario e de ovocitos maduros/ovario, o comprimento dos ovariolos, o tamanho e a largura dos ovocitos, a altura do epitelio folicular e a distância intertegular foram analisadas, mostrando que os representantes das tribos altamente eussociais Meliponini e Apini tem os maiores ovarios, enquanto as abelhas solitarias apresentam maiores ovocitos. Os resultados obtidos sugerem que ha uma tendencia para o aumento no tamanho do ovario e numero de ovariolos conforme ha um aumento no nivel de sociabilidade das abelhas.

Agrochemical-induced stress in stingless bees: peculiarities, underlying basis, and challenges

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.

Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection

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 °C but had different LD50 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.

Detection of a new pyrethroid resistance mutation (V410L) in the sodium channel of Aedes aegypti : a potential challenge for mosquito control

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.

Protocol for the in vitro rearing of honey bee (Apis mellifera L.) workers

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.

Histochemical and ultrastructural studies of the mosquito Aedes aegypti fat body: effects of aging and diet type.

Aedes aegypti is the principal vector of dengue world wide and a major vector of urban yellow fever. Despite its epidemiological importance, not much is known regarding cellular and structural changes in the fat body in this mosquito. Here, we applied light and transmission electron microscopies to investigate structural changes in the fat body of three groups of A. aegypti females: newly emerged, 18‐day‐old sugar‐fed, and 18‐day‐old blood‐fed. The fat body consists of a layer of cells attached to the abdomen integument, formed by trophocytes and oenocytes. Trophocytes are strongly positive for carbohydrates, while oenocytes are strongly positive for proteins and lipids. Ultrastructural analyses of trophocytes from newly emerged and 18‐day‐old blood‐fed indicate that these cells are rich in glycogen and free ribosomes. Many lipid droplets and protein granules, which are broken down after the blood meal, are also detected. In 18‐day‐old sugar‐fed, trophocytes display a disorganized cytoplasm filled with lipid droplets, and reduced numbers of free ribosomes, glycogen, rough endoplasmic reticulum (RER) and mitochondria. Following a blood meal, the RER and mitochondria display enlarged sizes, suggestive of increased activity. With regard to oenocytes, these cells display an electron‐dense cytoplasm and plasma membrane infoldings facing the hemolymph. As the A. aegypti female ages, trophocyte and oenocyte cell nuclei become larger but decrease in diameter after blood feeding. Our findings suggest that the trophocytes and oenocytes remodeling is likely involved in functional changes of fat body that take place during aging and following a blood meal in A. aegypti females. Microsc. Res. Tech. 2011.