Effect of neonicotinoid and fungicide strobilurin in neotropical solitary bee Centris analis.

The indiscriminate use of pesticides is one of the factors directly impacting bee populations. However, limited information is available on the pesticide effects on solitary bees, especially in Neotropical countries. In this scenario, this study evaluated the survival and histopathological effects caused by the neonicotinoid insecticide acetamiprid (7 ng/µL) and the fungicide azoxystrobin (10 ng/µL) in the midgut and parietal fat body of the solitary bee Centris analis. Female and male newly-emerged bees were orally exposed for 48 h to the pesticides, or alone or in combination, under laboratory conditions. The exposure to the insecticide reduced the survival of males, while the mixture reduced survival in both sexes. Acetamiprid promoted a reduction in the number of regenerative nests in the midgut, alterations of fat body cells by increasing carbohydrates in trophocytes, and reduction of oenocyte size, and increased the frequency of pericardial cells in the advanced activity stage. Both pesticides caused changes in HSP70 immunolabelling of midgut from males at the end of pesticide exposure. Comparatively, the effects on males were stronger than in females exposed to the same pesticides. Therefore, acetamiprid alone and in mixture with fungicide azoxystrobin can be harmful to males and females of Neotropical solitary bee C. analis showing lethal and sublethal effects at a concentration likely to be found in the environment.

Thiamethoxam toxicity on the stingless bee Friesiomelitta varia: LC50, survival time, and enzymatic biomarkers assessment.

Bees play a crucial role as pollinating insects in both natural and cultivated areas. However, the use of pesticides, such as thiamethoxam, has been identified as a contributing factor compromising bee health. The current risk assessment primarily relies on the model species Apis mellifera, raising concerns about the applicability of these assessments to other bee groups, including stingless bees. In this study, we investigated the acute toxicity of thiamethoxam on the stingless bee Frieseomelitta varia by determining the average lethal concentration (LC50) and mean lethal time (LT50). Additionally, we evaluated the enzymatic profile of Acetylcholinesterase (AChE), Carboxylesterase-3 (CaE-3), and Glutathione S-Transferase (GST), in the heads and abdomens of F. varia after exposure to thiamethoxam (LC50/10). The LC50 of thiamethoxam was determined to be 0.68 ng ai/µL, and the LT50 values were 37 days for the control group, 25 days at LC50/10, and 27 days at LC50/100. The thiamethoxam significantly decreased the survival time of F. varia. Furthermore, the enzymatic profile exhibited differences in CaE3 activity within one day in the heads and ten days in the abdomen. GST activity showed differences in the abdomen after one and five days of thiamethoxam exposure. These findings suggests that the abdomen is more affected than the head after oral exposure to thiamethoxam. Our study provides evidence of the toxicity of thiamethoxam at both the cellular and organismal levels, reinforcing the need to include non-Apis species in pollinator risk assessments. and provide solid arguments for bee protection.

Sublethal exposure to thiamethoxam and pyraclostrobin affects the midgut and Malpighian tubules of the stingless bee Frieseomelitta varia (Hymenoptera: Apidae: Meliponini).

The stingless bee Frieseomelitta varia Lepeletier 1836 (Hymenoptera: Apidae) is an essential pollinator in natural and agricultural ecosystems in the Neotropical region. However, these bees may be exposed to pesticides during foraging, which can affect both individuals and their colonies. One example comes from the use of pyraclostrobin (a fungicide) and thiamethoxam (an insecticide) for pest control in pepper crops, which F. varia visits. This study aimed to evaluate the isolated and combined sublethal effects of thiamethoxam (TMX) (0.000543 ng a.i./µL) and pyraclostrobin (PYR) (1.5 ng i.a./µL) on the morphology of the midgut and Malpighian tubules of F. varia workers. Results showed that both pesticides, regardless of the exposure time (through feeding during 48 h or 96 h), disturbed the morphology of the analyzed organs. Specifically, F. varia exposed orally to sublethal concentrations of thiamethoxam and pyraclostrobin, either alone or in combination, exhibited a higher rate of damage to the midgut (e.g., vacuolization, apocrine secretion, and cellular elimination) compared to the bees in the control groups, both after 48 h and 96 h of exposure. In Malpighian tubules, vacuolation is the only damage present. As the observed morphological alterations likely compromise the excretion and absorption functions, exposure to pyraclostrobin and thiamethoxam may lead to disturbances at both the individual and colony levels. These results highlight the urgent need for a future reassessment of the safety of fungicides and insecticides regarding their potential effects on bee populations.

Exposure of the stingless bee Melipona scutellaris to imidacloprid, pyraclostrobin, and glyphosate, alone and in combination, impair its walking activity and fat body morphology and physiology.

The stingless bee Melipona scutellaris performs buzz pollination, effectively pollinating several wild plants and crops with economic relevance. However, most research has focused on honeybees, leaving a significant gap in studies concerning native species, particularly regarding the impacts of pesticide combinations on these pollinators. Thus, this study aimed to evaluate the sublethal effects of imidacloprid (IMD), pyraclostrobin (PYR), and glyphosate (GLY) on the behavior and fat body cell morphology and physiology of M. scutellaris. Foragers were orally exposed to the different pesticides alone and in combination for 48 h. Bees fed with contaminated solution walked less, moved slower, presented morphological changes in the fat body, including vacuolization, altered cell shape and nuclei morphology, and exhibited a higher count of altered oenocytes and trophocytes. In all exposed groups, alone and in combination, the number of cells expressing caspase-3 increased, but the TLR4 number of cells expressing decreased compared to the control groups. The intensity of HSP70 immunolabeling increased compared to the control groups. However, the intensity of the immunolabeling of HSP90 decreased in the IMD, GLY, and I + G (IMD + GLY) groups but increased in I + P-exposed bees (IMD + PYR). Alternatively, exposure to PYR and P + G (PYR + GLY) did not affect the immunolabeling intensity. Our findings demonstrate the hazardous effects and environmental consequences of isolated and combined pesticides on a vital neotropical pollinator. Understanding how pesticides impact the fat body can provide crucial insights into the overall health and survival of native bee populations, which can help develop more environmentally friendly approaches to agricultural practices.

Profiling the neuroproteomics of honeybee brain: A clue for understanding the role of neuropeptides in the modulation of aggressivity.

The aggressivity is modulated in honeybee brain through a series of actions in cascade mode, with the participation of the neuropeptides AmAST A (59-76) and AmTRP (254-262). The aggressivity of honeybees was stimulated by injecting both neuropeptides in the hemocoel of the worker honeybees, which were submitted to behavioral assays of aggression. The brain of stinger individuals were removed by dissection and submitted to proteomic analysis; shotgun proteomic approach of honeybee brain revealed that both neuropeptides activate a series of biochemical processes responsible by production of energy, neuronal plasticity and cell protection. In addition to this, AmTRP (254-262) elicited the expression of proteins related to the processing of the potential of action and lipid metabolism; meanwhile AmAST A (59-76) elicited the metabolism of steroids and Juvenile hormone-related metabolism, amongst others. Apparently, the most complex biochemical process seems to be the regulation of ATP production, which occurs at two levels: i) by a subgroup of proteins common to the three experimental groups, which are over-/under-regulated through glycolysis, pyruvate pathway, Krebbs cycle and oxidative phosphorylation; ii) by a subgroup of proteins unique to the each experimental group, which seems to be regulated through Protein-Protein Interactions, where the protein network regulated by AmTRP (254-262) seems to be more complex than the other two experimental groups. SIGNIFICANCE: Recently we reported the effect of the neuropeptides AmAST A (59-76) and AmTRP (254-262) in the modulation of the aggressive behavior of the worker honeybees. Up to now it is known that the simple presence of the allatostatin and tachykinin-related-peptide in bee brain, is enough for inducing the aggressive behavior. However, nothing was known about how these neuropeptides perform their action, inducing the aggressive behavior. The results of the present study elucidated some of the metabolic pathways that were activated or inhibited to support the complex defensive behavior, which includes the aggressivity. These results certainly will impact the behavioral research of honeybees, since we are paving the way for understanding the molecular base of regulation, of individual /nest defense of honeybees.

Landscape and land use affect composition and nutritional values of bees' food.

Bees are primary pollinators across various terrestrial biomes and rely heavily on floral resources for sustenance. The composition of landscapes can influence bee foraging behavior, while human activities can directly affect both the composition and nutritional value of bee food. We aimed to assess how landscape structure and land use practices can impact the composition and nutritional value of food sources for two generalist social bee species, Apis mellifera and Scaptotrigona postica. Food samples were collected from twenty-five colonies of A. mellifera and thirteen of S. postica to examine how food composition and nutritional value may vary based on the extent of human land use and the composition of landscapes surrounding beekeeping sites. The pollen composition and nutritional value of A. mellifera were influenced by both land use practices and landscape heterogeneity. The number of patches determined total sugar and lipid content. Landscape heterogeneity affected pollen composition in S. postica, primarily due to the number of patches, while total sugar was affected by landscape diversity. Pollen nutritional value in S. postica was linked to land use, mainly meadow and vegetation, which influenced total sugar and dry matter. S. postica showed a higher sensitivity to land use changes compared to A. mellifera, which was more affected by landscape heterogeneity. Assuring landscape heterogeneity by preserving remaining forest patches around apiaries and meliponaries is crucial. Thoughtful land use planning is essential to support beekeeping activities and ensure an adequate quantity and quality of bee food resources.

Exposure to sublethal concentrations of imidacloprid, pyraclostrobin, and glyphosate harm the behavior and fat body cells of the stingless bee Scaptotrigona postica.

Pesticide use in agriculture threatens non-target insects such as bees. Considering the ecological and economic relevance of native bees, such as Scaptotrigona postica, and the insufficient studies on the effects of pesticides on their behavior and physiology, improving the current knowledge on this issue is essential. Therefore, this study investigated the sublethal effects of imidacloprid, pyraclostrobin, and glyphosate on the behavior and fat body cells of S. postica. Pesticide ingestion decreased the walking distance and mean velocity of bees compared to the control and solvent control groups. The oenocytes of the control groups were spherical, with central nuclei containing decondensed chromatin, and the trophocytes presented irregular morphology, with cells varying in shape and the cytoplasm filled with vacuoles and granules. However, bees exposed to pesticides showed extensive cytoarchitectural disruption in the fat body, such as vacuolization and shape changes in oenocytes and altered nuclei morphology in trophocytes. Moreover, pesticide exposure increased the number of atypical oenocytes and altered trophocytes, except for the PYR group, which showed a lower number of atypical oenocytes. Caspase-positive labeling significantly increased in all exposed bee groups. Alternatively, TLR4 labeling was significantly decreased in the exposed groups compared to the control groups. There was a significant increase in HSP90 immunolabeling in all exposed groups compared to the control. These findings reinforce the importance of research on the sublethal effects of low pesticide concentrations on key neotropical pollinators and prove that these toxic substances can impair their detoxification and immune defense.

Ultrastructural Changes in the Midgut of Brazilian Native Stingless Bee Melipona scutellaris Exposed to Fungicide Pyraclostrobin.

Melipona scutellaris is a Brazilian stingless bee that is important for pollinating wild flora and agriculture crops. Fungicides have been widely used in agriculture, and floral residues can affect forager bees. The goal of our study was to evaluate the effects of sublethal concentrations of pyraclostrobin on the midgut ultrastructure of M. scutellaris forager workers. The bees were collected from three non-parental colonies and kept under laboratory conditions. The bees were orally exposed continuously for five days to pyraclostrobin in syrup at concentrations of 0.125 ng a.i./µL (FG1) and 0.005 ng a.i./µL (FG2). The control bees (CTL) were fed a no-fungicide sucrose solution, and the acetone solvent control bees (CAC) received a sucrose solution containing acetone. At the end of the exposure, the midguts were sampled, fixed in Karnovsky solution, and routinely processed for transmission electron microscopy. Ultrastructural analysis demonstrated that both the fungicide concentrations altered the midgut, such as cytoplasmic vacuolization (more intense in FG1), the presence of an atypical nuclear morphology, and slightly dilated mitochondrial cristae in the bees from the FG1 and FG2 groups (both more intense in FG1). Additionally, there was an alteration in the ultrastructure of the spherocrystals (FG1), which could be the result of cellular metabolism impairment and the excretion of toxic metabolites in the digestive cells as a response to fungicide exposure. The results indicate that ingested pyraclostrobin induced cytotoxic effects in the midgut of native stingless bees. These cellular ultrastructural responses of the midgut are a prelude to a reduced survival rate, as observed in previous studies.

Variation in honey yield per hive of Africanized bees depending on the introducing time of young queens / Variação na produção de mel por colmeias de abelhas africanizadas em função da época de introdução de rainhas jovens

ABSTRACT: The objective of this research was to evaluate the honey production per hive and the egg laying rates of queens produced in 2007, 2008 and 2010. Thirty colonies initiated with a queen per colony at each climatic season were used during the three years. The years, started on January (summer), April (autumn), July (winter) and October (spring) and ended 12 months later, at the same periods related to each season of the later years. Honey supply were weighed before and after centrifugation to evaluate the quantity of the stored honey. Colonies with queens introduced during autumn and winter in the three years produced 57.2±6.0kg and 60.7±7.5kg of honey, respectively. In the first year of production activity, after the introduction of queens in the initial colonies, values were significantly higher than those obtained in colonies with queens introduced in the summer (39.3±7.6kg) and spring (41.8±3.7kg). Egg laying rates of queens were higher in spring (98.2±3.9%) and summer (88.4±7%), indicating greater food flow (flowerings) in these seasons compared to the averages in autumn (30.3±8.1%) and winter (24.5±7.2%). Produce and introduce queens of Africanized Apis mellifera in colonies initiated during autumn and winter was found to be economically feasible. Honey production of colonies initiated in these periods were higher and they had greater population stability in times of scarcity of flowerings.

RESUMO: O objetivo deste trabalho foi avaliar a produção de mel por colmeia e as taxas de postura de ovos das rainhas produzidas nos anos de 2007, 2008 e 2010. Foram utilizadas 30 colônias iniciadas com uma rainha/colônia por estação climática, durante os três anos. A cada ano, iniciou-se em: janeiro (verão), abril (outono), julho (inverno) e outubro (primavera) e encerrou-se doze meses depois, nos mesmos períodos referentes a cada estação dos anos posteriores. As melgueiras foram pesadas antes e depois da centrifugação para avaliar a quantidade do mel estocado. As colônias com rainhas introduzidas durante o outono e o inverno nos três anos produziram 57,2±6,0kg e 60,7±7,5kg de mel, respectivamente, no primeiro ano de atividade de produção após a introdução das rainhas nas colônias iniciais, valores significativamente superiores aos obtidos para as colônias com rainhas introduzidas no verão (39,3±7,6kg) e na primavera (41,8±3,7kg). As taxas de postura de ovos das rainhas foram mais altas na primavera e no verão (98,2±3,9% e 88,4±7%, respectivamente), indicando maior fluxo de alimento (floradas) nessas épocas, quando comparadas às médias do outono e inverno (30,3±8,1% e 24,5±7,2%, respectivamente). Constatou-se que é economicamente viável produzir rainhas e introduzir em colônias iniciais de Apis mellifera africanizada durante o outono e inverno. Além da produção de mel das colônias iniciadas nesses períodos ser superior, elas terão maior estabilidade populacional em épocas de escassez de floradas.

Cell markers for ecotoxicological studies in target organs of bees

Bees are important pollinators that, because of extensive deforestation of their natural habitats, now forage widely in agricultural areas. This interaction with human agricultural activity has led to a reduction in the number of bee species because of contact with widely used pesticides. However, little is known about the adverse effects that exposure to such agents has on bee tissues and organs. In this review, we discuss the morphological alterations induced by environmental contaminants in the midgut and Malpighian tubules of bees; these two organs are involved in the absorption and excretion of toxic compounds, respectively. We also discuss the role of heat shock proteins, also known as stress proteins, in the cellular response to chemical compounds, and the importance of cell death as an indicator of the toxicity of these compounds. The analysis of these two cellular markers may be useful for monitoring bees that forage in agricultural areas.

Estudo dos métodos laboratoriais utilizados no diagnóstico de alergia a Hymenoptera: análise crítica / Study of laboratorial methods for Hymenoptera allergy diagnosis: a critical analysis

Objetivo:Avaliar dados epidemiológicos de 138 pacientes alérgicosa Hymenoptera e analisar o valor da IgE específica (RAST) em 68 pacientes, considerando a sensibilidade (S), e especificidade (Es), a eficácia (Ef), o valor preditivo positivo (VPP) e negativo (VPN)deste método. Métodos: Avaliou-se 138 pacientes acompanhados em serviço especializado que apresentaram reaçöes a himenópteros (abelha, vespa ou formiga), sendo submetidos a um protocolo clínico. Em 68 pacientes realizou-se teste cutâneo para o inseto referido na anamnese, sendo considerados alérgicos os pacientes que possuíam história sugestiva de reaçäo sistêmica (grau I a IV) e teste cutâneo positivo. Realizou-se o RASTpara cada um dos insetos e calculou-se S, Es, Ef, VPP, e VPN deste método em relaçäo à história clínica e testes cutâneos. Resultados: Nos 138 pacientes (92M: 46F), o primeiro episódio de alergia ocorreu em média aos 20,11 anos (mediana = 16 anos). Cerca de 93,47 por cento dos pacientes reconheceram o inseto envolvido (32,60 por cento, vespa; 28,20 por cento, abelha; 26,80 por cento, formiga; 5,78 por cento, mais de um inseto). A análise do RAST com relaçäo aos pacientes alérgicos mostrou os seguintes resultados para formiga: S= 90,47 por cento, Es= 36,36 por cento, Ef=53,84 por cento, VPP= 40,42 por cento, e VPN= 88,88 por cento. Para vespa: S= 61,90 por cento, Es=69,23 por cento, Ef=66,66 por cento, VPP=61,90 por cento e VPN=69,23 por cento e para abelha: S= 78,57 por cento, Es=67,39 por cento, EF= 70,00 por cento, VPP= 42,30 por cento e VPN = 91,17 por cento. Conclusöes: As reaçöes alérgicas aos himenópteros ocorreram preferencialmente em jovens do sexo masculino. Quanto ao RAST, sua valorizaçäo deve considerar o inseto envolvido. Para as formigas, mostrou-se um exame de alta sensibilidade, no entanto, baixas especificidade e eficiência. Quanto às abelhas e vespas, os índices apontam a necessidade do desenävolvimento de novos extratos, principalmente em relaçäo às vespas, por sua grande diversidade de espécies em países tropicais. Como nos testes cutâneos, o isolamento do inseto e produçäo de alérgeno específico, tornará o RAST um exame mais fidedigno.