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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., Competing Interests: Declaration of competing interest The authors declare no competing financial interest. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD041422., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The concern about pesticide exposure to neotropical bees has been increasing in the last few years, and knowledge gaps have been identified. Although stingless bees, (e.g.: Melipona scutellaris), are more diverse than honeybees and they stand out in the pollination of several valuable economical crops, toxicity assessments with stingless bees are still scarce. Nowadays new approaches in ecotoxicological studies, such as omic analysis, were pointed out as a strategy to reveal mechanisms of how bees deal with these stressors. To date, no molecular techniques have been applied for the evaluation of target and/or non-target organs in stingless bees, such as the Malpighian tubules (Mt). Therefore, in the present study, we evaluated the differentially expressed genes (DEGs) in the Mt of M. scutellaris after one and eight days of exposure to LC 50/100 (0.000543 ng a.i./μL) of thiamethoxam (TMX). Through functional annotation analysis of four transcriptome libraries, the time course line approach revealed 237 DEGs (nine clusters) associated with carbon/energy metabolism and cellular processes (lysosomes, autophagy, and glycan degradation). The expression profiles of Mt were altered by TMX in processes, such as detoxification, excretion, tissue regeneration, oxidative stress, apoptosis, and DNA repair. Transcriptome analysis showed that cell metabolism in Mt was mainly affected after 8 days of exposure. Nine genes were selected from different clusters and validated by RT-qPCR. According to our findings, TMX promotes several types of damage in Mt cells at the molecular level. Therefore, interference of different cellular processes directly affects the health of M. scutellaris by compromising the function of Mt., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Eucalyptus plantations occupy 26 % of Portuguese forested areas. Its flowers constitute important sources for bees and beekeepers take advantage of this and keep their honey bee colonies within or near the plantations for honey production. Nonetheless, these plantations are susceptible to pests, such as the eucalyptus weevil Gonipterus platensis. To control this weevil, some plantations must be treated with pesticides, which might harm non-target organisms. This study aimed to perform a multifactorial assessment of the health status and development of Apis mellifera iberiensis colonies in two similar landscape windows dominated by Eucalyptus globulus plantations - one used as control and the other with insecticide treatment. In each of the two selected areas, an apiary with five hives was installed and monitored before and after a single application of the insecticide acetamiprid (40 g a.i./ha). Colony health and development, resources use, and pesticide residues accumulation were measured. The results showed that the application of acetamiprid in this area did not alter the health status and development of the colonies. This can be explained by the low levels of residues of acetamiprid detected only in pollen and bee bread samples, ~52 fold lower than the sublethal effect threshold. This could be attributed to the low offer of resources during and after the application event and within the application area, with the consequent foraging outside the sprayed area during that period. Since exposure to pesticides in such complex landscapes seems to be dependent on the spatial and temporal distribution of resources, we highlight some key monitoring parameters and tools that are able to provide reliable information on colony development and use of resources. These tools can be easily applied and can provide a better decision-taking of pesticide application in intensive production systems to decrease the risk of exposure for honey bees., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The increasing use of insecticides, promoted by the intensification of agriculture, has raised concerns about their influence on the decline of bee colonies, which play a fundamental role in pollination. Thus, it is fundamental to elucidate the effects of insecticides on bees. This study investigated the damage caused by a sublethal concentration of thiamethoxam - TMX (0.0227 ng/μL of feed) in the head and midgut of Africanized Apis mellifera, by analyzing the enzymatic biomarkers, oxidative stress, and occurrence of lipid peroxidation. The data showed that the insecticide increased acetylcholinesterase activity (AChE) and glutathione-S-transferase (GST), whereas carboxylesterase (CaE3) activity decreased in the heads. Our results indicate that the antioxidant enzymes were less active in the head because only glutathione peroxidase (GPX) showed alterations. In the midgut, there were no alkaline phosphatase (ALP) or superoxide dismutase (SOD) responses and a decrease in the activity of CaE was observed. Otherwise, there was an increase in GPX, and the TBARS (thiobarbituric acid reactive substances) assay also showed differences in the midgut. The TBARS (thiobarbituric acid reactive substances) assay also showed differences in the midgut. The results showed enzymes such as CaE3, GST, AChE, ALP, SOD, and GPX, as well as the TBARS assay, are useful biomarkers on bees. They may be used in combination as a promising tool for characterizing bee exposure to insecticides., (Copyright © 2021. Published by Elsevier Inc.)

Native stingless bees are key pollinators of native flora and important for many crops. However, the loss of natural fragments and exposure to pesticides can hinder the development of colonies and represent a high risk for them. Nevertheless, most studies are conducted with honeybees and there are not many studies on native species, especially in relation to the effects of fungicides on them. Therefore, the objective of this paper is to evaluate the effects of sublethal concentrations of pyraclostrobin, on Melipona scutellaris forager workers. These Brazilian native stingless bees were submitted to continuous oral exposure to three concentrations of pyraclostrobin in sirup: 0.125 ng a.i./µL (P1), 0.025 ng a.i./µL (P2), and 0.005 ng a.i./µL (P3). Histopathological and histochemical parameters of midgut, as well as survival rate were evaluated. All concentrations of fungicide showed an increase in the midgut lesion index and morphological signs of cell death, such as cytoplasmic vacuolizations, presence of atypical nuclei or pyknotic nuclei. Histochemical analyzes revealed a decreased marking of polysaccharides and neutral glycoconjugates both in the villi and in peritrophic membrane in all exposed-groups in relation to control-groups. P1 and P2 groups presented a reduction in total protein marking in digestive cells in relation to control groups. As a consequence of alteration in the midgut, all groups exposed to fungicide showed a reduced survival rate. These findings demonstrate that sublethal concentrations of pyraclostrobin can lead to significant adverse effects in stingless bees. These effects on social native bees indicate the need for reassessment of the safety of fungicides to bees., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bees are important pollinators that help to maintain the biodiversity of wild and cultivated plants. However, the increased and inappropriate use of agrochemicals has caused an imbalance in the populations of these insects visiting flowers for pollen and nectar collection. Therefore, new research methods for understanding the mechanisms of action of pesticides and their impacts on the brains of bees, such as neurotoxicity and cellular changes, in response to different active characteristics and dosages of insecticides are necessary. Thus, with the aim of developing tests with greater specificity at the level of cells or tissues, this study sought to standardize a method for the in vitro culture of the nervous tissue of Apis mellifera. For this purpose, the brains of six foragers bees were transferred to three different insect cell culture media and it supplementation with 10% foetal bovine serum (FBS): Grace, Schneider, Leibovitz, Grace + FBS, Schneider + FBS and Leibovitz + FBS media for each collection time. Nervous tissue was collected after 1, 6, 12 and 24 h of incubation in a humidified CO 2 incubator at 32 °C, and histological sections of the organs were analysed. The results showed that Leibovitz medium and Leibovitz medium + serum are potential culture media for the cultivation of nervous tissue, since they resulted in less tissue spacing and tissue disarrangement. Therefore, additional supplements are necessary to obtain an ideal medium for the cultivation of A.mellifera nervous tissue., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Thiamethoxam (TMX) is a neurotoxic insecticide widely used for insect pest control. TMX and other neonicotinoids are reported to be potential causes of honey bee decline. Due to its systematic action, TMX may be recovered in pollen, bee bread, nectar, and honey, which make bees likely to be exposed to contaminated diet. In this study, we used immunolabeling to demonstrate that sublethal concentrations of TMX decrease the protein levels of synapsin in the mushroom bodies (MBs) and the antennal lobes (ALs) of pupae and newly emerged worker bees that were exposed through the food to TMX during the larval phase. A decrease in the synapsin level was observed in the MBs of pupae previously exposed to 0.001 and 1.44 ng/µL and in newly emerged bees previously exposed to 1.44 ng/µL and no changes were observed in the optical lobes (OLs). In the ALs, the decrease was observed in pupae and newly emerged bees exposed to 1.44 ng/µL. Because the MBs and ALs are brain structures involved in stimuli reception, learning, and memory consolidation and because synapsin is important for the regulation of neurotransmitter release, we hypothesize that exposure to sublethal concentrations of TMX during the larval stage may cause neurophysiological disorders in honey bees., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The proboscis extension reflex (PER) is an unconditioned stimulus (US) widely used to access the ability of honeybees to correlate it with a conditioned stimulus (CS) during learning and memory acquisition. However, little is known about the biochemical/genetic changes in worker honeybee brains induced by the PER alone. The present investigation profiled the proteomic complement associated with the PER to further the understanding of the major molecular transformations in the honeybee brain during the execution of a US. In the present study, a quantitative shotgun proteomic approach was employed to assign the proteomic complement of the honeybee brain. The results were analyzed under the view of protein networking for different processes involved in PER behavior. In the brains of PER-stimulated individuals, the metabolism of cyclic/heterocyclic/aromatic compounds was activated in parallel with the metabolism of nitrogenated compounds, followed by the up-regulation of carbohydrate metabolism, the proteins involved with the anatomic and cytoskeleton; the down-regulation of the anatomic development and cell differentiation in other neurons also occurred., Significance: The assay of proboscis extension reflex is frequently used to access honeybees' ability to correlate an unconditioned stimulus with a conditioned stimulus (such as an odor) to establish learning and memory acquisition. The reflex behavior of proboscis extension was associated with various conditioned stimuli, and the biochemical/genetic evaluation of the changes occurring in honeybee brains under these conditions reflect the synergistic effects of both insect manipulations (training to answer to an unconditioned stimulus and training to respond to a conditioned stimulus). Little or no information is available regarding the biochemical changes stimulated by an unconditioned stimulus alone, such as the proboscis extension reflex. The present investigation characterizes the proteomic changes occurring in the brains of honeybee workers submitted to proboscis extension reflex. A series of metabolic and cellular processes were identified to be related to the reflex of an unconditioned stimulus. This strategy may be reproduced to further understand the processes of learning and memory acquisition in honeybees., (Copyright © 2016 Elsevier B.V. All rights reserved.)