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.

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.

The functional activity of the miR-1914-5p in lipid metabolism of the hepatocarcinoma cell line HepG2: a potential molecular tool for controlling hepatic cellular migration.

Hepatocellular carcinoma is one of the most common types of cancer in the world with high mortality rate and new therapies that control of fatty acid metabolism may limit the proliferation of cancer cells. In the last two decades, the non-coding RNAs have been considered as promising molecular tools to treat diseases, because they are able to modulate gene expression and the metabolic routes; however, deep investigation of their mechanistic behavior in pathologies must be performed. Thus, our aim was to evaluate the modulatory effect of the miR-1914-5p in controlling lipid metabolism in HepG2, a widely used human hepatocarcinoma cell line. The molecular and cellular analyses demonstrated that the functional inhibition of the investigated microRNA completely changed the cellular metabolism and behavior, compared to control groups. The in vitro inhibition of the miR-1914-5p increased the energy expenditure pointed in different analyses, decreasing cell doubling time and migration rate verified in wound healing and in the classical transwell chambers invasion assays, which makes the miR-1914-5p a candidate for further translational and preclinical studies to validate its function in controlling metastasis in liver cancer or even treat those diseases.

Enzymatic responses in the head and midgut of Africanized Apis mellifera contaminated with a sublethal concentration of thiamethoxam.

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.

In Situ Metabolomics of the Honeybee Brain: The Metabolism of l-Arginine through the Polyamine Pathway in the Proboscis Extension Response (PER).

The proboscis extension response (PER) reflex may be used to condition the pairing of an odor with sucrose, which is applied to the antennae, in experiments to induce learning, where the odor represents a conditioned stimulus, while sucrose represents an unconditioned stimulus. A series of studies have been conducted on honeybees, relating learning and memory acquisition/retrieval using the PER as a strategy for accessing their ability to exhibit an unconditioned stimulus; however, the major metabolic processes involved in the PER are not well known. Thus, the aim of this investigation is profiling the metabolome of the honeybee brain involved in the PER. In this study, a semiquantitative approach of matrix-assisted laser desorption ionization (MALDI) mass spectral imaging (MSI) was used to profile the most abundant metabolites of the honeybee brain that support the PER. It was reported that execution of the PER requires the metabolic transformations of arginine, ornithine, and lysine as substrates for the production of putrescine, cadaverine, spermine, spermidine, 1,3-diaminopropane, and γ-aminobutyric acid (GABA). Considering the global metabolome of the brain of honeybee workers, the PER requires the consumption of large amounts of cadaverine and 1,3-diaminopropane, in parallel with the biosynthesis of high amounts of spermine, spermidine, and ornithine. To exhibit the PER, the brain of honeybee workers processes the conversion of l-arginine and l-lysine through the polyamine pathway, with different regional metabolomic profiles at the individual neuropil level. The outcomes of this study using this metabolic route as a reference are indicating that the antennal lobes and the calices (medial and lateral) were the most active brain regions for supporting the PER.

Cellular and molecular effects of silymarin on the transdifferentiation processes of LX-2 cells and its connection with lipid metabolism.

Fibrosis process in the liver is a clinical condition established in response to chronic lesions and may be reversible in many situations. In this process, hepatic stellate cells (HSCs) activate and produce extracellular matrix compounds. During fibrosis, the lipid metabolism is also altered and contributes to the transdifferentiation of the HSCs. Thus, controlling lipid metabolism in HSCs is suggested as a method to control or reverse the fibrotic condition. In the search for therapies that modulate lipid metabolism and treat liver diseases, silymarin has been identified as a relevant natural compound to treat liver pathologies. The present study aimed to evaluate the cellular and molecular effects of silymarin in the transdifferentiation process of HSCs (LX-2) from activated phenotype to a more quiesced-like cells , also focusing on understanding the modulatory effects of silymarin on lipid metabolism of HSCs. In our analyses, 100 µM of silymarin reduced the synthesis of actin filaments in activated cells, the synthesis of the protein level of α-SMA, and other pro-fibrotic factors such as CTGF and PFGF. The concentration of 150 µM silymarin did not reverse the activation aspects of LX-2 cells. However, both evaluated concentrations of the natural compound protected the cells from the negative effects of dimethyl sulfoxide (DMSO). Furthermore, we evaluated lipid-related molecules correlated to the transdifferentiation process of LX-2, and 100 µM of silymarin demonstrated to control molecules associated with lipid metabolism such as FASN, MLYCD, ACSL4, CPTs, among others. In contrast, cellular incubation with 150 µM of silymarin increased the synthesis of long-chain fatty acids and triglycerides, regarding the higher presence of DMSO (v/v) in the solvent. In conclusion, silymarin acts as a hepatoprotective agent and modulates the pro-fibrogenic stimuli of LX-2 cells, whose effects depend on stress levels in the cellular environment.

Occurrence of virus, microsporidia, and pesticide residues in three species of stingless bees (Apidae: Meliponini) in the field.

Bees are important pollinators whose population has declined due to several factors, including infections by parasites and pathogens. Resource sharing may play a role in the dispersal dynamics of pathogens among bees. This study evaluated the occurrence of viruses (DWV, BQCV, ABPV, IAPV, KBV, and CBPV) and microsporidia (Nosema ceranae and Nosema apis) that infect Apis mellifera, as well as pesticide residues in the stingless bees Nannotrigona testaceicornis, Tetragonisca angustula, and Tetragona elongata sharing the same foraging area with A. mellifera. Stingless bees were obtained from 10 nests (two of N. testaceicornis, five of T. angustula, and three of T. elongata) which were kept in the field for 1 year and analyzed for the occurrence of pathogens. Spores of N. ceranae were detected in stingless bees but were not found in their midgut, which indicates that these bees are not affected, but may be vectors of the microsporidium. Viruses were found in 23.4% of stingless bees samples. APBV was the most prevalent virus (10.8%) followed by DWV and BQCV (both in 5.1% of samples). We detected glyphosate and its metabolites in small amounts in all samples. The highest occurrence of N. ceranae spores and viruses was found in autumn-winter and may be related to both the higher frequency of bee defecation into the colony and the low food resources available in the field, which increases the sharing of plant species among the stingless bees and honey bees. This study shows the simultaneous occurrence of viruses and spores of the microsporidium N. ceranae in asymptomatic stingless bees, which suggest that these bees may be vectors of pathogens.

The modulatory effect of triclosan on the reversion of the activated phenotype of LX-2 hepatic stellate cells.

Hepatic diseases leading to fibrosis affect millions of individuals worldwide and are a major public health challenge. Although, there have been many advances in understanding hepatic fibrogenesis, an effective therapy remains elusive. Studies focus primarily on activation of the hepatic stellate cells (HSCs), the principal fibrogenic cells in the liver; however, fewer numbers of studies have examined molecular mechanisms that deactivate HSC, controlling the profibrogenic phenotype. In the present study, we evaluated cellular and molecular actions of the chemical triclosan (TCS) in reverting activated HSCs to a quiesced phenotype. We demonstrated that the inhibition of the enzyme fatty acid synthase by TCS in activated HSCs promotes survival of the cells and triggers cellular and molecular changes that promote cellular phenotypic reversion, offering potentially new therapeutic directions.

Fungicide pyraclostrobin affects midgut morphophysiology and reduces survival of Brazilian native stingless bee Melipona scutellaris.

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.

Standardization of in vitro nervous tissue culture for honeybee: A high specificity toxicological approach.

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 CO2 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.

Is the Water Supply a Key Factor in Stingless Bees' Intoxication?

Water is an important resource for stingless bees, serving for both honey dilution and the composition of larval food inside nests, yet can be an important route of exposure to pesticides. Assuming bees can forage naturally on pesticide-contaminated or noncontaminated areas, we investigated whether water supply influences the choice between neonicotinoid-dosed or nondosed feeders and on mortality of the stingless bee, Melipona scutellaris (Latreille, Hymenoptera, Apidae). At the field concentration, there was no significant mortality; however, the bees were not able to distinguish the feeders. In the cages containing high-concentration feeders, with water supply, the bees preferred nondosed food, and with no water, the mortality increased. Considering that in the field it is common to find extrapolated concentrations, our work suggested that water may allow avoidance of high dosed food and minimize mortality.

Exposure to thiamethoxam during the larval phase affects synapsin levels in the brain of the honey bee.

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.

MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee ( Apis mellifera) Brain: Effect of Aggressiveness.

Aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown, and thus worker honeybees were stimulated to attack and to sting leather targets hung in front of the colonies. The aggressive individuals were collected and immediately frozen in liquid nitrogen; the heads were removed and sliced at sagittal plan. The brain slices were submitted to MALDI spectral imaging analysis, and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88-96) (APMGFQGMR-NH2), and AmTRP (254-262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of the honeybee brain during the aggressive behavior, possibly taking part in the neuromodulation of different aspects of this complex behavior. These results were biologically validated by performing aggressiveness-related behavioral assays using young honeybee workers that received 1 ng of AmAST A (69-76) or AmTRP (88-96) via hemocele. The young workers that were not expected to be aggressive individuals presented a complete series of aggressive behaviors in the presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in the honeybee brain with the aggressiveness of this insect.

Exposure to a sublethal concentration of imidacloprid and the side effects on target and nontarget organs of Apis mellifera (Hymenoptera, Apidae).

The use of insecticides has become increasingly frequent, and studies indicate that these compounds are involved in the intoxication of bees. Imidacloprid is a widely used neonicotinoid; thus, we have highlighted the importance of assessing its oral toxicity to Africanized bees and used transmission electron microscopy to investigate the sublethal effects in the brain, the target organ, and the midgut, responsible for the digestion/absorption of food. In addition, the distribution of proteins involved in important biological processes in the brain were evaluated on the 1st day of exposure by MALDI-imaging analysis. Bioassays were performed to determine the Median Lethal Concentration (LC50) of imidacloprid to bees, and the value obtained was 1.4651 ng imidacloprid/µL diet. Based on this result, the sublethal concentration to be administered at 1, 4 and 8 days was established as a hundredth (1/100) of the LC50. The results obtained from the ultrastructural analysis showed alterations in the midgut cells of bees as nuclear and mitochondrial damage and an increase of vacuoles. The insecticide caused spacing among the Kenyon cells in the mushroom bodies, chromatin condensation and loss of mitochondrial cristae. The MALDI-imaging analysis showed an increase in the expression of such proteins as vascular endothelial growth factor receptor, amyloid protein precursor and protein kinase C, which are related to oxygen supply, neuronal degeneration and memory/learning, and a decrease in the expression of the nicotinic acetylcholine receptor alpha 1, which is fundamental to the synapses. These alterations demonstrated that imidacloprid could compromise the viability of the midgut epithelium, as well as inhibiting important cognitive processes in individuals, and may be reflected in losses of the colony.

Antigenotoxicity and antimutagenicity of ethanolic extracts of Brazilian green propolis and its main botanical source determined by the Allium cepa test system.

Brazilian green propolis is a resinous substance prepared by bees from parts of the plant Baccharis dracunculifolia. As it possess several biological properties, this work assessed the cytotoxic/anticytotoxic, genotoxic/antigenotoxic and mutagenic/antimutagenic potential of ethanolic extracts of Brazilian green propolis (EEGP) and of B. dracunculifolia (EEBD), by means of the Allium cepa test system. The effects were evaluated by assessing the chromosomal aberrations (CA) and micronuclei (MN) frequencies on meristematic and F1 generation cells from onion roots. Chemical analyses performed with the extracts showed differences in flavonoid quality and quantity. No genotoxic or mutagenic potential was detected, and both extracts were capable of inhibiting cellular damage caused by methyl methanesulfonate (MMS) treatment, reducing the frequencies of CA and MN. By these data, we can infer that, independent of their flavonoid content, the extracts presented a protective effect in A. cepa cells against the clastogenicity of MMS.

Liver alterations in Oreochromis niloticus (Pisces) induced by insecticide imidacloprid: Histopathology and heat shock protein in situ localization.

Liver is very sensitive to environmental contaminants such as pesticides, it being the first target of toxicity of a substance. The objective of this study was to investigate the possible effects of the insecticide imidacloprid (IMI) on the liver of Oreochromis niloticus according concentrations used for growing sugarcane. A semi-quantitative analysis of histopathological alterations of IMI on liver was performed by light microscopy and cellular labeling of heat shock proteins (HSP70) by immunohistochemistry. The most common changes in liver at all concentrations of IMI were hydropic degeneration, pyknotic nuclei, and loss of cell limits. Steatosis and increased levels of HSP70 were detected in hepatocytes with the highest concentration of IMI. In conclusion, the tested concentrations of IMI induced histopathological changes in the liver of O. niloticus and active defence mechanisms to maintain the morphophysiological integrity of the liver. This insecticide has a toxicity potential for these fish, which is a non-target organism of its action.

Toxicity of Imidacloprid to the Stingless Bee Scaptotrigona postica Latreille, 1807 (Hymenoptera: Apidae).

The stingless bee Scaptotrigona postica is an important pollinator of native and cultivated plants in Brazil. Among the factors affecting the survival of these insects is the indiscriminate use of insecticides, including the neonicotinoid imidacloprid. This work determined the toxicity of imidacloprid as the topical median lethal dose (LD50) and the oral median lethal concentration (LC50) as tools for assessing the effects of this insecticide. The 24 and 48 h LD50 values were 25.2 and 24.5 ng of active ingredient (a.i.)/bee, respectively. The 24 and 48 h LC50 values were 42.5 and 14.3 ng a.i./µL of diet, respectively. Ours results show the hazard of imidacloprid and the vulnerability of stingless bees to it, providing relevant toxicological data that can used in mitigation programs to ensure the conservation of this species.

MALDI imaging analysis of neuropeptides in the Africanized honeybee (Apis mellifera) brain: effect of ontogeny.

The occurrence and spatial distribution of the neuropeptides AmTRP-5 and AST-1 in the honeybee brain were monitored via MALDI spectral imaging according to the ontogeny of Africanized Apis mellifera. The levels of these peptides increased in the brains of 0-15 day old honeybees, and this increase was accompanied by an increase in the number of in-hive activities performed by the nurse bees, followed by a decrease in the period from 15 to 25 days of age, in which the workers began to perform activities outside the nest (guarding and foraging). The results obtained in the present investigation suggest that AmTRP-5 acts in the upper region of both pedunculi of young workers, possibly regulating the cell cleaning and brood capping activities. Meanwhile, the localized occurrence of AmTRP-5 and AST-1 in the antennal lobes, subesophageal ganglion, upper region of the medulla, both lobula, and α- and ß-lobes of both brain hemispheres in 20 to 25 day old workers suggest that the action of both neuropeptides in these regions may be related to their localized actions in these regions, regulating foraging and guarding activities. Thus, these neuropeptides appear to have some functions in the honeybee brain that are specifically related to the age-related division of labor.

Side-effects of thiamethoxam on the brain andmidgut of the africanized honeybee Apis mellifera (Hymenopptera: Apidae).

The development of agricultural activities coincides with the increased use of pesticides to control pests, which can also be harmful to nontarget insects such as bees. Thus, the goal of this work was assess the toxic effects of thiamethoxam on newly emerged worker bees of Apis mellifera (africanized honeybee-AHB). Initially, we determined that the lethal concentration 50 (LC50 ) of thiamethoxam was 4.28 ng a.i./µL of diet. To determine the lethal time 50 (LT50 ), a survival assay was conducted using diets containing sublethal doses of thiamethoxam equal to 1/10 and 1/100 of the LC50. The group of bees exposed to 1/10 of the LC50 had a 41.2% reduction of lifespan. When AHB samples were analyzed by morphological technique we found the presence of condensed cells in the mushroom bodies and optical lobes in exposed honeybees. Through Xylidine Ponceau technique, we found cells which stained more intensely in groups exposed to thiamethoxam. The digestive and regenerative cells of the midgut from exposed bees also showed morphological and histochemical alterations, like cytoplasm vacuolization, increased apocrine secretion and increased cell elimination. Thus, intoxication with a sublethal doses of thiamethoxam can cause impairment in the brain and midgut of AHB and contribute to the honeybee lifespan reduction.

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.