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.

Breaking the cycle: Reforming pesticide regulation to protect pollinators.

Over decades, pesticide regulations have cycled between approval and implementation, followed by the discovery of negative effects on nontarget organisms that result in new regulations, pesticides, and harmful effects. This relentless pattern undermines the capacity to protect the environment from pesticide hazards and frustrates end users that need pest management tools. Wild pollinating insects are in decline, and managed pollinators such as honey bees are experiencing excessive losses, which threatens sustainable food security and ecosystem function. An increasing number of studies demonstrate the negative effects of field-realistic exposure to pesticides on pollinator health and fitness, which contribute to pollinator declines. Current pesticide approval processes, although they are superior to past practices, clearly continue to fail to protect pollinator health. In the present article, we provide a conceptual framework to reform cyclical pesticide approval processes and better protect pollinators.

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.

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.

Walker 256 Tumor Growth Suppression by Crotoxin Involves Formyl Peptide Receptors and Lipoxin A4.

We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A4. Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A4 and its natural analogue 15-epi-LXA4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A4 and 15-epi-LXA4, which might inhibit both tumor growth and formation of new vessels via FPRs.

Changes in Synapsin Levels in the Millipede Gymnostreptus olivaceus Schubart, 1944 Exposed to Different Concentrations of Deltamethrin.

Millipedes are ecologically important soil organisms and may also be an economically threatening species in rural and urban areas when population outbreaks occur. In order to control infestations commercial formulations of deltamethrin have been commonly applied, even though there are few studies about the effects of such insecticide on millipedes. This paper describes the effects of this insecticide on millipedes showing neurotoxic effects assessed by synapsin labeling and confocal microscopy. Deltamethrin concentrations related to the DL50 of the active ingredient and a field concentration were applied topically in the diplopod Gymnostreptus olivaceus to evaluate the behavior, mortality rate, and synapsin levels in the brain 12, 24, and 48h after contact with deltamethin. The insecticide caused mortality at the higher concentrations employed, in which no change was observed in neurotransmission in the survivors. In contrast, at field concentrations, deltamethrin did not cause any deaths, but triggered significant changes in synapsin levels. The results obtained form the synapsin labeling provide several interpretations suggesting that the isolated application of this tool must be associated with additional tools in order to evaluate biologically induced effects of deltamethrin in an accurate way. In addition, the feasibility of chemical control of millipedes with deltamethrin is questioned.

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.

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.

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.

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.

Effects of sublethal dose of fipronil on neuron metabolic activity of Africanized honeybees.

Fipronil is a neurotoxic insecticide that inhibits the gamma-aminobutyric acid receptor and can affect gustative perception, olfactory learning, and motor activity of the honeybee Apis mellifera. This study determined the lethal dose (LD50) and the lethal concentration (LC50) for Africanized honeybee and evaluated the toxicity of a sublethal dose of fipronil on neuron metabolic activity by way of histochemical analysis using cytochrome oxidase detection in brains from worker bees of different ages. In addition, the present study investigated the recovery mechanism by discontinuing the oral exposure to fipronil. The results showed that mushroom bodies of aged Africanized honeybees are affected by fipronil, which causes changes in metabolism by increasing the respiratory activity of mitochondria. In antennal lobes, the sublethal dose of fipronil did not cause an increase in metabolic activity. The recovery experiments showed that discontinued exposure to a diet contaminated with fipronil did not lead to recovery of neural activity. Our results show that even at very low concentrations, fipronil is harmful to honeybees and can induce several types of injuries to honeybee physiology.

Acute toxicity of fipronil to the stingless bee Scaptotrigona postica Latreille.

Fipronil is an insecticide widely used to control a great number of pests, thus the aim of this study was to determine the lethal dose and lethal concentration (LD(50) and LC(50)) of this insecticide to the stingless bees Scaptotrigona postica Latreille, 1807. The LD(50) and LC(50) values obtained after 24 h of exposition were of 0.54 ng a.i./bee and 0.24 ng a.i./µL diet, respectively. These values were considered highly toxic to stingless bees.

3D electrochemical device obtained by additive manufacturing for sequential determination of paraquat and carbendazim in food samples.

Pesticides are heavily employed compounds protecting crops, however, these compounds can be extremely harmful to human health. Once the monitoring of pesticides in foods is of great importance, in this work we propose a ready-to-use electrochemical sensor made with 3D printing technology, capable of detecting paraquat and carbendazim in sequential analysis. The proposed electrodes are lab-made and of easy obtention, composed of graphite on a polylactic acid matrix, and provided great results for the analysis of paraquat and carbendazim in honey, milk, juice, and water samples. The sequential analysis of paraquat and carbendazim was proposed, providing optimal analysis of both compounds individually when both are present in a mixture. Limits of detection of 0.01 and 0.03 µmol/L for paraquat and carbendazim, respectively. Recovery tests attested to the suitability of the method, ranging from 94.5 to 113.7 %, and the suitability of 3D printing for environmental and food samples analysis.

Surrogate species in pesticide risk assessments: Toxicological data of three stingless bees species.

Discussions about environmental risk reassessment of pesticides have grown in the last decades, especially in tropical and subtropical regions since the diversity of bee species in these places is quite different. Stingless bees are highly affected by pesticides, and toxicity information is necessary to include them in the regulatory process of countries that hosts a diversity of these species. Therefore, the present study aimed to evaluate the Median Lethal Concentration (LC50), estimate the Median Lethal Dose (LD50) and compared the sensitivity of three species of stingless bees exposed to the commercial formulation of the neonicotinoid thiamethoxam (TMX). The LD50 was estimated based on the LC50 determined in the present study (LC50 = 0.329 ng a.i./µL for Tetragonisca angustula; 0.624 ng a.i./µL for Scaptotrigona postica, and 0.215 ng a.i./µL for Melipona scutellaris). Considering these data, toxicity endpoints were used to fit species sensitive distribution curves (SSD) and determine the sensitivity ratio. The results showed that all the stingless bees tested are more sensitive to TMX than the Apis mellifera, the model organism used in ecotoxicological tests. Regarding the oral LC50, the most susceptible and most tolerant species were M. scutellaris > T. angustula > S. postica > A. mellifera. Following the same evaluated pattern, for the LD50 (considering the weight of the bees - ng a.i./g bee), we have: M. scutellaris > S. postica > T. angustula > A. mellifera, and without the weight considered (ng a.i./bee): T. angustula > M. scutellaris > S. postica > A. mellifera. The different sensitivities among stingless bee species highlight the importance of inserting more than one surrogate species with a variety of sizes in research and protocol development. Additionally, the research suggests the need to investigate patterns regarding the influence of body mass on pesticide sensitivity among stingless bee species.

Effects of chronic exposure to sublethal doses of neonicotinoids in the social wasp Polybia paulista: Survival, mobility, and histopathology.

Several studies have investigated the consequences of exposure to neonicotinoids in honeybees. Given the lack of studies concerning the consequences of exposure of social wasps to neonicotinoids, as well as the ecological importance of these insects, the objective of this study was to test the hypothesis that chronic exposure to sublethal concentrations of thiamethoxam decreases survival and mobility by causing damage to the brain and midgut of the social wasp Polybia paulista. The wasps were exposed to different concentrations of thiamethoxam, in order to obtain the mean lethal concentration (LC50), which was used as a reference for calculation of two sublethal concentrations (LC50/100 and LC50/10) employed in subsequent experiments. To calculate survival, groups of exposed (EW) and unexposed (UW) wasps were monitored until death, allowing calculation of the average lethal time. The EW and UW groups were evaluated after 12, 24, 48, and 72 h of exposure, considering their mobility and histopathological parameters of the midgut and brain. A lesion index based on semiquantitative analyses was used for comparison of histopathological damage. The results demonstrated that exposure to the LC50/10 led to a significantly shorter survival time of the P. paulista workers, compared to unexposed wasps. In addition, both sublethal concentrations decreased mobility and caused damage to the intestine (loss of brush border, presence of spherocrystals, loss of cytoplasmic material, and pyknosis) and the brain (loss of cell contact and pyknosis), regardless of the exposure time. The findings showed that, like bees, social wasps are nontarget insects susceptible to the detrimental consequences of neonicotinoid use, with exposure leading to impaired survival, locomotion, and physiology.

Neonicotinoid effects on tropical bees: Imidacloprid impairs innate appetitive responsiveness, learning and memory in the stingless bee Melipona quadrifasciata.

Together with other anthropogenic factors, pesticides play a major role in pollinator decline worldwide. Most studies on their influence on pollinators have focused on honey bees given the suitability of this insect for controlled behavioral testing and raising. Yet, studies on pesticide impact should also contemplate tropical species, which contribute a major part of biodiversity and which have remained so far neglected. Here we focused on the stingless bee Melipona quadrifasciata and asked if the widely used neonicotinoid imidacloprid disrupts its learning and memory capabilities. We fed stingless bees with 0.1, 0.5 or 1 ng of imidacloprid, tested their innate appetitive responsiveness and trained them to associate odors and sucrose reward using the olfactory conditioning of the proboscis extension response. The same experiments were performed on Africanized honey bees. One hour after intoxication, both species decreased their innate responsiveness to sucrose but the effect was more accentuated in stingless bees. In both species, learning and memory were affected in a dose-dependent manner. These results indicate that pesticides have dramatic consequences on tropical bee species and claim for rational policies regulating their use in the tropics.

Lethal and Sublethal Dose of Thiamethoxam and Its Effects on the Behavior of a Non-target Social Wasp.

In the last few years, with the increase in agricultural productivity, there has also been an increase in the use of insecticides to combat insects considered pests. However, these chemical compounds end up affecting nontarget insects that also interact with the crops. Studies have shown that social bees are among the insects that are suffering most from the effects of these compounds, resulting in negative ecological and economic impacts, considering that these insects provide pollination services in ecosystems. At the same time, social wasps also interact with plants, including cultivated ones, and perform ecological services similar to those of social bees, so it can be hypothesized that insecticides are also affecting social wasp colonies. Therefore, the purpose of this study was to evaluate contamination and sublethal effects of neonicotinoids on the mobility of the social wasp Protopolybia exigua (Saussure). In the first step, oral exposure experiments were performed to determine lethal and sublethal concentrations. In a second step, the wasps were exposed to sublethal concentrations, in order to evaluate the effects on their mobility. The results demonstrated that this species is more susceptible to exposure to neonicotinoids, compared to several bee species that have so far been studied, but lower than others. Exposure to sublethal concentrations can significantly reduce wasp mobility, which can have short-term consequences both for worker wasps and for the maintenance of their colonies.

Oral toxicity of fipronil insecticide against the stingless bee Melipona scutellaris (Latreille, 1811).

For a better evaluation of the model using Apis mellifera in toxicology studies with insecticides, the oral acute toxicity of the insecticide fipronil against the stingless bee Melipona scutellaris was determined. The results showed that fipronil was highly toxic to M. scutellaris, with a calculated LC(50) (48 h) value of 0.011 ng a.i./µL of sucrose solution and an estimated oral LD(50) (48 h) of 0.6 ng a.i./bee. Our results showed that M. scutellaris bee is more sensitive to fipronil than the model specie A. mellifera.