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.

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.

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.

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.

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.

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.

Fungivorous mites enhance the survivorship and development of stingless bees even when exposed to pesticides.

Stingless bees are the largest group of eusocial bees in the world. They play an essential role as crop pollinators and have been considered for inclusion in pesticide risk assessments (RAs). Beyond the mutualism involving stingless bee larvae and fungi, the fungivorous mite Proctotydaeus (Neotydeolus) alvearii proved to be interesting for studies of associations with stingless bees. Their presence is related to colony strength and health, showing a permanent-host-association level. Here, we tested whether the coexistence with P. (N.) alvearii affects stingless bee larvae survivorship and development, including when fed pesticide-dosed food. We chose dimethoate, the reference standard for toxicity tests, and thiamethoxam, widely used in neotropical crops and listed to be reassessed in RAs. Bees associated with the mites showed higher larval survivorship rates, even in the dosed ones, and revealed changes in the developmental time and body size. Our study represents the first approach to stingless bee responses to the coexistence of fungivorous mites inside brood cells, leading us to believe that these mites play a beneficial role in stingless bees, including when they are exposed to pesticides.

Transcriptomic analysis of Malpighian tubules from the stingless bee Melipona scutellaris reveals thiamethoxam-induced damages.

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 LC50/100 (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.

A systematic review of research conducted by pioneer groups in ecotoxicological studies with bees in Brazil: advances and perspectives.

Brazil presents the most threatened endemic or rare species among neotropical regions, with the Hymenoptera order, to which bees belong, classified as a high-risk category. In Brazil, the main cause of bee death is the indiscriminate use of pesticides. In this context, groups such as Bee Ecotoxicology and Conservation Laboratory (LECA in Portuguese) and Bees and Environmental Services (ASAs in Portuguese) have become a reference in studies evaluating the impacts of pesticides on bees since 1976. Thus, the objective of this review was to conduct a quantitative and qualitative review of the studies conducted by these groups to evaluate and compile the advances made over the years, identify potential knowledge gaps for future studies, and support the sensitivities of stingless bees when compared to the species Apis mellifera. The quantitative analyses showed that most studies were carried out in the genus Apis, under laboratory conditions. However, more recently (since 2003), studies have also focused on stingless bees and the neonicotinoid class of insecticides. The most relevant gaps identified were the lack of studies under field conditions and on bee biology. The qualitative analyses indicated that Brazilian stingless bees are more susceptible to pesticides than A. mellifera and require a much lower average dose, concentration, or lethal time to display morphological and behavioral damage or decreased lifespan. Thus, future studies should work towards establishing more representative protocols for stingless bees. Furthermore, public policies must be created for the protection and conservation of bees native to Brazil.

Optimization of in vitro culture of honeybee nervous tissue for pesticide risk assessment.

The most used pesticides have neurotoxic action on the neurotransmitter system of target and non-targeted insects, such as honeybees. However, honeybees have foremost importance worldwide, which has encouraged the development of tools to evaluate the action of specific pesticide molecules on their nervous system, providing accurate data on damage to their brain. In this sense, our study aimed to optimize in vitro honeybee nervous tissue culture to assess pesticide risks. To this end, six forager honeybee brains were dissected and transferred to different combinations of Leibovitz-15 (L-15) culture medium supplemented with Fetal Bovine Serum (FBS), Hank's Balanced Salt Solution (HBSS), and Insect Medium Supplement (IMS). Nervous tissues were collected after different incubation times (1, 6, 12, and 24 h) for morphology and Kenyon cell analyses. Our results showed that L-15 medium supplemented with HBSS and with HBSS plus FBS were the best media for culturing honey nervous tissue for 24 h, as they resulted in less tissue spacing and cell disarrangement. Therefore, they may be assessed in future ecotoxicological tests.

Is the social wasp Polybia paulista a silent victim of neonicotinoid contamination?

Neonicotinoids are among the chemicals most widely used against insects considered agricultural pests, although they may also affect nontarget species, as has been reported for social bees. Social wasps are recognized as efficient predators of larvae of other insects, including pest species, so they may have contact with insecticides, at least indirectly. However, to date, there have been no studies investigating the consequences for social wasps of the use of neonicotinoids. Therefore, the aim of this study was to obtain the topical and oral acute lethal mean doses of the neonicotinoid thiamethoxam, as well as to evaluate the effects of sublethal contamination, for the social wasp Polybia paulista. Foraging wasps were exposed orally and topically, with monitoring of their mortality after 24 and 48 h, in order to calculate the LD50 values. Other wasps were then exposed with sublethal doses, in order to evaluate the effects of contamination on their mobility. The results indicated that P. paulista workers are as sensitive to thiamethoxam contamination as several nontarget species studied previously. Exposure to a sublethal dose (LD10) negatively affected their mobility. It could be concluded that social wasp colonies may be as much affected by exposure to neonicotinoids as social bees. Further toxicological studies should be conducted, evaluating other parameters and different species, in order to understand the extent of the problems faced by these insects due to the use of neonicotinoids.

Removal of the pesticide thiamethoxam from sugarcane juice by magnetic nanomodified activated carbon.

The removal of the neonicotinoid and systemic pesticide thiamethoxam (TMX) from water and sugarcane juice by magnetic nanomodified activated carbon (AC-NP) is proposed. This adsorbent was synthesized and characterized by FTIR, XRD, and SEM, and TMX was quantified by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). The AC-NP was efficiently synthesized using a co-precipitation method and the impregnation of magnetite (NP) in the activated carbon (AC) was assessed by the crystalline planes found in the AC-NP structure shown in the XRD diffractograms. The AC-NP FTIR analysis also indicated predominant bands of Fe-O stretching of the magnetite at 610-570 cm-1. Functional groups in AC and AC-NP were identified by absorption bands at 3550 and 1603 cm-1, characteristic of O-H and C = C, respectively. The TMX adsorption kinetics in sugarcane juice was the pseudo-second-order type with r2 = 0.9999, indicating a chemical adsorption process. The experimental sorption capacity (SCexp) for both TMX (standard) and TMX-I (insecticide) by AC-NP were 13.44 and 42.44 mg/g, respectively. Seven non-linear isotherm models (Langmuir, Freundlich, Dubinin-Radushkevich, Toth, Hill, Sips, and Redlich-Peterson) were fitted to the experimental adsorption data of TMX and TMX-I by AC-NP. Considering the standard error (SE), Freundlich, Langmuir, and Sips were the most appropriate models to describe the TMX adsorption, and Hill's best adjusted to TMX-I experimental data. The chromatographic method was highly satisfactory due to its high selectivity and recovery (91-103%). The efficiency of AC-NP in the sorption of TMX was confirmed by the excellent values of SCexp and sorption kinetics.

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.

A high quality method for hemolymph collection from honeybee larvae.

The drastic decline of bees is associated with several factors, including the immune system suppression due to the increased exposure to pesticides. A widely used method to evaluate these effects on these insects' immune systems is the counting of circulating hemocytes in the hemolymph. However, the extraction of hemolymph from larvae is quite difficult, and the collected material is frequently contaminated with other tissues and gastrointestinal fluids, which complicates counting. Therefore, the present work established a high quality and easily reproducible method of extracting hemolymph from honeybee larvae (Apis mellifera), the extraction with ophthalmic scissors. Extraction methods with the following tools also were tested: 30G needle, fine-tipped forceps, hypodermic syringe, and capillaries tubes. The hemolymph was obtained via an incision on the larvae's right side for all methods, except for the extraction with ophthalmic scissors, in which the hemolymph was extracted from the head region. To assess the purity of the collected material, turbidity analyses of the samples using a turbidimeter were proposed, tested, and evaluated. The results showed that the use of ophthalmic scissors provided the clearest samples and was free from contamination. A reference range between 22,432.35 and 24,504.87 NTU (nephelometric turbidity units) was established, in which the collected samples may be considered of high quality and free from contamination.