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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.

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 (LC

Managed honey bees are daily exposed in agricultural settings or wild environments to multiple stressors. Currently, fungicide residues are increasingly present in bees’ pollen and nectar and can harm colonies’ production and survival. Therefore, our study aimed to evaluate the effects of the fungicide pyraclostrobin on the fat body and pericardial cells of Africanized honey bees. The foragers were divided into three experimental treatment groups and two controls: pyraclostrobin 0.125 ng/µL (FG1), 0.025 ng/µL (FG2), 0.005 ng/µL (FG3), untreated control (CTL), and acetone control (CAC). After five days of oral exposure (ad libitum), the bees were dissected and prepared for histopathological and morphometric analysis. The FG1-treated bees showed extensive cytoarchitecture changes in the fat body and pericardial cells, inducing cell death. Bees from the FG2 group showed disarranged oenocytes, peripheral vacuolization, and pyknotic nuclei of pericardial cells, but the cytoarchitecture was not compromised as observed in FG1. Additionally, immune system cells were observed through the fat body in the FG1 group. Bees exposed to FG3 demonstrated only oenocytes vacuolization. A significant decrease in the oenocyte’s surface area for bees exposed to all pyraclostrobin concentrations was observed compared to the CTL and CAC groups. The bees from the FG1 and FG2 treatment groups presented a reduced surface area of pericardial cells compared to the controls and the FG3 group. This study highlighted the harmful effects of fungicide pyraclostrobin concentrations at the individual bee cellular level, potentially harming the colony level on continuous exposure.

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

Made available in DSpace on 2021-06-25T10:37:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The development of new materials for electrochemical sensors has been received attention due to the search for sustainability. However, the use of in natura materials from vegetable and animal origin is still little explored for this purpose. Therefore, this work aims to use beeswax as biofilm, as an environmentally friendly and sustainable platform for incorporating carbon black nanoparticles. The electrode was prepared by a simple dispersion of beeswax and carbon black on the surface of the glassy carbon electrode. The proposed modified electrode was used to determine the herbicide paraquat (PQ) in 3 different samples of Apis mellifera honey. The presence of carbon black decreases the electrical resistance when compared with the bare electrode. The beeswax-carbon black film considerably increased the sensitivity of the PQ determination measures at a concentration range between 5.0 × 10−7 and 7.5 × 10−6 mol L−1. Samples with PQ contents between 7.5 × 10−7 and 7.5 × 10−6 mol L−1 presented recovery values from 97.2 to 114%. Also, the proposed sensor showed good repeatability and reproducibility for successive measurements. This suggests the use of the proposed electrode is feasible as an alternative strategy in the monitoring of herbicides in honey samples. Department of Nature Sciences Mathematics and Education Federal University of São Carlos Institute of Chemistry University of Campinas Center of Social Insects Studies Institute of Biosciences São Paulo State University (Unesp) Center of Social Insects Studies Institute of Biosciences São Paulo State University (Unesp) FAPESP: 2013/22127-2 FAPESP: 2017/21097-3 FAPESP: 2019/00473-2 FAPESP: 2019/01844-4 CNPq: 303338/2019-9 CAPES: Finance Code 001

Made available in DSpace on 2022-04-29T08:34:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In recent years, the importance of bee's biodiversity in the Neotropical region has been evidencing the relevance of including native bees in risk assessments. Therefore, the sublethal effects of the insecticide thiamethoxam on the survival and morphological parameters of the stingless bee Melipona scutellaris were investigated in the present study. Cells from both non-target organs (Malpighian tubules and midgut) and target organs (brain) were analyzed for morphological alterations using light microscopy and transmission electron microscopy. The findings showed that when M. scutellaris foragers were exposed to a sublethal concentration of thiamethoxam (LC50/100 = 0.000543 ng a. i./μL), longevity was not reduced but brain function was affected, even with the non-target organs attempting to detoxify. The cellular damage in all the organs was mostly reflected in irregular nuclei shape and condensed chromatin, indicating cell death. The most frequent impairments in the Malpighian tubules were loss of microvilli, disorganization of the basal labyrinth, and cytoplasmic loss. These characteristics are related to an attempt by the cells to increase the excretion process, probably because of the high number of toxic molecules that reach the Malpighian tubules and need to be secreted. In general, damages that compromise the absorption of nutrients, excretion, memory, and learning processes, which are essential for the survival of M. scutellaris, were found. The present results also fill in gaps on how these bees respond to thiamethoxam exposure and will be useful in future risk assessments for the conservation of bee biodiversity. Department of General and Applied Biology Institute of Biosciences São Paulo State University (UNESP) Department of Entomology and Acarology Luiz de Queiroz College of Agriculture (ESALQ) Department of Biostatistics Institute of Biosciences e IBB São Paulo State University (UNESP) Department of General and Applied Biology Institute of Biosciences São Paulo State University (UNESP) Department of Biostatistics Institute of Biosciences e IBB São Paulo State University (UNESP) FAPESP: 2012/13370-8 FAPESP: 2013/21832-4 FAPESP: 2013/21833-0 FAPESP: 2015/20380-8 FAPESP: 2017/05953-7 FAPESP: 2017/21097-3 FAPESP: 2018/20435-5 FAPESP: 2020/03527-3

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.

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.

Made available in DSpace on 2019-10-06T16:06:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Universidade Estadual Paulista (UNESP) Centro de Estudos de Insetos Sociais (CEIS) Instituto de Biociências Campus Rio Claro Universidade Federal de São Carlos (UFSCar) Departamento de Biologia Laboratório de Ecotoxicologia e Biomarcadores em Animais (LEBA) Campus Sorocaba Universidade Federal de São Carlos (UFSCar) Departamento de Ciências da Natureza Matemática e Educação Centro de Ciências Agrárias Campus Araras Universidade Estadual Paulista (UNESP) Centro de Estudos de Insetos Sociais (CEIS) Instituto de Biociências Campus Rio Claro FAPESP: 2012/01498-0 FAPESP: 2012/13370-8 FAPESP: 2012/50197-2

Made available in DSpace on 2022-05-01T07:58:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. São Paulo State University (Unesp) Institute of Biosciences, Av. 24A, 1515. CEP: 13506-900 NUPEFEN – Núcleo de pesquisas em Educação Física Estética e Nutrição Claretiano University Center, Avenida Santo Antônio Maria Claret, 1724. CEP: 13503-257 São Paulo State University (Unesp) Institute of Biosciences, Av. 24A, 1515. CEP: 13506-900 FAPESP: 2012/13370-8 (TCR) FAPESP: 2013/21833-0 (LM) FAPESP: 2014/23197-7 (PD) FAPESP: 2017/21097-3 (OM)

Made available in DSpace on 2021-06-25T10:35:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 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. Universidade Estadual Paulista (UNESP) - “Júlio de Mesquita Filho” Instituto de Biociências (IB) Departamento de Biologia Centro de Estudos de Insetos Sociais (CEIS) Universidade Federal de São Carlos (UFSCar) Departamento de Biologia (DBio) NuPECA (Núcleo de Pesquisa em Ecotoxicologia e Conservação de Abelhas) Laboratório de Ecotoxicologia e Análise de Integridade Ambiental (LEIA) Universidade Estadual Paulista (UNESP) - “Júlio de Mesquita Filho” Instituto de Biociências (IB) Departamento de Biologia Centro de Estudos de Insetos Sociais (CEIS) FAPESP: 2016/15743-7 FAPESP: 2017/21097-3 CNPq: 400540/21097-3

Made available in DSpace on 2020-12-10T19:57:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-16 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Univ Fed Vicosa, Programa Posgrad Entomol, Vicosa, MG, Brazil EMBRAPA, Lab Genet Mol, Juiz De Fora, MG, Brazil Univ Fed Vicosa, Dept Biol Geral, Vicosa, MG, Brazil SAA SP, Lab Especializado Sanidade Apicola LASA IB APTA, Pindamonhangaba, SP, Brazil Univ Sao Paulo, Dept Biol Celular Mol & Bioagentes Patogen, Ribeirao Preto, SP, Brazil Univ Fed Sao Carlos, Dept Ciencias Nat Matemat & Educ, Araras, SP, Brazil Univ Estadual Paulista, Ctr Estudos Insetos Sociais, Dept Biol, Rio Claro, SP, Brazil Univ Estadual Paulista, Ctr Estudos Insetos Sociais, Dept Biol, Rio Claro, SP, Brazil

Background Toxicological studies evaluating the possible harmful effects of pesticides on bees are important and allow the emergence of protection and pollinator conservation strategies. This study aimed to evaluate the effects of exposure to a sublethal concentration of imidacloprid (LC50/100 : 0.014651 ng imidacloprid µL-1 diet) on the distribution of certain proteins identified in the brain of Apis mellifera worker bees using a MALDI-imaging approach. This technique enables proteomic analysis of tissues in situ by monitoring the spatiotemporal dynamics of the biochemical processes occurring at a specific time in specific brain neuropils. For this purpose, foraging bees were exposed to an 8-day diet containing a sublethal concentration of imidacloprid corresponding to the LC50/100 . Bees were collected on day 8 of exposure, and their brains analyzed using protein density maps. Results The results showed that exposure to imidacloprid led to a series of biochemical changes, including alterations in synapse regulation, apoptosis regulation and oxidative stress, which may adversely impair the physiology of these colony bees. Conclusion Worker bee contact with even tiny amounts of imidacloprid had potent effects leading to the overexpression of a series of proteins related to important cellular processes that were possibly damaged by the insecticide. © 2018 Society of Chemical Industry.

The proboscis extension reflex (PER) applied to restrained individuals is an important method to assess the sucrose responsiveness under laboratory conditions. Several authors have used the PER bioassay to assess behavioral effects of pesticides. A lot of them reported the difficult to use this method with non-Apis bees showing that this basic technique cannot be applied for all bees. The aim of this study was to evaluate the sucrose responsiveness of two brazilian stingless bees, Melipona scutellaris and Scaptotrigona postica using two different protocols: the traditional one and the new one where bees have free movements. In both cases, the bees were anesthetized (freezing) and inserted into plastic tubes with the tip cut out. After a starvation period were offered an increasing concentration of sucrose-water solution (w/v). Between the solutions, were offered water. With the traditional method, the sucrose responsiveness were observed only in M. scutellaris bees (12.5% of tested bees) in just one sucrose concentration (75%). Using the methodology with free movements, both species showed sucrose responsiveness in all concentrations (25%, 50% and 75%) tested. The number of M. scutellaris bees that had sucrose response ranged from 53.7% to 76.2 % depending on the sucrose concentration. And the number of S. postica ranged from 54% to 79%. These results showed that using the methodology with free movements the sucrose responsiveness can be assessment non-Apis bees.

Made available in DSpace on 2022-04-29T08:30:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-10 The present paper describes a new way to construct composite electrodes by using beeswax as an alternative binder and graphite powder as the conductive phase. Beeswax can be an attractive binder since it is a biodegradable and renewable material. Also, it attributes to the composite material a good rigidity after dry at room temperature. The monitoring of hydrogen peroxide (H2O2) in honey is extremely important due to its antibacterial properties, which are vital for the maintenance of the hive. The determination of H2O2 by electrochemical techniques is widely noted in the literature, mainly using redox mediators or catalysts, such as Prussian blue (PB). Therefore, the proposed composite electrode was prepared with beeswax and graphite in n-hexane, and the PB was electrochemically synthesized by cyclic voltammetry. The constructed electrode showed a low resistance of electronic transfer (60 Ω), high electroactive surface area, and high electronic transference constant (1.73×10−3 cm s−1) when compared with other composite electrodes. The presence of PB on composite electrode allowed the amperometric determination of H2O2 with good linearity (R2 = 0.997) at a concentration range of 1.0−180 µmol L−1 H2O2, showing a sensitivity of 0.184 µAL µmol−1. Consequently, it was possible to determine H2O2 accurately at three concentration levels in wild honey samples (RSD = 98.5−102%), suggesting the use of this alternative method for the quality control analysis of honey. Department of Nature Sciences Mathematics and Education Federal University of São Carlos Institute of Chemistry University of Campinas Department of Chemistry Federal University of Paraná Center of Social Insects Studies São Paulo State University Institute of Biosciences Center of Social Insects Studies São Paulo State University Institute of Biosciences

Made available in DSpace on 2022-04-29T08:36:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Among the more than twenty thousand bee species currently described, Apis mellifera stands out for its high economic relevance due to crop pollination. In recent decades, many studies have registered a decline in bee populations associating multiple factors, including pesticides. These molecules can arrive through residues present in the nectar and pollen, which can be ingested by the larvae. However, studies that contemplate the effects of fungicides on larvae and adults are rare. Thus, the objective of the study was to evaluate the effects of the exposure to pyraclostrobin in the larval phase and verify the late effects in newly emerged bees. The larvae were subjected to repeated exposure (third to the sixth day of the bioassay). The concentrations of pyraclostrobin consumption were 127.58 ng/larva (PT1), 13.16 ng/larva (PT2), and 4.24 ng/larva (PT3), both based on residues found in the field like as pollen and bee bread. The effects on larval mortality, pupation, emergence and survival rates, and median lethal time of newly emerged worker bees were evaluated. We also evaluated cell death and chitin marking of the peritrophic matrix in the intestine of larvae and newly emerged bees. No adverse effects were observed on the post-embryonic development of the larvae and survival time of the newly emerged workers, showing tolerance to the fungicide. However, the intestine epithelium of larvae (PT1 and PT2) and adults (PT1) showed immunostaining for cell death and increased intensity of chitin marking in the peritrophic matrix, indicating a late effect of the pyraclostrobin fungicide. Thus, the ingestion of the fungicide in the larval stage can impair the bee development and the health and performance of the colony. In this perspective, studies that consider the effects of fungicides in the different stages of bee development are crucial for a better interpretation of the risks of exposure. Universidade Estadual Paulista (UNESP) - “Júlio de Mesquita Filho” Instituto de Biociências (IB) Departamento de Biologia Centro de Estudos de Insetos Sociais (CEIS) Universidade Federal de São Carlos (UFSCar) Departamento de Biologia (DBio) Laboratório de Ecotoxicologia e Análise de Integridade Ambiental (LEIA) Universidade Estadual Paulista (UNESP) - “Júlio de Mesquita Filho” Instituto de Biociências (IB) Departamento de Biologia Centro de Estudos de Insetos Sociais (CEIS) FAPESP: 2016/15743-7 FAPESP: 2017/21097-3 CNPq: 400540/21097-3

Bees are recognized worldwide for their social, economic, and environmental value. In recent decades they have been seriously threatened by diseases and high levels of pesticide use. The susceptibility of bees to insecticides makes them an important terrestrial model for assessing environmental quality, and various biomarkers have been developed for such assessments. The present study aimed to evaluate the activity of the enzymes acetylcholinesterase (AChE), carboxylesterase (CaE), and glutathione-S-transferase (GST) in Africanized honeybees exposed to fipronil. The results showed that fipronil at a sublethal dose (0.01 ng/bee) modulates the activity of CaE in all isoforms analyzed (CaE-1, CaE-2, and CaE-3) in both newly emerged and aged bees, and does not affect the activity of AChE or GST. The recovery of the bees after fipronil exposure was also investigated, and these results demonstrated that even the cessation of fipronil ingestion might not lead to complete recovery of individual bees. Even at low doses, fipronil was shown to cause changes in the activity of key enzymes in bees. The possible consequences of these changes are discussed. Environ Toxicol Chem 2017;36:1652-1660. © 2016 SETAC.

Made available in DSpace on 2020-12-12T01:50:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Biotechnology and Biological Sciences Research Council Securing food supply for a growing population is a major challenge and heavily relies on the use of agrochemicals to maximize crop yield. It is increasingly recognized, that some neonicotinoid insecticides have a negative impact on non-target organisms, including important pollinators such as the European honeybee Apis mellifera. Toxicity of neonicotinoids may be enhanced through simultaneous exposure with additional pesticides, which could help explain, in part, the global decline of honeybee colonies. Here we examined whether exposure effects of the neonicotinoid thiamethoxam on bee viability are enhanced by the commonly used fungicide carbendazim and the herbicide glyphosate. We also analysed alternative splicing changes upon pesticide exposure in the honeybee. In particular, we examined transcripts of three genes: (i) the stress sensor gene X box binding protein-1 (Xbp1), (ii) the Down Syndrome Cell Adhesion Molecule (Dscam) gene and iii) the embryonic lethal/abnormal visual system (elav) gene, which are important for neuronal function. Our results showed that acute thiamethoxam exposure is not enhanced by carbendazim, nor glyphosate. Toxicity of the compounds did not trigger stress-induced, alternative splicing in the analysed mRNAs, thereby leaving dormant a cellular response pathway to these man-made environmental perturbations. Universidade Estadual Paulista (UNESP) Instituto de Biociências Centro de Estudos de Insetos Sociais School of Biosciences College of Life and Environmental Sciences University of Birmingham Research Center on Animal Cognition Center for Integrative Biology Toulouse University CNRS UPS School of Biosciences University of Nottingham MRC Centre for Molecular Bacteriology and Infection and Department of Life Sciences Imperial College London Ground Floor Flowers Building South Kensington Campus Universidade Estadual Paulista (UNESP) Instituto de Biociências Centro de Estudos de Insetos Sociais Biotechnology and Biological Sciences Research Council: BB/K006827/1

Made available in DSpace on 2020-12-12T01:48:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Universidade Estadual Paulista ‘Júlio de Mesquita Filho’(UNESP) Departamento de Biologia Centro de Estudos de Insetos Sociais (CEIS) Programa de Pós-Graduação em Biologia Celular e Molecular, Campus Rio Claro Universidade Estadual de Campinas (UNICAMP) Instituto de Biologia Grupo de Genética e Genômica da Conservação Programa de Pós-Graduação em Genética e Biologia Molecular Universidade Federal de São Carlos Centro de Ciências Agrárias Departamento de Ciências da Natureza Matemática e Educação Grupo de Abelhas e Serviços Ambientais, Campus Araras Universidade Estadual Paulista ‘Júlio de Mesquita Filho’(UNESP) Departamento de Biologia Centro de Estudos de Insetos Sociais (CEIS) Programa de Pós-Graduação em Biologia Celular e Molecular, Campus Rio Claro CAPES: 001 FAPESP: 2013/09555–5 FAPESP: 2014/12488–0 FAPESP: 2017/21097–3 CAPES: 88887.318482/2019–00

Securing food supply for a growing population is one of the current major challenges and heavily relies on the use of agrochemicals to maximize crop yield. Neonicotinoids are globally one of the most widely used insecticides. It is increasingly recognized, that neonicotinoids have a negative impact on non-target organisms, including important pollinators such as the European honey bee Apis mellifera. Toxicity of neonicotinoids may be enhanced through simultaneous exposure with additional pesticides, which could help explain, in part, the global decline of honey bee colonies. Here we examined whether exposure effects of the neonicotinoid Thiamethoxam are enhanced by the commonly used fungicide Carbendazim and the herbicide Glyphosate. For the first time, we also analysed alternative splicing changes upon pesticide exposure in the honey bee. In particular, we examined transcripts of three genes: i) the stress sensor gene X box binding protein-1 (Xbp1), ii) the Down Syndrome Cell Adhesion Molecule (Dscam) gene and iii) the embryonic lethal/abnormal visual system (elav) gene, both important genes for neuronal function. Our results indicate that neonicotinoid toxicity applied at sub-lethal doses is not enhanced by Carbendazim nor Glyphosate. Likewise, toxicity of these compounds did not impact on the complex process of spliceosomal-directed joining of exons and non-spliceosomal intron excision in the analysed mRNAs.

Made available in DSpace on 2020-12-10T19:40:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-12 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Univ Estadual Paulista, Dept Biol, Inst Biociencias, Campus Rio Claro, Rio Claro, SP, Brazil Univ Estadual Paulista, Ctr Estudos Insetos Sociais, Inst Biociencias, Rio Claro, SP, Brazil Univ Estadual Paulista, Dept Biol, Inst Biociencias, Campus Rio Claro, Rio Claro, SP, Brazil Univ Estadual Paulista, Ctr Estudos Insetos Sociais, Inst Biociencias, Rio Claro, SP, Brazil CNPq: 474060/2012-8 FAPESP: 2013/21186-5 FAPESP: 2018/05286-3

Submitted by Vivian Rosa Storti (vivian.storti@unesp.br) on 2019-08-01T13:54:12Z No. of bitstreams: 1 Chemosphere nanopesticide.pdf: 3823193 bytes, checksum: b33dd69cd645b32b0e732e6c07d466c2 (MD5) Approved for entry into archive by Luana Priscila Costa (luana@reitoria.unesp.br) on 2019-08-01T14:18:24Z (GMT) No. of bitstreams: 1 Chemosphere nanopesticide.pdf: 3823193 bytes, checksum: b33dd69cd645b32b0e732e6c07d466c2 (MD5) Made available in DSpace on 2019-08-01T14:18:24Z (GMT). No. of bitstreams: 1 Chemosphere nanopesticide.pdf: 3823193 bytes, checksum: b33dd69cd645b32b0e732e6c07d466c2 (MD5) Previous issue date: 2019 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Nanotechnology has the potential to overcome the challenges of sustainable agriculture, and nano-pesticides can control agricultural pests and increase farm productivity with little environmental impact. However, it is important to evaluate their toxicity on non-target organisms, such as honeybees (Apismellifera) that forage on crops. The aims of this study were to develop a nanopesticide that was based onsolid lipid nanoparticles (SLNs) loaded with pyrethrum extract (PYR) and evaluate its physicochemicalproperties and short-term toxicity on a non-target organism (honeybee). SLNþPYR was physico-chemically stable after 120 days. SLNþPYR had afinal diameter of 260.8±3.7 nm and a polydispersionindex of 0.15±0.02 nm, in comparison with SLN alone that had a diameter of 406.7±6.7 nm and apolydispersion index of 0.39±0.12 nm. SLNþPYR had an encapsulation efficiency of 99%. The survivalanalysis of honeybees indicated that PYR10ng presented shorter longevity than those in the control group(P 0.01). Empty nanoparticles and PYR10ng caused morphological alterations in the bees’midguts, whereas pyrethrum-loaded nanoparticles had no significant effect on digestive cells, so are consideredsafer, at least in the short term, for honeybees. These results are important in understanding the effects ofnanopesticides on beneficial insects and may decrease the environmental impacts of pesticides Preprint Universidade Estadual Paulista (UNESP) “Júlio de Mesquita Filho”, Instituto de Ciência e Tecnologia de Sorocaba, Laboratório de Nanotecnologia Ambiental Universidade Federal de São Carlos (UFSCar), Campus Sorocaba, Departamento de Biologia (CCHB), Laboratório de Fisiologia da Conservação e Laboratório de Ecotoxicologia e Biomarcadores em Animais Universidade Estadual Paulista (UNESP)“Júlio de Mesquita Filho”, Campus Rio Claro, Departamento de Biologia, Centro de Estudos de Insetos Sociais (CEIS) Faculdade de Medicina São Leopoldo Mandic, Campus Araras FAPESP: 2017/21004-5

Made available in DSpace on 2018-12-11T17:07:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-12-01 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. UNESP-São Paulo State University UNIARARAS-Hermínio Ometto Foundation Jd. Universitário UNESP/CEIS-Centre for the Study of Social Insects, Rio Claro, Bela Vista Department of Biology UFSCar-Federal University of São Carlos UNESP-São Paulo State University UNESP/CEIS-Centre for the Study of Social Insects, Rio Claro, Bela Vista

Made available in DSpace on 2018-12-11T17:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-06-01 The ethanolic extract of propolis, especially the Brazilian green type, is widely and mainly used for therapeutic purposes despite the lack of knowledge about its effects and its cellular mode of action. This type of propolis, derived from Baccharis dracunculifolia (alecrim-do-campo), has been extensively commercialized and the consumers use it to enhance health. This work aimed to assess the genotoxic/mutagenic and antigenotoxic/antimutagenic potentials of the ethanolic extracts of Brazilian green propolis and of B. dracunculifolia, on mammalian cells. It was not observed genotoxic and mutagenic effects by both extracts. After evaluate the exposure of the cells to each extract with a recognized mutagen, simultaneously, the results showed a significant reduction on DNA damage. The experiment carried out with a pre-incubation period was more effective than without incubation test, showing that the tested extracts were able to inactivate the mutagen before it could react with the DNA. Departamento de Biologia Instituto de Biociências UNESP - Univ. Estadual Paulista, Avenida 24-A, 1515, CP199 Departamento de Ciências Biológicas Centro de Ciências Humanas e Naturais UFES - Universidade Federal do Espírito Santo, Avenida Fernando Ferrari, 514 Departamento de Ciências Farmacêuticas Centro de Ciências da Saúde UFES - Universidade Federal do Espírito Santo, Avenida Marechal Campos, 1468 Departamento de Biologia Instituto de Biociências UNESP - Univ. Estadual Paulista, Avenida 24-A, 1515, CP199

The social Hymenoptera increasingly arouse the attention of researchers due to the great economic and ecological importance to the species belonging to this order. The processes of pollination performed by these insects, the biological control, the structures of nests and colonies, the social organization, the accidents arising from their stings and the pharmacological potential of venoms synthesized by them, represent some of the features that make these individuals, targets of important studies. The sting apparatus and venom represent fundamental defense mechanisms of these insects. In order to characterize the convoluted and the venom gland of these species and gain an understanding of the relationship between the components, this study aimed to analyze through morphology and histochemical techniques the venom glands of the wasps, Polistes versicolor , Agelaia palipes palipes and Polybia paulista , and also the convoluted gland - a structure found in the reservoir, establishing the defining characteristics of these structures. In addition to the morphological features described about these structures, our results showed the presence of secretion produced by the convoluted gland cells which indicates that this structure contributes to the final composition of the venom produced and stored in the reservoir.

Made available in DSpace on 2020-12-12T02:42:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Currently, Brazil has a full framework for pesticide risk assessment established for Apis mellifera, based on the North American approach. However, the use of Apis mellifera as model-organism as a surrogate for Brazilian native species of stingless bees has been questioned. Assessments on other stages of development than adult individual are essential. Our study aimed to standardize in vitro larval rearing method for the stingless bee species Scaptotrigona postica and Tetragonisca angustula, comparing the results to those obtained for M. scutellaris (previously described), for proposing the most suitable one for using in toxicological larval tests. We used the most efficient method for determining the toxicity of dimethoate on S. postica larvae. We presented the first comparative approach of responses to in vitro larval rearing methods among native bee species from Neotropical region, for use in risk assessment. Our results showed that S. postica was the most suitable native species to be proposed as model-organism. In addition, our results are also very useful for a ring test to validate the method, in accordance to OECD. São Paulo State University (UNESP) Institute of Biosciences, Social Insect Study Center 24A Avenue 1515 Technological Institute of Vale - Boaventura da Silva St, 955 Center of Agrarian Sciences Federal University of São Carlos, Anhanguera Road Km 174 São Paulo State University (UNESP) Institute of Biosciences, Social Insect Study Center 24A Avenue 1515 CAPES: 001 FAPESP: 2016/00328-4 FAPESP: 2017/21097-3 CNPq: 400540/2018-5

Toxicological studies evaluating the possible harmful effects of pesticides on bees are important and allow the emergence of protection and pollinator conservation strategies. This study aimed to evaluate the effects of exposure to a sublethal concentration of imidacloprid (LCThe results showed that exposure to imidacloprid led to a series of biochemical changes, including alterations in synapse regulation, apoptosis regulation and oxidative stress, which may adversely impair the physiology of these colony bees.Worker bee contact with even tiny amounts of imidacloprid had potent effects leading to the overexpression of a series of proteins related to important cellular processes that were possibly damaged by the insecticide. © 2018 Society of Chemical Industry.

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.

Although the importance of bees as the pollinators responsible for maintaining gene flow for many native and cultivated plants in ecosystems around the world is recognized, much of their biodiversity and behavior remains to be discovered. Stingless bees are considered key pollinators for several plant species in tropical and subtropical ecosystems and they also provide pollination services for economically important agricultural crops. Many countries are using the honey bee (Apis mellifera Linnaeus, 1758, Hymenoptera: Apidae) as a surrogate to evaluate the risk of pesticides to all species of bees. However, there is uncertainty regarding the extent to which honey bees can serve as surrogates for non-Apis bee species in the risk assessment for pesticides. This paper provides a short overview of the life history traits relevant in risk assessment of stingless bees. It summarizes what is known about stingless bee exposure to pesticides compared to that of honey bees and presents criteria for potential candidate species from Brazil for use in pesticide risk assessment in tropical environments. This paper also identifies gaps in knowledge of bee biology and pesticide exposure routes not covered by the current honey bee exposure assessment paradigm. Based on these gaps, research is needed on life history traits, estimates of nectar and pollen consumption, mud, resin, and water collection and available protocols to adequately assess toxic effects of pesticides to stingless bees. This review is part of a series of papers on the risk of exposure of non-Apis bees to pesticides.

Made available in DSpace on 2018-11-26T16:16:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Several investigations have revealed the toxic effects that neonicotinoids can have on Apis mellifera, while few studies have evaluated the impact of these insecticides can have on the larval stage of the honeybee. From the lethal concentration (LC50) of thiamethoxam for the larvae of the Africanized honeybee, we evaluated the sublethal effects of this insecticide on morphology of the brain. After determine the LC50 (14.34 ng/mu L of diet) of thiamethoxam, larvae were exposed to a sublethal concentration of thiamethoxam equivalent to 1.43 ng/mu L by acute and subchronic exposure. Morphological and immunocytochemistry analysis of the brains of the exposed bees, showed condensed cells and early cell death in the optic lobes. Additional dose-related effects were observed on larval development. Our results show that the sublethal concentrations of thiamethoxam tested are toxic to Africanized honeybees larvae and can modulate the development and consequently could affect the maintenance and survival of the colony. These results represent the first assessment of the effects of thiamethoxam in Africanized honeybee larvae and should contribute to studies on honey bee colony decline. (C) 2015 Elsevier Ltd. All rights reserved. Univ Estadual Paulista, Dept Biol, BR-13506900 Rio Claro, SP, Brazil Univ Fed Uberlandia, Inst Ciencias Agr, BR-38400 Uberlandia, MG, Brazil Univ Fed Sao Carlos, Dept Biol, Sorocaba, SP, Brazil Univ Estadual Paulista, Dept Biol, BR-13506900 Rio Claro, SP, Brazil FAPESP: 2009/06701-5 FAPESP: 2013/09419-4 FAPESP: 2012/50197-2

BACKGROUND Although ants are common insects in agricultural ecosystems, few studies have considered how xenobiotics might induce physiological and morphological alterations in these insects. This study aimed to verify the neurotoxic action of sublethal doses of fipronil on the mushroom bodies of brains from the leaf-cutting ant Atta sexdens rubropilosa through immunocytochemistry analysis for the protein synapsin. RESULTS The LD50 value was established as 1.42 ng ant−1, and the sublethal doses used were LD50/10 and LD50/100. Synapsin labelling was more evident in the brains extracted from ants exposed to the insecticide, specifically in the regions of glia in the mushroom bodies, compared with the control group. It was possible to measure the intensity of emitted fluorescence in the areas of the mushroom bodies, and a statistical test showed differences between the control group and the treatment group. CONCLUSION Thus, it is concluded that sublethal doses of the insecticide fipronil intensified synapsin immunostaining, suggesting an increased release of neurotransmitters, which may be linked to neurotoxicity and overexcitation. These sublethal doses may have two different effects: compromising the operation and maintenance of the colony and leading to the establishment of resistance in insects. © 2015 Society of Chemical Industry

Propolis is a substance produced by bees from plant sources located in the vicinity of the bee hives and it has a wide spectrum of biological properties. Brazilian propolis stands out due to its unique characteristics. In the 1990s, some Brazilian beekeepers started specializing in propolis production due to growing commercial demand for this product. In the districts where propolis production is plentiful, propolis harvests have become the main product and honey is not even harvested. Nowadays, harvesting techniques have improved and productivity increased substantially. Here, we report on characteristics of propolis and the evolution of propolis harvest in Brazil.

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.

The indiscriminate use of pesticides has become a serious environmental concern. Insecticide imidacloprid (IMI) is the second most widely used pesticides worldwide. In 2010, 1.934 tons of IMI were sold in Brazil, mostly to be used in sugarcane crops. Several studies have detected its presence in the aquatic environment constituting a risk to non-target organisms. The aquatic animals are organisms used for environmental biomonitoring worldwide. They are considered excellent genetic models to detect environmental mutagens. Among animal species, the fish Oreochromis niloticus, commonly known as Nile tilapia, has been used to evaluate DNA damage. The present study therefore evaluated the effect of IMI on the genetic material of Oreochromis niloticus (Pisces) erythrocytes exposed to different concentration (250; 125 and 62.5 μg/L) of IMI used in growing sugarcane. The effect of the IMI was measured using the comet assay and the micronucleus (MN) test, assays that detected genotoxic damage. The results in the comet assay demonstrated that the concentrations tested induced primary damage to DNA. They also proved the occurrence of MN and nuclear abnormalities at the higher concentration used in the micronuclei and other nuclear abnormalities test. The insecticide IMI induced primary DNA damage at all concentrations and damage at the chromosomal level at the highest concentration tested. The results of this study show the potential risk of IMI in a non-target organism.

Made available in DSpace on 2018-12-11T17:15:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Departamento de Biologia Centro de Estudos de Insetos Sociais UNESP – Univ. Estadual Paulista campus de Rio Claro Instituto de Biociências Departamento de Biologia Centro de Estudos de Insetos Sociais UNESP – Univ. Estadual Paulista campus de Rio Claro Instituto de Biociências FAPESP: 2012/13370-8 FAPESP: 2012/50197-2 FAPESP: 2014/14070-3

Made available in DSpace on 2018-12-11T17:32:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Under laboratory conditions, the effects of thiamethoxam were investigated in larvae, pupae and emerging honey bees after exposure at larval stages with different concentrations in the food (0.00001 ng/μL, 0.001 ng/μL and 1.44 ng/μL). Thiamethoxam reduced the survival of larvae and pupae and consequently decreased the percentage of emerging honey bees. Thiamethoxam induced important physiological disturbances. It increased acetylcholinesterase (AChE) activity at all developmental stages and increased glutathione-S-transferase (GST) and carboxylesterase para (CaEp) activities at the pupal stages. For midgut alkaline phosphatase (ALP), no activity was detected in pupae stages, and no effect was observed in larvae and emerging bees. We assume that the effects of thiamethoxam on the survival, emergence and physiology of honey bees may affect the development of the colony. These results showed that attention should be paid to the exposure to pesticides during the developmental stages of the honey bee. This study represents the first investigation of the effects of thiamethoxam on the development of A. mellifera following larval exposure. UNESP Universidade Estadual Paulista Departamento de Biologia INRA Laboratoire de Toxicologie Environnementale UR 406 Abeilles & Environnement INRA UR 546 Biostatistique et Processus Spatiaux UFLA Universidade Federal de Lavras, Lavras UFSCar Universidade Federal de São Carlos INRA Laboratoire de L'Environnement et de L'Alimentation de La Vendée, La Roche sur Yon UNESP Universidade Estadual Paulista Departamento de Biologia FAPESP: 2013/21634-8

Made available in DSpace on 2018-12-11T17:13:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The use of insecticides on crops can affect non-target insects, such as bees. In addition to the adult bees, larvae can be exposed to the insecticide through contaminated floral resources. Therefore, this study aimed to investigate the possible effects of the exposure of A. mellifera larvae to a field concentration of thiamethoxam (0.001 ng/μL thiamethoxam) on larval and pupal survival and on the percentage of adult emergence. Additionally, its cytotoxic effects on the digestive cells of midgut, Malpighian tubules cells and Kenyon cells of the brain of newly emerged A. mellifera bees were analyzed. The results showed that larval exposure to this concentration of thiamethoxam did not influence larval and pupal survival or the percentage of adult bee emergence. However, this exposure caused ultra-structural alterations in the target and non-target organs of newly emerged bees. The digestive cell of bees that were exposed to the insecticide exhibited a basal labyrinth without long and thin channels and compromised mitochondria. In Malpighian tubules cells, disorganized basal labyrinth, dilated mitochondria with a deformed shape and a loss of cristae, and disorganized microvilli were observed. The results showed that the exposed bees presented Kenyon cells with alterations in the nucleus and mitochondria. These alterations indicate possible tissue degeneration, demonstrating the cytotoxicity of thiamethoxam in the target and non-target organs of newly emerged bees. Such results suggest cellular organelle impairment that can compromise cellular function of the midgut cells, Malpighian tubules cells and Kenyon cells, and, consequently, can compromise the longevity of the bees of the whole colony. UNESP - Univ. Estadual Paulista campus de Rio Claro Instituto de Biociências Departamento de Biologia Centro de Estudos de Insetos Sociais UNESP - Univ. Estadual Paulista campus de Rio Claro Instituto de Biociências Departamento de Biologia Centro de Estudos de Insetos Sociais FAPESP: 2012/13370-8 FAPESP: 2012/50197-2 FAPESP: 2015/09691-1

Fipronil is a phenylpyrazole insecticide that is widely used in Brazilian agriculture for pest control. Although honeybees are not targets of fipronil, studies indicate that this pesticide can be harmful to honeybees. To assess the effects of fipronil in the brain of Africanized Apis mellifera workers, this study focused on the toxico-proteome profiling of the brain of newly emerged and aged honeybee workers that were exposed to a sub-lethal dose (10 pg fipronil per day. i.e. (1)/100 of LD50/bee/day during 5 days) of the insecticide. Proteomic analysis identified 25 proteins that were differentially up-regulated or down-regulated when the fipronil-exposed and non-exposed groups were compared. These proteins are potentially related to pathogen susceptibility, neuronal chemical stress, neuronal protein misfolding, and occurrence of apoptosis, ischemia, visual impairment, damaged synapse formation, brain degeneration, memory and learning impairment. The exposure of honeybees to a very low dose of fipronil, even for a short period of time (5 days), was sufficient to cause a series of important neuroproteomic changes in the brains of honeybees.

BACKGROUND Studies on stingless bees are scarce, and little is known about these insects, especially regarding the effects of contamination by neurotoxic insecticides, which can cause damage to important structures of the insect brain. This study evaluated the morphological changes in the intrinsic neurons of the protocerebral mushroom bodies (Kenyon cells) of the stingless bee Scaptotrigona postica after exposure to different doses of fipronil, using light microscopy and transmission electron microscopy. This region of the brain was selected for analysis because of its importance as a sensory integration centre. RESULTS In both oral and topical treatments, Kenyon cells presented pyknotic profiles, suggesting cell death. Statistical analysis showed significant differences among doses and exposure times. Transmission electron microscopy revealed changes in the nucleus and cellular organelles. Depending on the dose, the characteristics observed suggested apoptotosis or necrosis. CONCLUSION This study demonstrates the toxic effects of fipronil. An increase in the number of pyknotic profiles of Kenyon cells of mushroom bodies was observed even at the sublethal doses of 0.27 ng AI bee−1 and 0.24 ng AI µL−1 in the topical and oral treatments respectively. Also, differences in the number of pyknotic profiles were dose and time dependent. © 2014 Society of Chemical Industry

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 β-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.

Due to its expansion, agriculture has become increasingly dependent on the use of pesticides. However, the indiscriminate use of insecticides has had additional effects on the environment. These products have a broad spectrum of action, and therefore the insecticide affects not only the pests but also non-target insects such as bees, which are important pollinators of agricultural crops and natural environments. Among the most used pesticides, the neonicotinoids are particularly harmful. One of the neonicotinoids of specific concern is thiamethoxam, which is used on a wide variety of crops and is toxic to bees. Thus, this study aimed to analyze the effects of this insecticide in the midgut and Malpighian tubule cells of Africanized Apis mellifera. Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam equal to 1/10 of LC50 (0.0428 ng a.i./l L of diet). The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide. Microsc. Res. Tech. 77:274–281, 2014. © 2014 Wiley Periodicals, Inc.

Urban pests are known to infest health service facilities, especially in a warm, wet neotropical climate. In practice, pest control services for health service facilities are generally outsourced and in most cases are not properly monitored. To address this problem, an integrated pest management (IPM) programme was developed at a hospital in the municipality of Botucatu, state of Sao Paulo, Brazil, by the infection control committee. The method of control applied used spreadsheet pest reports to monitor the presence of vectors. These reports were forwarded to the committee and used to implement several actions of integrated pest control. In 2011, a total of 183 occurrences were recorded by the infection control committee, 5% lower than the number of incidents recorded in 2010, the first year of integrated pest control. In 2012, the total number of occurrences decreased to 159 events, demonstrating the IPM effects. Three years after the implementation of the programme, the predominant pests in the hospital were scorpions (30%), ants (19%) and spiders (16%), with Tityus serrulatus, Paratrechina longicornis and Loxosceles sp as the predominant species, respectively. The presence of scorpions in a hospital environment is not documented in the literature, although these animals are common in urban ecosystems. These results demonstrate that integrated pest control in hospitals is effective and should be conducted on a continuous basis. We suggest the use of this approach by all health services located in neotropical environments.

Made available in DSpace on 2020-12-10T19:38:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) During foraging, bees are exposed to sublethal doses of insecticides, which can cause morphological changes to various organs, such as the midgut, Malpighian tubules, and mushroon body. Thus, the purpose of this study was to establish a scoring system to evaluate these alterations based on the damage caused and its reversibility. Therefore, a grade 1 score indicates a minimal and easily reversible lesion, increased apocrine secretion, increased cell elimination into the lumen, and a larger quantity of spherocrystals; grade 2 was assigned to moderate and typically reversible injuries, such as changes in the brush border, vacuolation/loss of cytoplasmic material, presence/height of the brush border, and cell swelling; and grade 3 was assigned to serious and irreversible, loss of cell nests of regenerative cells, pyknosis, and loss of contact between Kenyon cells. In addition, frequency values were assigned since the alterations can occur at different frequencies according to the insecticide and the bees exposed; the frequency ranges from 0 to 6, with 0 representing the absence of an alteration and 6 representing a high frequency occurrence. Based on the analyses, we conclude that each change causes morphological damage, which may or may not be irreversible and could affect the health of the colony. (C) 2019 Elsevier Ltd. All rights reserved. Univ Estadual Paulista, Dept Biol, Ctr Estudos Insetos Socials, Ave 24-A,1-515, BR-13506900 Rio Claro, SP, Brazil Univ Fed Sao Carlos, Ctr Ciencias Agr, Dept Ciencias Nat Matemat & Educ, Campus Araras,Rodovia Anhanguera SP-330,Km 174, BR-13600970 Araras, SP, Brazil Univ Fed Sao Carlos, Dept Biol, Ctr Ciencias & Tecnol Sustentabilidade, Campus Sorocaba,Rodovia Joao Leme dos Santos, BR-18052780 Sorocaba, SP, Brazil Univ Estadual Paulista, Dept Biol, Ctr Estudos Insetos Socials, Ave 24-A,1-515, BR-13506900 Rio Claro, SP, Brazil CAPES: 001 FAPESP: 2014/12488-0 FAPESP: 2013/09555-5 FAPESP: 2017/21097-3

Departamento de Biologia UNESP-Univ. Estadual Paulista, Campus de Rio Claro Centro de Estudos de Insetos Sociais, Avenida 24-A, n.1515 Bela Vista, Rio Claro, Sao Paulo, 13506-900

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./lL 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 life

Made available in DSpace on 2018-12-11T17:03:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-16 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Center of the Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP) Laboratory for Proteomics and Protein Engineering Carlos Chagas Institute Center of the Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP)

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.