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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

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

Made available in DSpace on 2022-04-29T08:35:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 Greener materials have been used in the construction of electrochemical devices, becoming one of the main strands of this scientific development area. Thin films based on natural and renewable sources, such as propolis, allow the development of biocomposites. Hence, we have proposed the use of a thin film based on propolis for the stable and homogeneous immobilization of carbon nanotubes (CNTs), which increased the electrical conductivity of the base electrode (glassy carbon electrode) by almost 500 thousand times. The CNTs/propolis biofilm proportion was optimized by multivariate analysis, providing a decrease in the number of experiments, the demand for reagents, and analysis time. The biocomposite thin film was used for the spontaneous preconcentration and anchoring of metal ions, and the electrochemical synthesis of redox mediators and catalysts. This qualifies the proposed strategies for the development of eco-friendly electrochemical devices. Department of Nature Sciences Mathematics and Education Federal University of São Carlos Institute of Chemistry University of Campinas Center of Social Insects Studies São Paulo State University Institute of Biosciences Center of Social Insects Studies São Paulo State University Institute of Biosciences

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 2021-06-25T11:10:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Biotechnology and Biological Sciences Research Council Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Maximizing crop yields relies on the use of agrochemicals to control insect pests. One of the most widely used classes of insecticides are neonicotinoids that interfere with signalling of the neurotransmitter acetylcholine, but these can also disrupt crop-pollination services provided by bees. Here, we analysed whether chronic low dose long-term exposure to the neonicotinoid thiamethoxam alters gene expression and alternative splicing in brains of Africanized honey bees, Apis mellifera, as adaptation to altered neuronal signalling. We find differentially regulated genes that show concentration-dependent responses to thiamethoxam, but no changes in alternative splicing. Most differentially expressed genes have no annotated function but encode short Open Reading Frames, a characteristic feature of anti-microbial peptides. As this suggested that immune responses may be compromised by thiamethoxam exposure, we tested the impact of thiamethoxam on bee immunity by injecting bacteria. We show that intrinsically sub-lethal thiamethoxam exposure makes bees more vulnerable to normally non-pathogenic bacteria. Our findings imply a synergistic mechanism for the observed bee population declines that concern agriculturists, conservation ecologists and the public. Institute of Biosciences São Paulo State University (Unesp) School of Biosciences College of Life and Environmental Sciences University of Birmingham, Edgbaston School of Biosciences University of Nottingham, Sutton Bonington School of Veterinary Medicine and Science University of Nottingham, Sutton Bonington Institute of Biosciences São Paulo State University (Unesp)

Made available in DSpace on 2021-06-25T10:44:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 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. Centro de Estudos de Insetos Sociais - Ceis Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP-SP) Departamento de Ciências da Natureza Matemática e Educação Universidade Federal de São Carlos (UFSCar-SP) Centro de Estudos de Insetos Sociais - Ceis Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP-SP)

Made available in DSpace on 2021-06-25T15:03:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-28 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Hepatocellular carcinoma is one of the most common types of cancer in the world with high mortality rate and new therapies that control of fatty acid metabolism may limit the proliferation of cancer cells. In the last two decades, the non-coding RNAs have been considered as promising molecular tools to treat diseases, because they are able to modulate gene expression and the metabolic routes; however, deep investigation of their mechanistic behavior in pathologies must be performed. Thus, our aim was to evaluate the modulatory effect of the miR-1914-5p in controlling lipid metabolism in HepG2, a widely used human hepatocarcinoma cell line. The molecular and cellular analyses demonstrated that the functional inhibition of the investigated microRNA completely changed the cellular metabolism and behavior, compared to control groups. The in vitro inhibition of the miR-1914-5p increased the energy expenditure pointed in different analyses, decreasing cell doubling time and migration rate verified in wound healing and in the classical transwell chambers invasion assays, which makes the miR-1914-5p a candidate for further translational and preclinical studies to validate its function in controlling metastasis in liver cancer or even treat those diseases. Univ Estadual Paulista, Dept Biol Geral & Aplicada, Inst Biociencias, Lab Biol Mol, Campus Rio Claro, BR-13506900 Rio Claro, SP, Brazil Univ Estadual Paulista, Ctr Estudos Insetos Sociais, Inst Biociencia, Rio Claro, SP, Brazil Univ Estadual Paulista, Dept Biol Geral & Aplicada, Inst Biociencias, Lab Biol Mol, Campus Rio Claro, BR-13506900 Rio Claro, SP, Brazil Univ Estadual Paulista, Ctr Estudos Insetos Sociais, Inst Biociencia, Rio Claro, SP, Brazil FAPESP: FAPESP-2013/21186-5 FAPESP: 2018/05286-3

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 2020-12-12T02:19:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-01 ASCRS Research Foundation Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The honeybee has economic importance both for the commercial value of bee products and for its role in the pollination of agricultural crops. Despite the fact that the fungicides are widely used in agriculture, studies comparing the effects of this group of pesticides on bees are still scarce. There are many gaps preventing the understanding of bees’ responses to exposure to fungicides, including the influence of the age of the exposed workers. However, this study aimed to compare the effects of residual concentrations of pyraclostrobin on young and old bees of Africanized Apis mellifera. The parameters analyzed were the survival rates, as well as the histopathological and histochemical changes in midgut of orally exposed workers to different sublethal concentrations of this strobilurin fungicide: 0.125 ng a.i./μL (C1), 0.025 ng a.i./μL (C2) e 0.005 ng a.i./μL (C3). The results showed a significant decrease in the longevity only for old bees exposed to the three concentrations of pyraclostrobin. After the five-day exposure period, the fungicide induced sublethal effects in the midgut only from the old bees. These effects were the increase both in cytoplasmic vacuolization of digestive cells and morphological changes in the nests of regenerative cells, which reflected in the higher lesion index of organ for groups C1 and C2. Additionally, there was a reduction in total protein staining in the intestinal epithelium in C1 and C2. At the same exposure period, the midgut of young bees presented only a reduction in the staining of neutral polysaccharides in the group C1. Concluding, old workers are more sensitive to the fungicide than young workers. This study showed different responses according to worker age, which can affect the maintenance of colony health. Future studies should take into account the age of the workers to better understand the effects of fungicides on 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) 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 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

Made available in DSpace on 2020-12-12T01:55:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-07 The proboscis extension response (PER) reflex may be used to condition the pairing of an odor with sucrose, which is applied to the antennae, in experiments to induce learning, where the odor represents a conditioned stimulus, while sucrose represents an unconditioned stimulus. A series of studies have been conducted on honeybees, relating learning and memory acquisition/retrieval using the PER as a strategy for accessing their ability to exhibit an unconditioned stimulus; however, the major metabolic processes involved in the PER are not well known. Thus, the aim of this investigation is profiling the metabolome of the honeybee brain involved in the PER. In this study, a semiquantitative approach of matrix-assisted laser desorption ionization (MALDI) mass spectral imaging (MSI) was used to profile the most abundant metabolites of the honeybee brain that support the PER. It was reported that execution of the PER requires the metabolic transformations of arginine, ornithine, and lysine as substrates for the production of putrescine, cadaverine, spermine, spermidine, 1,3-diaminopropane, and γ-aminobutyric acid (GABA). Considering the global metabolome of the brain of honeybee workers, the PER requires the consumption of large amounts of cadaverine and 1,3-diaminopropane, in parallel with the biosynthesis of high amounts of spermine, spermidine, and ornithine. To exhibit the PER, the brain of honeybee workers processes the conversion of l-arginine and l-lysine through the polyamine pathway, with different regional metabolomic profiles at the individual neuropil level. The outcomes of this study using this metabolic route as a reference are indicating that the antennal lobes and the calices (medial and lateral) were the most active brain regions for supporting the PER. Department of Biology Center of the Study of Social Insects Institute of Biosciences University of São Paulo State (UNESP) Department of Biology Center of the Study of Social Insects Institute of Biosciences University of São Paulo State (UNESP)

Made available in DSpace on 2020-12-12T02:11:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Laboratório Nacional de Biociências 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 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. Centro de Estudos de Insetos Sociais-CEIS Instituto de Biociências-Programa de Pós Graduação em Biologia Celular e Molecular Universidade Estadual Paulista 'Júlio de Mesquita Filho' (UNESP-SP) Grupo de Genética e Genômica da Conservação Instituto de Biologia Programa de Pós Graduação em Genética e Biologia Molecular Universidade Estadual de Campinas (UNICAMP-SP) Departamento de Ciências da Natureza Matemática e Educação Centro de Ciências Agrárias Universidade Federal de São Carlos (UFSCar-SP) Centro de Estudos de Insetos Sociais-CEIS Instituto de Biociências-Programa de Pós Graduação em Biologia Celular e Molecular Universidade Estadual Paulista 'Júlio de Mesquita Filho' (UNESP-SP) FAPESP: 17/21097-3 FAPESP: 2016/15743-7 CNPq: 400540/2018-5 CAPES: 88887.318480/2019-00 CAPES: 88887.464706/2019-00

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

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

Made available in DSpace on 2019-10-06T16:19:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Among the factors that contribute to the reduction of honeybee populations are the pesticides. These chemical compounds reach the hive through forager bees, and once there, they can be ingested by the larvae. We evaluated the effects of repeated larval exposure to neonicotinoid insecticide, both in isolation and in combination with strobilurin fungicide, at environmentally relevant doses. The total consumption of the contaminated diet was 23.63 ng fungicide/larvae (pyraclostrobin) and 0.2364 ng insecticide/larvae (clothianidin). The effects on post-embryonic development were evaluated over time. Additionally, we assessed the survival pattern of worker bees after emergence, and the pesticides’ effects on the behavior of newly emerged workers and young workers. Young bees that were exposed to the fungicide and those subjected to co-exposure to both pesticides during larval phase showed behavioral changes. The insecticide, both in isolation and in combination with fungicide reduced the bees’ longevity; this effect of larval exposure to pesticides was stronger in bees that were exposed only to the insecticide. Although the larvae did not have sensitivity to exposure to pesticides, they showed later effects after emergence, which may compromise the dynamics of the colony, contributing to the reduction of the populations of bees in agroecosystems. Universidade Federal de São Carlos (UFSCar) Campus Sorocaba Programa de Pós-Graduação em Biotecnologia e Monitoramento Ambiental CCTS. Rodovia João Leme dos Santos km 110 Itinga Universidade Federal de São Carlos (UFSCar) Campus Sorocaba Departamento de Biologia CCHB. Rodovia João Leme dos Santos km 110 Itinga Universidade Estadual Paulista (UNESP) – “Júlio de Mesquita Filho” Campus Rio Claro Programa de Pós-Graduação em Biologia Celular e Molecular. Av. 24 A 1515 Jardim Bela Vista Department of Entomology and Acarology Luiz de Queiroz College of Agriculture (ESALQ) University of São Paulo (USP), Av. Pádua Dias 11 Department of Agricultural Statistics and Experimentation Luiz de Queiroz College of Agriculture (ESALQ) University of São Paulo (USP), Av. Pádua Dias 11 Universidade Estadual Paulista (UNESP) – “Júlio de Mesquita Filho” Campus Rio Claro Programa de Pós-Graduação em Biologia Celular e Molecular. Av. 24 A 1515 Jardim Bela Vista