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Metabolic associated fatty liver disease (MAFLD) is considered an indicator of metabolic syndrome, which affects millions of people around the world and no effective treatment is currently available. MAFLD involves a wide spectrum of liver damage, that initiates from steatosis (fatty live) and may progress to more complex pathophysiology. Then, details in lipid metabolism controlling should be explored aiming to control the fatty liver. In this context, the miR-1914-5p can be considered a potential biotechnology tool to control lipid metabolism in hepatic cells. This miRNA finds potential mRNA binding sequences in more than 100 molecules correlated with energy production and lipid metabolism pointed in bioinformatic platforms. The present study addressed the miR-1914-5p effects in hepatic HepG2/LX-2 co-cultured cells in a in vitro steatotic environment stablished by the addition of 400 μM of a mixture of oleic and palmitic acids. The analyses demonstrated that the inhibition of the miRNA reduced energetic metabolites such as total lipids, triglycerides, cholesterol and even glucose. In addition, the miR-inhibitor-transfected cells did not present any deleterious effect in cellular environment by controlling reactive oxygen species production (ROS), mitochondrial membrane potential (ΔΨm) and even the pro-inflammatory environment. Moreover, the functional effect of the investigated miR, suggested its close connection to the modulation of Sirt-1-PGC1-α pathway, a master switch metabolic route that controlls cellular energetic metabolism. Our assays also suggested a synergistic effect of this miR-1914-5p in cell metabolism, which should be considered as a strong candidate to control steatotic environment in future clinical trials.

Abstract 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.

Melipona scutellaris is a Brazilian stingless bee that is important for pollinating wild flora and agriculture crops. Fungicides have been widely used in agriculture, and floral residues can affect forager bees. The goal of our study was to evaluate the effects of sublethal concentrations of pyraclostrobin on the midgut ultrastructure of M. scutellaris forager workers. The bees were collected from three non-parental colonies and kept under laboratory conditions. The bees were orally exposed continuously for five days to pyraclostrobin in syrup at concentrations of 0.125 ng a.i./µL (FG1) and 0.005 ng a.i./µL (FG2). The control bees (CTL) were fed a no-fungicide sucrose solution, and the acetone solvent control bees (CAC) received a sucrose solution containing acetone. At the end of the exposure, the midguts were sampled, fixed in Karnovsky solution, and routinely processed for transmission electron microscopy. Ultrastructural analysis demonstrated that both the fungicide concentrations altered the midgut, such as cytoplasmic vacuolization (more intense in FG1), the presence of an atypical nuclear morphology, and slightly dilated mitochondrial cristae in the bees from the FG1 and FG2 groups (both more intense in FG1). Additionally, there was an alteration in the ultrastructure of the spherocrystals (FG1), which could be the result of cellular metabolism impairment and the excretion of toxic metabolites in the digestive cells as a response to fungicide exposure. The results indicate that ingested pyraclostrobin induced cytotoxic effects in the midgut of native stingless bees. These cellular ultrastructural responses of the midgut are a prelude to a reduced survival rate, as observed in previous studies.

Abstract 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.

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.

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.

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 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.

Brazil has the highest biodiversity of native stingless bees in the world. However, Brazilian regulations are based on protocols standardized by the Organization for Economic Cooperation and Development (OECD), which uses Apis mellifera as a model organism. The safety of the use of an exotic species as a substitute for a native species is a problem that concerns members of the academy and the government agencies responsible for studies of this nature in the neotropical regions where there are occurrences of stingless bee species. Regarding the exposure of larvae to pesticides, several indicators suggest that the same rearing method for A. mellifera cannot be applied to stingless bees, mainly because of their different feeding systems. Thus, it is necessary to establish an in vitro rearing method for native social bees. We developed a larval rearing method for the stingless bee species Melipona scutellaris and evaluated parameters such as the defecation rate, pupation, emergence, mortality and morphometry of the newly emerged workers. The control was represented by the morphometry of individuals that emerged from natural combs (in vivo). In addition, we determined the average lethal concentration (LC50) of the insecticide dimethoate, the standard active ingredient used for the validation of toxicity tests. Procedures conducted prior to the in vitro bioassays allowed us to obtain the actual dimensions of the rearing cells for making acrylic plates for use in establishing how much each larva consumes during its development, that is, determining how much larval food should be placed in every artificial cell. Tests performed with M. scutellaris indicated an average of 80.2% emergence of individuals relative to the larvae, 92.61% relative to the pupae and a mean of 7.42% larval mortality. The mean of the intertegular distance, head width and wing asymmetry parameters were not significantly different between individuals from the in vitro and in vivo rearing methods. The LC50 value determined was 27.48 ng dimethoate / μL diet. The method described for M. scutellaris showed development rates above OECD standards, which requires at least 75% emergence, and produced newly emerged workers with similar dimensions to those produced under natural conditions; thus these results enable their use as a rearing protocol for this species (or genus) and, consequently, their use in toxicity tests. The results produced with M. scutellaris are the first steps for a proposed toxicity test protocol for stingless bee larvae that can be standardized and included as a protocol in the OECD.

[This corrects the article DOI: 10.1371/journal.pone.0213109.].

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

Resumo: Nas últimas décadas, o declínio de populações de abelhas silvestres e o colapso de colônias de Apis mellifera têm preocupado pesquisadores e apicultores. O objetivo deste artigo de revisão foi compilar alguns dos estudos mais relevantes relacionados às possíveis causas desses problemas - como nutrição, manejo, patógenos, parasitas e efeitos de agrotóxicos -, além de apresentar um breve histórico da síndrome "colony collapse disorder" (CCD) nos Estados Unidos e na Europa. Abordaram-se com mais detalhes os estudos desenvolvidos no Brasil, principalmente sobre agrotóxicos utilizados na agricultura e patógenos e parasitas que acometem as colônias de A. mellifera africanizada. Casos de enfraquecimento, declínio e colapso têm sido registrados, principalmente nos estados de São Paulo e Santa Catarina, que somam grandes perdas. Resultados das ocorrências analisadas indicaram que as mortalidades em massa registradas no Brasil não estavam associadas a patógenos ou a parasitas. Somente dois casos com características semelhantes às descritas para CCD foram registrados, porém sem causas definidas. Para que se conheçam as causas de enfraquecimentos e perdas de colônias de abelhas, é urgente a implementação de programas oficiais de levantamento sistemático da sanidade apícola, associados a pesquisas com foco nas avaliações dos possíveis impactos da fragmentação de habitats e das práticas agrícolas sobre as comunidades de abelhas.

ABSTRACT: The objective of this research was to evaluate the honey production per hive and the egg laying rates of queens produced in 2007, 2008 and 2010. Thirty colonies initiated with a queen per colony at each climatic season were used during the three years. The years, started on January (summer), April (autumn), July (winter) and October (spring) and ended 12 months later, at the same periods related to each season of the later years. Honey supply were weighed before and after centrifugation to evaluate the quantity of the stored honey. Colonies with queens introduced during autumn and winter in the three years produced 57.2±6.0kg and 60.7±7.5kg of honey, respectively. In the first year of production activity, after the introduction of queens in the initial colonies, values were significantly higher than those obtained in colonies with queens introduced in the summer (39.3±7.6kg) and spring (41.8±3.7kg). Egg laying rates of queens were higher in spring (98.2±3.9%) and summer (88.4±7%), indicating greater food flow (flowerings) in these seasons compared to the averages in autumn (30.3±8.1%) and winter (24.5±7.2%). Produce and introduce queens of Africanized Apis mellifera in colonies initiated during autumn and winter was found to be economically feasible. Honey production of colonies initiated in these periods were higher and they had greater population stability in times of scarcity of flowerings.

Due to the current practice of intensive pesticide use in Brazil on crops with flowers that are attractive to bees, biological information about Brazilian native bees is required in order for public authorities that are responsible for environmental safety to use them for calculations of risk assessments. Thus, the present study aimed to obtain biological data on stingless bees: Melipona scutellaris, Scaptotrigiona postica and Tetragonisca angustula. The food consumed by larvae and by adults and the mass of forager workers were obtained. The results provide essential inputs for the risk assessment of stingless bee exposure to pesticides., combined with information about the concentrations of these substances in crops with flowers that are attractive to bees, may be used in risk calculations.

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

Muitas substâncias de origem vegetal podem ser tóxicas ou apresentar potencial inseticida. Com o objetivo de diminuir a problemática da poluição ambiental alguns estudos vêm tentando substituir os inseticidas artificiais pelos inseticidas botânicos. Ricinus communis (Euphorbiaceae) apresenta uma grande variedade de substâncias sendo a ricinina o principal componente tóxico. Considerando que as abelhas são insetos benéficos por atuarem como agentes polinizadores das plantas, este estudo teve por objetivo avaliar o efeito tóxico da ricinina para as operárias e larvas de Apis mellifera (Linnaeus, 1758) (Hymenoptera, Apidae) e Scaptotrigona postica (Latreille, 1907) (Hymenoptera, Meliponini). Para isso, foram realizados testes de ingestão em operárias confinadas recebendo ricinina incorporada à dieta e testes de aplicação tópica com a substância solubilizada em metanol e aplicada no pronoto das abelhas com auxílio de uma microseringa. Para as larvas foram realizados testes de ingestão e calculada sua taxa de mortalidade. Os resultados mostram atividade tóxica significativa (p < 0.0001) da ricinina nas abelhas adultas das duas espécies para a concentração 0,1% nos testes de ingestão e 0,2% nos testes de aplicação tópica. Outrossim, uma elevada porcentagem de larvas foi afetada negativamente pela ricinina logo nos primeiros dias de vida para todas as concentrações testadas (0,25%, 0,5% e 1%).Many substances of vegetal origin can be toxic or present an insecticidal potential. With the aim of decreasing the environment pollution problem, a few studies are trying to substitute synthetic insecticides with botanical ones. Ricinus communis (Euphorbiaceae) presents a great variety of substances, being the ricinine the main toxic component. Considering that bees are useful as pollinator agents of plants, this study evaluates toxicity potential of ricinine on workers and larvae of Apis mellifera (Linnaeus, 1758) (Hymenoptera, Apidae) and Scaptotrigona postica (Latreille, 1907) (Hymenoptera, Meliponini). In order to determine ricinine toxicity, ingestion tests were carried out with isolated workers bees that received ricinine on its diet. Furthermore, for topic tests, solutions of ricinine in methanol were applied on pronotum of worker bees with an "Agla" brand micrometer syringe outfit. For larvae of bees, ingestion tests were used and mortality rates were calculated. According to the results, it was detected a significant toxic activity (p < 0.0001) of ricinine on the workers of the two bee species at concentrations of 0.1% in the ingestion tests and 0.2% in the topic tests. For all concentrations (0.25%, 0.5% and 1%), a high percentage of larvae bees was negatively affected by ricinine on the first days of life.

The bee species Melipona scutellaris (L.) is native to Brazil, stingless and belongs to the Apidae family. In Brazil, stingless bees are responsible for 40 % to 90% of tree species pollination, depending on the considered ecosystem. However, their survival has been threatened since the country has been standing out as the biggest pesticides consumer in the world. Many of the pesticides used are considered toxic to bees, including imidacloprid (all existing studies were conducted with the Apis mellifera (L.) species). Although the bees are not the target of these substances, they are highly vulnerable to contamination. Thereby, the objective of this study was to establish the mean lethal dose (LD50) and the mean lethal concentration (LC50) of imidacloprid in the M. scutellaris fodder. In order to carry out this experiment, bees were collected and the test was performed according to OECD's protocol (1998a, 1998b), developed for A. mellifera. For the determination of LD50 and LC50, data was analyzed through the Probit method, using the BioStat software. The topical LD50 established in this study was 2.41 ng/bee for 24 hours and 1.29 ng/bee for 48 hours. The oral LC50 was 2.01 ng i.a./μL for 24 hours and 0.81 ng i.a./μL for 48 hours. This data showed that native bees of M. scutellaris species are more sensitive to this pesticide than A. mellifera. Thus, it is important to establish management methods which take this higher susceptibility into account to protect native species.

The lengths of the mouthparts of bees, the glossa, paraglossa, stipes, galea, labial palpus, maxillary palpus, cardo, lorum, mentum and prementum, were studied in Caucasian and Africanized bees and in their F1 descendants. Only the lengths of the paraglossa, stipe, galea, mentum, prementum and maxillary palpus differed significantly between these two bee types. These six variables were studied in the F1 descendants of two types of crosses, i.e., Caucasian queens x Africanized males (cross 1) and Africanized queens x Caucasian males (cross 2). Multidimensional analyses were also performed and the generalized Mahalanobis distances (D2) between the F1 descendants and the parental lines were determined. There was an apparent dominance of Africanized bees in both unidimensional and multidimensional analyses. Correlation analysis showed that bees with longer glossae collected more food (sugar syrup) and flew more slowly from the colony to the food source.

Uma análise sobre a produção de cria de mel e a variação do ganho de peso em colméias de abelhas africanizadas foi realizada em Limeira e Rio Claro (SP, Brasil) respectivamente. Os dados mostraram que houve uma redução na área de cria na época do florescimento da laranja, provavelmente relacionado com o estoque de mel. Como para a variação de peso, pode-se dizer que houve um ganho de peso das colméias no final de Maio e também no final de Novembro.

Uma análise sobre a produção de cria de mel e a variação do ganho de peso em colméias de abelhas africanizadas foi realizada em Limeira e Rio Claro (SP, Brasil) respectivamente. Os dados mostraram que houve uma redução na área de cria na época do florescimento da laranja, provavelmente relacionado com o estoque de mel. Como para a variação de peso, pode-se dizer que houve um ganho de peso das colméias no final de Maio e também no final de Novembro.

Victims of massive bee attacks become extremely ill, presenting symptoms ranging from dizziness and headache to acute renal failure and multiple organ failure that can lead to death. Previous attempts to develop specific antivenom to treat these victims have been unsuccessful. We herein report a F(ab)(´)(2)-based antivenom raised in horse as a potential new treatment for victims of multiple bee stings. The final product contains high specific IgG titers and is effective in neutralizing toxic effects, such as hemolysis, cytotoxicity and myotoxicity. The assessment of neutralization was revised and hemolysis, the primary toxic effect of these stings, was fully neutralized in vivo for the first time.