Effects of fipronil on the stingless bee Melipona scutellaris

Dissertação de mestrado em Segurança Alimentar, apresentada à Faculdade de Farmácia da Universidade de CoimbraO Alecrim (Rosmarinus officinalis L.) é uma espécie aromática medicinal pertencente à família das Lamiaceae largamente reconhecida pela sua utilização em culinária e nas indústrias farmacêutica, alimentar e cosmética. Esta é uma planta perene adaptando-se adequadamente ao clima predominantemente Mediterrâneo de Portugal, sendo caraterizada por possuir teores elevados de óleos essenciais. Os óleos essenciais são compostos de baixo peso molecular, capazes de atravessarem a barreira hemato-encefálica, normalmente isentos de toxicidade e que apresentam diversas propriedades biológicas, como por exemplo, propriedades antioxidantes, anti-inflamatórias e antimicrobianas. Doenças causadas por microorganismos infeciosos, nomeadamente fungos e bactérias têm representado um sério risco para as populações em todo o mundo, principalmente devido ao fato de desenvolverem resistências aos antimicrobianos sintéticos. Por outro lado, a oxidação assim como a contaminação microbiana têm sido causas relevantes de deterioração dos alimentos, originando grandes perdas económicas e problemas de saúde pública. Desta forma, a procura de agentes antimicrobianos naturais tem suscitado grande interesse na comunidade médica e científica. O stresse oxidativo associado a inflamação crónica foi recentemente reconhecido como um fator chave para as alterações fisiopatológicas observadas em uma ampla gama de doenças, tais como a disfunção cerebral, cancro, doenças cardiovasculares, doenças hepáticas e o declínio do sistema imunológico. Deste modo, moléculas detentoras de propriedades anti-inflamatorias e simultaneamente antioxidantes apresentam elevado potencial terapêutico no tratamento destas patologias. Neste contexto, no presente trabalho pretende-se avaliar as potencialidades biológicas do óleo essencial de R. Officinalis oriundo de Portugal, sendo possível estudar as suas atividades antifúngicas, contra várias estirpes de leveduras e fungos filamentosos patogénicos para o homem e outros animais, incluindo a inibição do tubo germinativo em Candida albicans, um importante fator de virulência. Também a atividade antibacteriana contra estirpes de bactérias patogénicas de origem alimentar foi avaliada, tendo por base a mínima concentração inibitória (MIC) e a mínima concentração letal (MLC). O potencial anti-inflamatório do óleo essencial de R. officinalis foi também avaliado num modelo in vitro representativo da inflamação periférica, utilizando a linha celular de macrófagos (Raw 264.7) estimulada com o agonista do recetor Toll-like 4, o lipopolissacarídeo (LPS). Na presença de produtos bacterianos, como o LPS, os macrófagos produzem mediadores pro-inflamatórios, nomeadamente o óxido nítrico (NO), responsável xiv pela amplificação da resposta inflamatória. Concominatemente testou-se a citotoxidade do óleo essencial na mesma linha celular na presença de LPS, bem como a sua citotoxicidade em diversas linhas celulares humanas do epitélio alveolar (A549), queratinócitos (HaCaT), hepatócitos (Hep G2) e intestinais (Caco-2), com o objetivo de identificar concentrações do óleo essencial bioativas e isentas de toxicidade. Para o presente trabalho avaliou-se ainda a atividade antioxidante do óleo essencial pelo método de substância reativas do ácido tiobarbitúrico (TBARS), na presença e na ausência de um indutor de peroxidação lipídica (ABAP). Os ensaios da avaliação da atividade antifúngica mostraram que o Cryptococcus neoformans foi a levedura que apresentou maior sensibilidade ao óleo essencial com valores de MIC de 0,64 μL/mL. Os resultados da inibição do tubo germinativo em Candida albicans, um fator importante de patogenicidade na invasão de tecidos, demonstraram que o óleo essencial inibiu totalmente a formação do tubo germinativo na concentração de 0,64 μL/mL e na concentração de 0,08 μL/mL foi ainda observada uma elevada percentagem de inibição (63,2%) em comparação com as células não tratadas. O fato do óleo inibir a sua formação, torna-o um promissor agente antifúngico com potencial utilização na indústria farmacêutica. Entre os fungos dermatofitos testados, o Epidermophyton floccosum foi o que apresentou maior sensibilidade ao óleo essencial com um MIC de 0,64 μL/mL, sendo esta concentração não citotóxica para os queratinócitos. Este fato evidencia a potencial aplicação tópica do óleo no tratamento de infeções causadas por dermatófitos. Com base nos valores de MIC obtido, verificou-se que, a Listeria monocytogenes e a Staphylococcus aureus foram as estirpes mais sensíveis ao óleo essencial com um MIC de 2,5 μL/mL. O estudo da atividade anti-inflamatória do óleo, avaliada pela inibição da produção do NO, demonstrou que o mesmo não apresenta bioatividade quando testado em concentrações não tóxicas. O óleo essencial demonstrarou ainda ausência de toxicidade para a maioria das célulasestudadas quando utilizado em concentrações iguais ou inferiores a0,64 μL/mL. Na linha celular de intestino humano o óleo essencial não apresentou citotoxidade significativa para concentrações inferiores a 0,32 μL/mL. Relativamente à avaliação da atividade antioxidante, o óleo essencial demonstrou inibição significativa da peroxidação lipídica na ausência do indutor (ABAP). Em síntese, o presente trabalho permite concluir que o óleo essencial de R. officinalis apresenta potencial terapêutico como fungicida no tratamento tópico de dermatofitoses, quando utilizada em concentrações não superiores a 0,64 μL/mL. Adicionalmente o óleo xv essencial poderá ser utilizado como um antioxidante natural na preservação dos alimentos, sendo a sua aplicação por via oral segura para concentrações inferiores a 0,32 μL/mLThe Rosemary (Rosmarinus officinalis L.) is a medicinal aromatic plant of the Lamiaceae family widely recognized for its use in cooking and in the pharmaceutical, food and cosmetic industries. This is a adequately perennial adapted species to the mediterranean climate of Portugal, being characterized by having a high content of essential oils. Essential oils are low molecular weight compounds capable of crossing the blood-brain barrier generally free of toxicity and which have different biological properties such as antioxidant, anti-inflammatory and antimicrobial. Diseases caused by infectious microorganisms, including fungi and bacteria represents a serious risk to populations all over the world, mainly because of developing resistance to synthetic antimicrobials. Moreover, the oxidation as well as microbial contamination have been important causes of food spoilage, causing large economic loss and public health problems. Thus, the demand for natural antimicrobials has aroused great interest in the medical and scientific community. Oxidative stress associated with chronic inflammation has recently been recognized as a key factor in the pathophysiologic changes seen in a wide range of diseases such as cerebral dysfunction, cancer, cardiovascular disease, liver disease and decline of the immune system. In this case molecules possessing anti-inflammatory properties and antioxidant simultaneously have a high therapeutic potential in the treatment of these pathologies. It was intended with this work to evaluate the biological potential of essential oils derived from R. officinalis coming from Portugal and also study its antifungal activity against various strains of yeast and filamentous fungi, both pathogenic to humans and other animals, including inhibition of germ tube in Candida albicans, an important virulence factor. Also the antibacterial activity against strains of food borne pathogenic bacteria was evaluated based on the minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC). The anti-inflammatory potential of the R. officinalis essential oil was also evaluated using an in vitro model representing a peripheral inflammation, using the macrophage cell line (RAW 264.7) stimulated with receptor agonist of Toll-like 4, lipopolysaccharide (LPS). In the presence of bacterial products such as LPS, the macrophages produce pro-inflammatory mediators including nitric oxide (NO), responsible for amplification of the inflammatory response. In parallel we tested the cytotoxicity of the essential oil in the same cell line and with the presence of LPS as well as their cytotoxicity in several human cell lines alveolar epithelial (A549), keratinocytes (HaCaT), hepatocytes (HepG2) and intestinal (Caco-2 ) with xviii the objective of identifying bioactive concentrations of the essential oil and free from toxicity. Also with the present study we evaluated the antioxidant activity of the essential oil by the method of reactive substance of thiobarbituric acid (TBARS) in the presence and absence of an inducing lipid peroxidation (ABAP). The tests on the evaluation of the antifungal activity showed that the Cryptococcus neoformans was the yeast that showed higher sensitivity to essential oil with MIC values of 0.64 μL/mL. The results of the inhibition of Candida albicans germ tube in a major pathogenic factor in tissue invasion, demonstrated that the essential oil completely inhibited germ tube formation in a concentration of 0.64 μL/mL and the concentration of 0.08 μL/mL was observed even a high percentage of inhibition (63.2%) compared to untreated cells. The fact that the oil inhibit its formation, makes it a promising antifungal agent with the potential use in the pharmaceutical industry. Among the dermatophytes fungi tested, Epidermophyton floccosum was introduced more sensitive to the essential oil with a MIC of 0.64 μL/mL, which is non-cytotoxic concentration to keratinocytes. This fact highlights the potential oil topical application in the treatment of infections caused by dermatophytes. Based on MIC values obtained, it was found that, Listeria monocytogenes and Staphylococcus aureus strains were more sensitive to the essential oil with an MIC of 2.5 μL/mL. The study oil anti-inflammatory activity as assessed by inhibition of NO production, has demonstrated that it shows no bioactivity when tested at non-toxic concentrations. The essential oil also demonstrated the absence of toxicity to most cells studied when used in concentrations equal or inferior to 0.64 uL / mL. In the human intestine cell line essential oil showed no significant cytotoxicity at concentrations below 0.32 μL / mL. Regarding the evaluation of antioxidant activity, the essential oil showed significant inhibition of lipid peroxidation in the absence of inducer (ABAP). In summary, this study shows that the essential oil of R. officinalis has therapeutic potential as a fungicide in the topical treatment of dermatophytosis, when used in concentrations not exceeding the 0.64 μL / mL. Additionally, the essential oil may be used as a natural antioxidant in food preservation, and their application for safe orally at less than 0.32 μL/mL

Biological Data of Stingless Bees with Potential Application in Pesticide Risk Assessments

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

Determination of fipronil LD50 for the brazilian bee Melipona scutellaris

To better understand the sensitivity of the models represented by Apis mellifera L., 1758 in toxicology studies of insecticides to bees, the aim of this study was to determine the LD50 of fipronil by topical application on the stingless bees Melipona scutellaris Latreille, 1811. Foraging bees were collected at the nest entrance and in laboratory anesthetized with CO2 for applying 1.0 μL of fipronil solution on the pronotum. Each group of treatments was made with thirteen bees divided in three cages, while in the control treatments the bees received only acetone. During the assay, the behavior and the number of dead bees were registered. The results showed that the insecticide fipronil applied topically was harmful to M. scutellaris and for A. mellifera, where the LD50 for 48 hours was 0.41 ng a.i./bee or 4.1 ng a.i./ g of bee. Comparing the LD50 values here obtained with the stingless bee M. scutellaris and those of A. mellifera in literature, we can conclude that the native bees are more sensitive to fipronil than the allochtonous bee, suggesting that further studies should be accomplished to determine the real hazard of pesticides to natives bees. Keywords: stingless bees, pesticides, phenylpyrazole, toxicity, LD5

Acute thiamethoxam toxicity in honeybees is not enhanced by common fungicide and herbicide and lacks stress-induced changes in mRNA splicing

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