Toxicity and larvicidal activity on Aedes aegypti of citronella essential oil submitted to enzymatic esterification / Toxicidade e atividade larvicida sobre Aedes aegypti de óleo essencial de citronella submetido à esterificação enzimática

Abstract The essential oil of citronella (Cymbopogon winterianus) has several biological activities, among them the insect repellent action. Some studies showed that cinnamic acid esters can be applied as natural pesticides, insecticides and fungicides. In this context, the objective of the present work was to evaluate the production of esters from citronella essential oil with cinnamic acid via enzymatic esterification. Besides, the essential oil toxicity before and after esterification against Artemia salina and larvicidal action on Aedes aegypti was investigated. Esters were produced using cinnamic acid as the acylating agent and citronella essential oil (3:1) in heptane and 15 wt% NS 88011 enzyme as biocatalysts, at 70 °C and 150 rpm. Conversion rates of citronellyl and geranyl cinnamates were 58.7 and 69.0% for NS 88011, respectively. For the toxicity to Artemia salina LC50 results of 5.29 μg mL-1 were obtained for the essential oil and 4.36 μg mL-1 for the esterified oils obtained with NS 88011. In the insecticidal activity against Aedes aegypti larvae, was obtained LC50 of 111.84 μg mL-1 for the essential oil of citronella and 86.30 μg mL-1 for the esterified oils obtained with the enzyme NS 88011, indicating high toxicity of the esters. The results demonstrated that the evaluated samples present potential of application as bioinsecticide.

Resumo O óleo essencial de citronela (Cymbopogon winterianus) possui diversas atividades biológicas, entre elas a ação repelente a insetos. Alguns estudos mostraram que os ésteres do ácido cinâmico podem ser aplicados como pesticidas naturais, inseticidas e fungicidas. Nesse contexto, o objetivo do presente trabalho foi avaliar a produção de ésteres a partir do óleo essencial de citronela com ácido cinâmico via esterificação enzimática. Além disso, foi investigada a toxicidade do óleo essencial antes e após a esterificação contra Artemia salina e a ação larvicida sobre Aedes aegypti. Os ésteres foram produzidos utilizando ácido cinâmico como agente acilante e óleo essencial de citronela (3: 1) em heptano e 15% em peso da enzima NS 88011 como biocatalisadores, a 70 ° C e 150 rpm. As taxas de conversão de cinamatos de citronelil e geranil foram 58,7 e 69,0% para NS 88011, respectivamente. Para a toxicidade sobre Artemia salina foram obtidos CL50 de 5,29 μg mL-1 para o óleo essencial e 4,36 μg mL-1 para os óleos esterificados com NS 88011. Na atividade inseticida contra larvas de Aedes aegypti, obteve-se CL50 de 111,84 μg mL-1 para o óleo essencial de citronela e 86,30 μg mL-1 para os óleos esterificados com a enzima NS 88011, indicando alta toxicidade dos ésteres. Os resultados demonstraram que as amostras avaliadas apresentam potencial de aplicação como bioinseticida.

Toxicity and larvicidal activity on Aedes aegypti of citronella essential oil submitted to enzymatic esterification / Toxicidade e atividade larvicida sobre Aedes aegypti de óleo essencial de citronella submetido à esterificação enzimática

The essential oil of citronella (Cymbopogon winterianus) has several biological activities, among them the insect repellent action. Some studies showed that cinnamic acid esters can be applied as natural pesticides, insecticides and fungicides. In this context, the objective of the present work was to evaluate the production of esters from citronella essential oil with cinnamic acid via enzymatic esterification. Besides, the essential oil toxicity before and after esterification against Artemia salina and larvicidal action on Aedes aegypti was investigated. Esters were produced using cinnamic acid as the acylating agent and citronella essential oil (3:1) in heptane and 15 wt% NS 88011 enzyme as biocatalysts, at 70 °C and 150 rpm. Conversion rates of citronellyl and geranyl cinnamates were 58.7 and 69.0% for NS 88011, respectively. For the toxicity to Artemia salina LC50 results of 5.29 μg mL-¹ were obtained for the essential oil and 4.36 μg mL-¹ for the esterified oils obtained with NS 88011. In the insecticidal activity against Aedes aegypti larvae, was obtained LC50 of 111.84 μg mL-¹ for the essential oil of citronella and 86.30 μg mL-¹ for the esterified oils obtained with the enzyme NS 88011, indicating high toxicity of the esters. The results demonstrated that the evaluated samples present potential of application as bioinsecticide.

O óleo essencial de citronela (Cymbopogon winterianus) possui diversas atividades biológicas, entre elas a ação repelente a insetos. Alguns estudos mostraram que os ésteres do ácido cinâmico podem ser aplicados como pesticidas naturais, inseticidas e fungicidas. Nesse contexto, o objetivo do presente trabalho foi avaliar a produção de ésteres a partir do óleo essencial de citronela com ácido cinâmico via esterificação enzimática. Além disso, foi investigada a toxicidade do óleo essencial antes e após a esterificação contra Artemia salina e a ação larvicida sobre Aedes aegypti. Os ésteres foram produzidos utilizando ácido cinâmico como agente acilante e óleo essencial de citronela (3: 1) em heptano e 15% em peso da enzima NS 88011 como biocatalisadores, a 70 ° C e 150 rpm. As taxas de conversão de cinamatos de citronelil e geranil foram 58,7 e 69,0% para NS 88011, respectivamente. Para a toxicidade sobre Artemia salina foram obtidos CL50 de 5,29 μg mL-¹ para o óleo essencial e 4,36 μg mL-¹ para os óleos esterificados com NS 88011. Na atividade inseticida contra larvas de Aedes aegypti, obteve-se CL50 de 111,84 μg mL-¹ para o óleo essencial de citronela e 86,30 μg mL-¹ para os óleos esterificados com a enzima NS 88011, indicando alta toxicidade dos ésteres. Os resultados demonstraram que as amostras avaliadas apresentam potencial de aplicação como bioinseticida.

Toxicity and larvicidal activity on Aedes aegypti of citronella essential oil submitted to enzymatic esterification

Abstract The essential oil of citronella (Cymbopogon winterianus) has several biological activities, among them the insect repellent action. Some studies showed that cinnamic acid esters can be applied as natural pesticides, insecticides and fungicides. In this context, the objective of the present work was to evaluate the production of esters from citronella essential oil with cinnamic acid via enzymatic esterification. Besides, the essential oil toxicity before and after esterification against Artemia salina and larvicidal action on Aedes aegypti was investigated. Esters were produced using cinnamic acid as the acylating agent and citronella essential oil (3:1) in heptane and 15 wt% NS 88011 enzyme as biocatalysts, at 70 °C and 150 rpm. Conversion rates of citronellyl and geranyl cinnamates were 58.7 and 69.0% for NS 88011, respectively. For the toxicity to Artemia salina LC50 results of 5.29 g mL-1 were obtained for the essential oil and 4.36 g mL-1 for the esterified oils obtained with NS 88011. In the insecticidal activity against Aedes aegypti larvae, was obtained LC50 of 111.84 g mL-1 for the essential oil of citronella and 86.30 g mL-1 for the esterified oils obtained with the enzyme NS 88011, indicating high toxicity of the esters. The results demonstrated that the evaluated samples present potential of application as bioinsecticide.

Resumo O óleo essencial de citronela (Cymbopogon winterianus) possui diversas atividades biológicas, entre elas a ação repelente a insetos. Alguns estudos mostraram que os ésteres do ácido cinâmico podem ser aplicados como pesticidas naturais, inseticidas e fungicidas. Nesse contexto, o objetivo do presente trabalho foi avaliar a produção de ésteres a partir do óleo essencial de citronela com ácido cinâmico via esterificação enzimática. Além disso, foi investigada a toxicidade do óleo essencial antes e após a esterificação contra Artemia salina e a ação larvicida sobre Aedes aegypti. Os ésteres foram produzidos utilizando ácido cinâmico como agente acilante e óleo essencial de citronela (3: 1) em heptano e 15% em peso da enzima NS 88011 como biocatalisadores, a 70 ° C e 150 rpm. As taxas de conversão de cinamatos de citronelil e geranil foram 58,7 e 69,0% para NS 88011, respectivamente. Para a toxicidade sobre Artemia salina foram obtidos CL50 de 5,29 g mL-1 para o óleo essencial e 4,36 g mL-1 para os óleos esterificados com NS 88011. Na atividade inseticida contra larvas de Aedes aegypti, obteve-se CL50 de 111,84 g mL-1 para o óleo essencial de citronela e 86,30 g mL-1 para os óleos esterificados com a enzima NS 88011, indicando alta toxicidade dos ésteres. Os resultados demonstraram que as amostras avaliadas apresentam potencial de aplicação como bioinseticida.

Multiple metals and agricultural use affects oxidative stress biomarkers in freshwater Aegla crabs / Presença de múltiplos metais e agricultura afetam biomarcadores de estresse oxidativo em caranguejos de água doce (Aegla)

Metals and agrochemicals are among the main aquatic contaminants, being able to trigger oxidative stress in exposed organisms. The objective of this work was to evaluate the correlation between the level of oxidative stress biomarkers in Aegla crabs (Crustacea, Anomura) with (i) the set of metals present in the streams sediment and (ii) with land uses of three hydrographic basins. The study was carried out in streams (≤ 2nd order) of hydrographic basins in southern Brazil (Basins of Rio Suzana, Rio Ligeirinho-Leãozinho and Rio Dourado). In these streams were quantified the land uses and Cu, Cr, Cd, Fe, Mn and Zn concentrations in the sediment. The enzymes Catalase (CAT) and Glutathione Reductase (GR), as well as the level of membrane lipid peroxidation (TBARS), were analyzed in adult females. The PCA analysis showed that the distribution of metals was different between the basins. Cd, Cr and Fe were correlated positively with CAT and negatively with TBARS and GR. The Dourado basin had the lowest concentrations of these three metals and the highest levels of TBARS. However, in Dourado basin there is predominance of agriculture land use, and TBARS was positively correlated with agricultural land use. Besides in Dourado basin, GR activity was higher than in the others basins, indicating a compensatory response in relation to CAT inhibition. The basins of Suzana and Ligeirinho-Leãozinho rivers had lower TBARS values, which may be due to the induction of CAT in response to metals accumulated in sediment. In summary, this work indicates that in the basins with a higher concentration of toxic metals there is an adaptive response of CAT induction, which reduces TBARS in Aegla. On the other hand, in the basin with lower metallic contamination, TBARS occurrence was primarily influenced by agricultural land use.

Os metais e agroquímicos estão entre os principais contaminantes aquáticos, podendo desencadear estresse oxidativo em organismos expostos. O objetivo deste trabalho foi avaliar uma possível correlação entre o nível de biomarcadores de estresse oxidativo em Aegla (Crustacea, Anomura) com (i) o conjunto de metais presentes no sedimento e (ii) com os usos da terra, em três bacias hidrográficas distintas. O estudo foi realizado em riachos (≤ 2ª ordem) de bacias hidrográficas do Sul do Brasil (Bacias do Rio Suzana, do Rio Ligeirinho-Leãozinho e do Rio Dourado), as quais foram caracterizadas em função do percentual de usos da terra e do nível de Cu, Cr, Cd, Fe, Mn e Zn no sedimento. As enzimas Catalase (CAT) e Glutationa Redutase (GR), bem como o nível de peroxidação lipídica das membranas (TBARS), foram analisadas em fêmeas adultas. Uma análise de PCA mostrou que a distribuição de metais foi distinta entre as bacias. Cd, Cr e Fe no sedimento correlacionaram positivamente com a CAT e negativamente com TBARS e GR. Entretanto, a bacia do Dourado apresentou os menores níveis destes três metais e os maiores níveis de TBARS, o que pode ser justificado pelo predomínio da agricultura nesta bacia, já que o TBARS correlacionou positivamente com o percentual de uso agrícola. Nesta bacia, a atividade da GR foi mais alta do que nas outras, indicando uma resposta compensatória em relação a inibição da CAT. As bacias do rio Suzana e rio Ligeirinho-Leãozinho apresentaram valores menores de TBARS, o que pode decorrer da indução da CAT em função dos metais acumulados no sedimento. Em síntese, este trabalho indica que nas bacias com maior concentração de metais tóxicos ocorre uma resposta adaptativa de indução da CAT, o que reduz os níveis de TBARS em Aegla. Por outro lado, na bacia com menor contaminação metálica os níveis de TBARS foram primariamente influenciados pelo uso agrícola.

Multiple metals and agricultural use affects oxidative stress biomarkers in freshwater Aegla crabs / Presença de múltiplos metais e agricultura afetam biomarcadores de estresse oxidativo em caranguejos de água doce (Aegla)

Metals and agrochemicals are among the main aquatic contaminants, being able to trigger oxidative stress in exposed organisms. The objective of this work was to evaluate the correlation between the level of oxidative stress biomarkers in Aegla crabs (Crustacea, Anomura) with (i) the set of metals present in the streams sediment and (ii) with land uses of three hydrographic basins. The study was carried out in streams (≤ 2nd order) of hydrographic basins in southern Brazil (Basins of Rio Suzana, Rio Ligeirinho-Leãozinho and Rio Dourado). In these streams were quantified the land uses and Cu, Cr, Cd, Fe, Mn and Zn concentrations in the sediment. The enzymes Catalase (CAT) and Glutathione Reductase (GR), as well as the level of membrane lipid peroxidation (TBARS), were analyzed in adult females. The PCA analysis showed that the distribution of metals was different between the basins. Cd, Cr and Fe were correlated positively with CAT and negatively with TBARS and GR. The Dourado basin had the lowest concentrations of these three metals and the highest levels of TBARS. However, in Dourado basin there is predominance of agriculture land use, and TBARS was positively correlated with agricultural land use. Besides in Dourado basin, GR activity was higher than in the others basins, indicating a compensatory response in relation to CAT inhibition. The basins of Suzana and Ligeirinho-Leãozinho rivers had lower TBARS values, which may be due to the induction of CAT in response to metals accumulated in sediment. In summary, this work indicates that in the basins with a higher concentration of toxic metals there is an adaptive response of CAT induction, which reduces TBARS in Aegla. On the other hand, in the basin with lower metallic contamination, TBARS occurrence was primarily influenced by agricultural land use.

Os metais e agroquímicos estão entre os principais contaminantes aquáticos, podendo desencadear estresse oxidativo em organismos expostos. O objetivo deste trabalho foi avaliar uma possível correlação entre o nível de biomarcadores de estresse oxidativo em Aegla (Crustacea, Anomura) com (i) o conjunto de metais presentes no sedimento e (ii) com os usos da terra, em três bacias hidrográficas distintas. O estudo foi realizado em riachos(≤ 2ª ordem) de bacias hidrográficas do Sul do Brasil (Bacias do Rio Suzana, do Rio Ligeirinho-Leãozinho e do Rio Dourado), as quais foram caracterizadas em função do percentual de usos da terra e do nível de Cu, Cr, Cd, Fe, Mn e Zn no sedimento. As enzimas Catalase (CAT) e Glutationa Redutase (GR), bem como o nível de peroxidação lipídicadas membranas (TBARS), foram analisadas em fêmeas adultas. Uma análise de PCA mostrou que a distribuição de metais foi distinta entre as bacias. Cd, Cr e Fe no sedimento correlacionaram positivamente com a CAT e negativamente com TBARS e GR. Entretanto, a bacia do Dourado apresentou os menores níveis destes três metais e os maiores níveis de TBARS, o que pode ser justificado pelo predomínio da agricultura nesta bacia, já que o TBARS correlacionou positivamente com o percentual de uso agrícola. Nesta bacia, a atividade da GR foi mais alta do que nas outras, indicando uma resposta compensatória em relação a inibição da CAT. As bacias do rio Suzana e rio Ligeirinho-Leãozinho apresentaram valores menores de TBARS, o que pode decorrer da indução da CAT em função dos metais acumulados no sedimento. Em síntese, este trabalho indica que nas bacias com maior concentração de metais tóxicos ocorre uma resposta adaptativa de indução da CAT, o que reduz os níveis de TBARS em Aegla. Por outro lado, na bacia com menor contaminação metálica os níveis de TBARS foram primariamente influenciados pelo uso agrícola.

Multiple metals and agricultural use affects oxidative stress biomarkers in freshwater Aegla crabs

Abstract Metals and agrochemicals are among the main aquatic contaminants, being able to trigger oxidative stress in exposed organisms. The objective of this work was to evaluate the correlation between the level of oxidative stress biomarkers in Aegla crabs (Crustacea, Anomura) with (i) the set of metals present in the streams sediment and (ii) with land uses of three hydrographic basins. The study was carried out in streams ( 2nd order) of hydrographic basins in southern Brazil (Basins of Rio Suzana, Rio Ligeirinho-Leãozinho and Rio Dourado). In these streams were quantified the land uses and Cu, Cr, Cd, Fe, Mn and Zn concentrations in the sediment. The enzymes Catalase (CAT) and Glutathione Reductase (GR), as well as the level of membrane lipid peroxidation (TBARS), were analyzed in adult females. The PCA analysis showed that the distribution of metals was different between the basins. Cd, Cr and Fe were correlated positively with CAT and negatively with TBARS and GR. The Dourado basin had the lowest concentrations of these three metals and the highest levels of TBARS. However, in Dourado basin there is predominance of agriculture land use, and TBARS was positively correlated with agricultural land use. Besides in Dourado basin, GR activity was higher than in the others basins, indicating a compensatory response in relation to CAT inhibition. The basins of Suzana and Ligeirinho-Leãozinho rivers had lower TBARS values, which may be due to the induction of CAT in response to metals accumulated in sediment. In summary, this work indicates that in the basins with a higher concentration of toxic metals there is an adaptive response of CAT induction, which reduces TBARS in Aegla. On the other hand, in the basin with lower metallic contamination, TBARS occurrence was primarily influenced by agricultural land use.

Resumo Os metais e agroquímicos estão entre os principais contaminantes aquáticos, podendo desencadear estresse oxidativo em organismos expostos. O objetivo deste trabalho foi avaliar uma possível correlação entre o nível de biomarcadores de estresse oxidativo em Aegla (Crustacea, Anomura) com (i) o conjunto de metais presentes no sedimento e (ii) com os usos da terra, em três bacias hidrográficas distintas. O estudo foi realizado em riachos ( 2ª ordem) de bacias hidrográficas do Sul do Brasil (Bacias do Rio Suzana, do Rio Ligeirinho-Leãozinho e do Rio Dourado), as quais foram caracterizadas em função do percentual de usos da terra e do nível de Cu, Cr, Cd, Fe, Mn e Zn no sedimento. As enzimas Catalase (CAT) e Glutationa Redutase (GR), bem como o nível de peroxidação lipídica das membranas (TBARS), foram analisadas em fêmeas adultas. Uma análise de PCA mostrou que a distribuição de metais foi distinta entre as bacias. Cd, Cr e Fe no sedimento correlacionaram positivamente com a CAT e negativamente com TBARS e GR. Entretanto, a bacia do Dourado apresentou os menores níveis destes três metais e os maiores níveis de TBARS, o que pode ser justificado pelo predomínio da agricultura nesta bacia, já que o TBARS correlacionou positivamente com o percentual de uso agrícola. Nesta bacia, a atividade da GR foi mais alta do que nas outras, indicando uma resposta compensatória em relação a inibição da CAT. As bacias do rio Suzana e rio Ligeirinho-Leãozinho apresentaram valores menores de TBARS, o que pode decorrer da indução da CAT em função dos metais acumulados no sedimento. Em síntese, este trabalho indica que nas bacias com maior concentração de metais tóxicos ocorre uma resposta adaptativa de indução da CAT, o que reduz os níveis de TBARS em Aegla. Por outro lado, na bacia com menor contaminação metálica os níveis de TBARS foram primariamente influenciados pelo uso agrícola.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes) / Avaliação de oito protocolos na extração de DNA genômico de Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.

Toxicity and larvicidal activity on Aedes aegypti of citronella essential oil submitted to enzymatic esterification.

The essential oil of citronella (Cymbopogon winterianus) has several biological activities, among them the insect repellent action. Some studies showed that cinnamic acid esters can be applied as natural pesticides, insecticides and fungicides. In this context, the objective of the present work was to evaluate the production of esters from citronella essential oil with cinnamic acid via enzymatic esterification. Besides, the essential oil toxicity before and after esterification against Artemia salina and larvicidal action on Aedes aegypti was investigated. Esters were produced using cinnamic acid as the acylating agent and citronella essential oil (3:1) in heptane and 15 wt% NS 88011 enzyme as biocatalysts, at 70 °C and 150 rpm. Conversion rates of citronellyl and geranyl cinnamates were 58.7 and 69.0% for NS 88011, respectively. For the toxicity to Artemia salina LC50 results of 5.29 µg mL-1 were obtained for the essential oil and 4.36 µg mL-1 for the esterified oils obtained with NS 88011. In the insecticidal activity against Aedes aegypti larvae, was obtained LC50 of 111.84 µg mL-1 for the essential oil of citronella and 86.30 µg mL-1 for the esterified oils obtained with the enzyme NS 88011, indicating high toxicity of the esters. The results demonstrated that the evaluated samples present potential of application as bioinsecticide.

Multiple metals and agricultural use affects oxidative stress biomarkers in freshwater Aegla crabs.

Metals and agrochemicals are among the main aquatic contaminants, being able to trigger oxidative stress in exposed organisms. The objective of this work was to evaluate the correlation between the level of oxidative stress biomarkers in Aegla crabs (Crustacea, Anomura) with (i) the set of metals present in the streams sediment and (ii) with land uses of three hydrographic basins. The study was carried out in streams (≤ 2nd order) of hydrographic basins in southern Brazil (Basins of Rio Suzana, Rio Ligeirinho-Leãozinho and Rio Dourado). In these streams were quantified the land uses and Cu, Cr, Cd, Fe, Mn and Zn concentrations in the sediment. The enzymes Catalase (CAT) and Glutathione Reductase (GR), as well as the level of membrane lipid peroxidation (TBARS), were analyzed in adult females. The PCA analysis showed that the distribution of metals was different between the basins. Cd, Cr and Fe were correlated positively with CAT and negatively with TBARS and GR. The Dourado basin had the lowest concentrations of these three metals and the highest levels of TBARS. However, in Dourado basin there is predominance of agriculture land use, and TBARS was positively correlated with agricultural land use. Besides in Dourado basin, GR activity was higher than in the others basins, indicating a compensatory response in relation to CAT inhibition. The basins of Suzana and Ligeirinho-Leãozinho rivers had lower TBARS values, which may be due to the induction of CAT in response to metals accumulated in sediment. In summary, this work indicates that in the basins with a higher concentration of toxic metals there is an adaptive response of CAT induction, which reduces TBARS in Aegla. On the other hand, in the basin with lower metallic contamination, TBARS occurrence was primarily influenced by agricultural land use.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes).

The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Characterization of oxidative stress biomarkers in a freshwater anomuran crab / Caracterização de biomarcadores de estresse oxidativo em caranguejos anomuros de água doce

Abstract In general, environmental responses at level of populations or communities are preceded by alterations at lower biological levels which can be efficiently detected by the analysis of biomarkers. We analyzed the oxidative biomarkers TBARS and Catalase in Aegla singularis, a freshwater crustacean highly sensitive to environmental changes. The objective was to address if are differences in these biomarkers related to the gender as well if they are influenced by seasonal or water physicochemical variables. The results showed differences in biomarkers profile related to the gender. In female crabs were not sensitive to seasonal variations throughout the study period. However, in males the biomarkers evaluated were higher in the winter as compared to remaining seasons and showed tendency of negative correlation with water temperature and pH. This study highlights that gender, seasonal variations and physicochemical variables can influence oxidative stress biomarkers in A. singularis. Female crabs probably are better suited as a model for biomarker application in environmental studies, because their insensibility to seasonal variations can facilitate the observations of responses related specifically to environmental disturbances.

Resumo Em geral, as respostas ambientais ao nível de populações ou comunidades são precedidas pelas alterações nos níveis biológicos inferiores que podem ser eficientemente detectados pela análise de biomarcadores. Neste trabalho, foram analisados os biomarcadores oxidativos TBARS e Catalase em Aegla singularis, um crustáceo de água doce altamente sensível às mudanças ambientais. O objetivo foi investigar se há diferenças nestes biomarcadores relacionados com o gênero, bem como se eles são influenciados por parâmetros sazonais ou físico-químicos. Os resultados mostraram diferenças no perfil de biomarcadores relacionados com o gênero. Caranguejos fêmeas não foram sensíveis a variações sazonais ao longo do período de estudo. Nos machos, os biomarcadores avaliadas apresentaram níveis mais altos no inverno, em comparação com as demais estações e mostraram uma tendência de correlação negativa com a temperatura e pH da água. Este estudo destaca que o sexo, variações sazonais e variáveis físico-químicas podem influenciar os biomarcadores de estresse oxidativo em A. singularis. As fêmeas de A. singularis provavelmente são mais adequadas como um modelo para aplicação destes biomarcadores em estudos ambientais, uma vez que sua insensibilidade às variações sazonais podem facilitar as observações das respostas relacionadas especificamente com perturbações ambientais.

Characterization of oxidative stress biomarkers in a freshwater anomuran crab.

In general, environmental responses at level of populations or communities are preceded by alterations at lower biological levels which can be efficiently detected by the analysis of biomarkers. We analyzed the oxidative biomarkers TBARS and Catalase in Aegla singularis, a freshwater crustacean highly sensitive to environmental changes. The objective was to address if are differences in these biomarkers related to the gender as well if they are influenced by seasonal or water physicochemical variables. The results showed differences in biomarkers profile related to the gender. In female crabs were not sensitive to seasonal variations throughout the study period. However, in males the biomarkers evaluated were higher in the winter as compared to remaining seasons and showed tendency of negative correlation with water temperature and pH. This study highlights that gender, seasonal variations and physicochemical variables can influence oxidative stress biomarkers in A. singularis. Female crabs probably are better suited as a model for biomarker application in environmental studies, because their insensibility to seasonal variations can facilitate the observations of responses related specifically to environmental disturbances.

Toxicity of clove essential oil and its ester eugenyl acetate against Artemia salina / Toxicidade do óleo essencial de cravo e seu éster acetato de eugenila contra Artemia salina

Abstract The production of compounds via enzymatic esterification has great scientific and technological interest due to the several inconveniences related to acid catalysis, mainly by these systems do not fit to the concept of “green chemistry”. Besides, natural products as clove oil present compounds with excellent biological potential. Bioactives compounds are often toxic at high doses. The evaluation of lethality in a less complex animal organism can be used to a monitoring simple and rapid, helping the identification of compounds with potential insecticide activity against larvae of insect vector of diseases. In this sense, the toxicity against Artemia salina of clove essential oil and its derivative eugenyl acetate obtained by enzymatic esterification using Novozym 435 as biocatalyst was evaluated. The conversion of eugenyl acetate synthesis was 95.6%. The results about the evaluation of toxicity against the microcrustacean Artemia salina demonstrated that both oil (LC50= 0.5993 µg.mL–1) and ester (LC50= 0.1178 µg.mL–1) presented high toxic potential, being the eugenyl acetate almost 5 times more toxic than clove essential oil. The results reported here shows the potential of employing clove oil and eugenyl acetate in insecticide formulations.

Resumo A produção de compostos via esterificação enzimática possui grande interesse científico e tecnológico devido às inúmeras inconveniências relacionadas com a catálise ácida, principalmente por estes sitemas não se adequarem ao atual termo “tecnologias limpas”. Além disso, produtos naturais como o óleo de cravo, apresentam compostos com excelentes potenciais biológicos. Compostos bioativos são quase sempre tóxicos em altas doses. A avaliação da letalidade em um organismo animal menos complexo pode ser usada para um monitoramento simples e rápido, servindo também para a identificação de compostos com potencial atividade inseticida contra larvas de insetos vetores de doenças. Neste sentido, foi determinada a toxicidade frente a Artemia salina do óleo essencial de cravo e do seu derivado acetato de eugenila obtido por esterificação enzimática com lipase Novozym 435. A conversão da reação de síntese de acetato de eugenila foi de 95,6%. Os resultados referentes à avaliação da toxicidade frente ao microcrustáceo Artemia salina demonstraram que tanto o óleo (LC50= 0,5993 µg.mL–1) quanto o éster (LC50= 0,1178 µg.mL–1) apresentam elevado potencial toxicológico, sendo que o éster apresenta aproximadamente 5 vezes mais toxicidade em relação ao óleo. Estes resultados demonstram o potencial emprego do óleo de cravo e de acetato de eugenila em formulações de inseticidas.

Toxicity of clove essential oil and its ester eugenyl acetate against Artemia salina.

The production of compounds via enzymatic esterification has great scientific and technological interest due to the several inconveniences related to acid catalysis, mainly by these systems do not fit to the concept of "green chemistry". Besides, natural products as clove oil present compounds with excellent biological potential. Bioactives compounds are often toxic at high doses. The evaluation of lethality in a less complex animal organism can be used to a monitoring simple and rapid, helping the identification of compounds with potential insecticide activity against larvae of insect vector of diseases. In this sense, the toxicity against Artemia salina of clove essential oil and its derivative eugenyl acetate obtained by enzymatic esterification using Novozym 435 as biocatalyst was evaluated. The conversion of eugenyl acetate synthesis was 95.6%. The results about the evaluation of toxicity against the microcrustacean Artemia salina demonstrated that both oil (LC50= 0.5993 µg.mL-1) and ester (LC50= 0.1178 µg.mL-1) presented high toxic potential, being the eugenyl acetate almost 5 times more toxic than clove essential oil. The results reported here shows the potential of employing clove oil and eugenyl acetate in insecticide formulations.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.

Antioxidant and genotoxic properties of Maytenus dasyclada: a comparative study in relation to Maytenus reference species.

In these work the in vitro antioxidant activity and the in vivo genotoxicity of M. dasyclada was compared to the reference species M. aquifolium and M. ilicifolia. M. dasyclada showed in vitro antioxidant activity comparable to M. aquifolium but lower than M. ilicifolia, being that a inverse Pearson correlation between DPPH IC50 values and total phenolic content was detected (-0.932). The carbonyl content of M. dasyclada and M. aquifolium extracts promoted a similar increase in protein oxidation in vivo, while M. ilicifolia no altered the carbonyl levels. The comet assay demonstrated that the three analyzed species promoted a low and similar level of genotoxicity; which is compatible with DNA damage induced by other medicinal plants and is partially recovered by a co-treatment with vitamin C. The data showed M. dasyclada as antioxidant activity in vitro, and that its genotoxic and pro-oxidant effects in vivo are comparable to the Maytenus reference species.

Insecticidal and repellent activity of the essential oil of Cinnamomum camphora var. linaloolifera Y. Fujita (Ho-Sho) and Cinnamomum camphora (L. ) J Presl. var. hosyo (Hon-Sho) on Sitophilus zeamaisMots. (Coleoptera, Curculionedae) / Atividade inseticida e repelente do óleo essencial de Cinnamomum camphora var. linalooliferaY. Fujita (Ho-Sho) e Cinnamomum camphora (L. ) J Presl. var. hosyo (Hon-Sho) sobre Sitophilus zeamais Mots. (Coleoptera, Curculionedae).

ABSTRACT: The aim of this work was to evaluate the insecticidal and repellency activity of the essential oil of Cinnamomum camphora var. linaloolifera Y. Fujita (Ho-Sho) and Cinnamomum camphora (L.) J Presl.var. hosyo (Hon-Sho), against the Sitophilus zeamais in maize grains. The essential oils were obtained by hydrodistillation and analyzed by GC-MS.The insecticidal activity was determined by the toxicity of different concentrations of essential oils during 24 hours of contact with the insects, in the absence of feed substrate. The Bioassays of repellency were conducted with lethal doses (LD50,LD25,and LD12.5) obtained from insecticidal bioassay. In order to compare the treatments the preference index (PI) was employed. The analysis of the essential oils of Cinnamomum camphora leaves indicated 68% of camphor and 9% of linalool for the variation Hon-Sho and 95% of linalool to the variation Ho-Sho. The variation Ho-Sho presented greatest insecticidal activity than the variation Hon-Sho against the Sitophilus zeamais, with LD50 of 0.35 µL/cm2, whereas in the variation Hon-Sho the ratewas 0.48 µL/cm2. However, considering only the concentrations of linalool and camphor of Ho-Sho and Hon-Sho, the lethal doses' evaluation of these compounds were similar. The values of the preference index ranged from -0.3 to -0.8 for thevariation Ho-Sho and -0.2 to -0.7 for the variation Hon-Sho. The essential oils evaluated in this work showed repellent activity against Sitophiluszeamais in vitro and in trials performed in mini-silos

RESUMO: Atividade inseticida e repelente do óleo essencial de Cinnamomum camphora var. linaloolifera Y. Fujita (Ho-Sho) e Cinnamomum camphora (L.) J Presl. var. hosyo (Hon-Sho) sobre Sitophilus zeamais Mots. (Coleoptera, Curculionedae). O objetivo deste trabalho foi avaliar a atividade inseticida e de repelência dos óleos essenciais de Cinnamomum camphora var. linaloolifera Y. Fujita (Ho-Sho) e Cinnamomum camphora (L.) J Presl. var. hosyo (Hon-Sho) contra Sitophilus zeamais em grãos de milho. Os óleos essenciais foram obtidos por hidrodestilação e analisados por CG-EM. A atividade inseticida foi determinada pela toxicidade de diferentes concentrações dos óleos essenciais durante 24 horas de contato com os insetos, na ausência de substrato alimentar. Os bioensaios de repelência foram realizados com as doses letais (DL50, DL25, e DL12,5) obtidas do bioensaio inseticida. Para comparar os tratamentos foi utilizado o índice de preferência (PI). A análise de óleos essenciais de folhas de Cinnamomum camphora indicou a presença de 68% de cânfora e 9% de linalol na var. Hon-Sho e de 95% de linalol na var. Ho-Sho. A var. Ho-Sho apresentou maior toxicidade que var. Hon-Sho contra Sitophilus zeamais, com DL50 de 0,35 µL/cm2,enquanto que na var. Hon-Sho foi de 0,48 µL/cm2. No entanto, considerando apenas as concentrações de linalol e cânfora de Ho-Sho e Hon-Sho, a avaliação das doses letais destes compostos foram semelhantes. Os valores do índice de preferência variaram de -0,3 a -0,8 para a var. Ho-Sho e -0,2 para -0,7 para a var. Hon-Sho. Os óleos essenciais avaliados neste trabalho apresentaram atividade repelente contra Sitophilus zeamais in vitro e em experimentos realizados em mini-silos

Essential oil of Cinnamodendron dinisii Schwanke for the control of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) / Óleo essencial de Cinnamodendron dinisii Schwanke para controle de Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)

ABSTRACT The use of natural compounds is a less aggressive alternative for the control of insects in stored grains, in relation to synthetic chemical agents. Plants with insecticidal properties can be used as a source of these compounds to the direct application in pest control. In this work, the essential oil of Cinnamodendron dinisii was chemically characterized and tested regarding its insecticidal and repellent effect on the control of Sitophilus zeamais in stored grains. The essential oil was obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry (GC-MS). The insecticidal potential was evaluated through the maintenance of the insects during 24 hours in contact with several doses of the oil, in the absence of feed substrate. The Bioassays of repellency were conducted with lethal doses (LD5, LD25, LD50 and LD95) obtained from insecticidal bioassay. In order to compare the treatments, the preference index (PI) was used. The essential oil of C. dinisii had insecticidal activity against S. zeamais, causing a linear and crescent mortality with LD of 0.04, 0.17, 0.34 and 0.63 µL/cm2, respectively. The repellency ranged from 55.4% to 85.2%, using the LD values previously mentioned. The DL5 was neutral regarding repellence (PI index -0,09), but from DL25 on, the PI index was between -0.1 and -1.0, indicating repellence activity.

RESUMO O uso de compostos naturais é uma alternativa menos agressiva para o controle de insetos em grãos armazenados em relação aos agentes químicos sintéticos. Plantas com propriedades inseticidas podem ser usadas como fonte destes compostos para a aplicação direta no controle de pragas. No presente trabalho, o óleo essencial de Cinnamodendron dinisii foi caracterizado quimicamente e testado em relação ao seu efeito repelente inseticida no controle de Sitophilus zeamais em grãos armazenados. O óleo essencial foi obtido por hidrodestilação e analisado por cromatografia gasosa-espectrometria de massa (GC-MS). O potencial inseticida foi avaliado pela manutenção dos insetos durante 24 horas em contato com várias doses de óleo, na ausência de substrato alimentar. Os bioensaios de repelência foram realizados com as doses letais (DL5, DL25, DL50 e DL95) obtidas do bioensaio inseticida. Para comparar os tratamentos foi utilizado o índice de preferência (PI). O óleo essencial de C. dinisii apresentou atividade inseticida sobre S. zeamais, causando mortalidade linear e crescente com DL de 0,04, 0,17, 0,34 e 0,63 µL/cm2, respectivamente. A repelência variou entre 55,4% até 85,2%, utilizando os valores de DL acima mencionados. A DL5mostrou-se neutra em relação à repelência (índice PI -0,09), mas a partir de DL25 o índice PI foi entre -0,1 e -1,0, indicando atividade de repelência.

Comparison between Oligoryzomys nigripes and O. flavescens by RAPD and genetic diversity in O. nigripes (Rodentia, Cricetidae).

The genus of Oligoryzomys includes species of small size, morphologically similar, which may impede taxonomic identification, mainly between O. flavescens (Waterhouse, 1837) and O. nigripes (Olfers, 1818). The main objective of this work was to investigate whether the RAPD markers are capable of genetically differentiating the specimens O. nigripes and O. flavescens, coming from Rio Grande do Sul (RS) and Santa Catarina (SC) states, and also to estimate the genetic variability among populations of O. nigripes, with the Uruguay River as a geographical barrier. For this purpose, samples were collected in fragments of forests situated in the North of RS, at FLONA (Floresta Nacional de Passo Fundo) and in fragments from SC, close to the Uruguay River. The karyotyping of two samples for each species was carried out and compared using the RAPD technique together with non- karyotyped individuals. Samples of O. nigripes presented 2n = 62; NA = 82, with submetacentric arms on the largest chromosomes, while samples of O. flavescens showed 2n = 64; NA = 66, with the largest chromosomes presenting acrocentric morphology, making such a result the main difference between the species. The analysis was able to detect two distinct groups, being the first one with karyotyped O. flavescens and the second with karyotyped O. nigripes. Identification afforded 211 loci, among them 181 (85.78%) polymorphic. The Jaccard similarity coefficient was in the range of 0.45 to 0.87. The UPGMA and Main Coordinate Analysis techniques demonstrated the existence of heterogeneous genetics among populations, but did not separate them completely in terms of geographical standards, and they are not influenced by the Uruguay River, which did not act as an efficient barrier.

Synthesis of eugenol esters by lipase-catalyzed reaction in solvent-free system.

Enzymatic esterification of eugenol is a matter of great scientific and technological interest due to the well-known drawbacks of the chemical-catalyzed route as well as the potential use of produced compounds as natural antimicrobials. This work reports the maximization of eugenil acetate production by esterification of eugenol and acetic anhydride in a solvent-free system using Novozym 435 as catalyst. The antimicrobial activity of eugenol and eugenil acetate was also determined. The operating conditions that maximized eugenil acetate production were 50 °C, eugenol to acetic anhydride of 1:3, 150 rpm, and 5.5 wt% of enzyme, with a conversion of 99 %. A kinetic study was performed to assess the influence of substrates molar ratio, enzyme concentration, and temperature on eugenil acetate yield. Results show that an excess of anhydride, low enzyme concentration (1 wt%), and 60 °C afforded nearly complete conversion after 6 h of reaction. The highest antimicrobial activity of eugenil acetate was observed against Acinetobacter sp. (48.66 mm) at concentration of 20 µL. Results indicate that the esterification of eugenol improved its antimicrobial properties. New experimental data on enzymatic esterification of eugenol and acetic anhydride are reported in this work, showing a promising perspective to overcome the inconvenient of the chemical-catalyzed route for obtaining antimicrobial natural compounds.