Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation

Abstract Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13) and adults (n = 12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh)

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.

Identificación de un polimorfismo del gen Est9 relacionado con resistencia a piretroides en Rhipicephalus (Boophilus) microplus / Identification of a gene polymorphism Est9 associated with resistance to pyrethroids in Rhipicephalus (Boophilus) microplus

Objetivo. Mediante procedimientos de PCR-RFLP, detectar un polimorfismo en el gen Est9 de garrapatas Rhipicephalus (Boophilus) microplus resistentes a piretroides en Ibagué, Colombia, determinando el grado de asociación entre los fenotipos y los genotipos resultantes. Materiales y métodos. El ADN de 30 teleoginas R. (Boophilus) microplus fenotípicamente susceptibles, resistentes o medianamente resistentes a piretroides en una prueba de Drummond modificada, fue amplificado por PCR con cebadores específicos para obtener un fragmento de 372 pb del gen Est9, que fue sometido a digestión con la enzima EcoRI para estudiar los RFLPs generados y poder diferenciar los respectivos genotipos. El grado de asociación entre los fenotipos y los genotipos resultantes se determinó mediante la prueba exacta de Fisher. Resultados. Luego de digerir el fragmento con la endonucleasa, se generaron dos segmentos en teleoginas con algún nivel de resistencia, mientras en las teleoginas susceptibles no hubo división del fragmento de 372 pb, demostrándose así la presencia de una mutación puntual y los genotipos homocigoto natural, homocigoto mutante y heterocigoto. Las diferencias altamente significativas (p<0.01) entre teleoginas susceptibles y aquellas con algún nivel de resistencia, mostraron una relación directa entre el genotipo y el fenotipo con un nivel de confianza de p=0.0009852. Conclusiones. Se comprobó, por primera vez en Colombia, la presencia de una mutación puntual en el gen Est9 de garrapatas R. (Boophilus) microplus resistentes a piretroides, sugiriendo la necesidad de realizar estudios para detectar alteraciones moleculares en otros genes relacionados con quimioresistencia.

Objective. Using PCR-RFLP procedures, to detect a polymorphism of the Est9 gene in Rhipicephalus (Boophilus) microplus ticks resistant to pyrethroids in Ibague, Colombia, determining the degree of association between phenotypes and resulting genotypes. Materials and methods. The DNA of 30 engorged R. (Boohilus) microplus phenotypically susceptible females, resistant or moderately resistant to pyrethroids in a modified Drummond test was amplified by PCR with specific primers. We obtained a 372 bp fragment of the Est9 gene which was digested with the EcoRI enzyme to study RFLPs generated in order to differentiate the respective genotypes. The degree of association between phenotypes and resulting genotypes was determined by Fisher’s exact test. Results. After digesting the fragment with the endonuclease, two segments were generated in engorged females with some level of resistance, while, in those susceptible, there was no fragment division. The presence of a mutation of 372 pb was demonstrated, as well as the natural homozygous genotypes, mutant homozygous and heterozygous. The highly significant differences (p<0.01) between engorged susceptible females and those with some level of resistance, revealed a direct relationship between genotype and phenotype with a confidence level of p = 0.0009852. Conclusions. For the first time in Colombia, the presence of a mutation in the Est9 gene of R. (B.) microplus tick resistant to pyrethroids was found, suggesting the need for studies to detect molecular alterations in other genes associated with chemical resistance.

Susceptibilidad de Aedes aegypti a DDT, deltametrina y lambdacialotrina en Colombia / Susceptibility of Aedes aegypti to DDT, deltamethrin, and lambda-cyhalothrin in Colombia

OBJETIVOS: Evaluar el estado de susceptibilidad a insecticidas piretroides deltametrina y lambdacialotrina y al organoclorado DDT, e identificar los mecanismos bioquímicos asociados con resistencia en 13 poblaciones naturales de Aedes aegypti recolectadas en localidades de Colombia donde el dengue es un grave problema de salud pública. MÉTODOS: Se recolectaron y criaron en condiciones controladas formas inmaduras de diferentes criaderos naturales del vector para cada localidad. Con la generación F2 se realizaron bioensayos utilizando las metodologías OMS 1981 (papeles impregnados) y CDC 1998 (botellas impregnadas). En las poblaciones con mortalidades compatibles con disminución de la susceptibilidad, se midieron los niveles de esterasas no específicas (ENE), oxidasas de función mixta (OFM) y acetilcolinesterasa modificada (ACEM) mediante pruebas colorimétricas. RESULTADOS: Todas las poblaciones del mosquito evaluadas evidenciaron resistencia al organoclorado DDT. En cuanto a los piretroides, se encontró resistencia generalizada a lambdacialotrina pero no a deltametrina. Los mecanismos bioquímicos de resistencia evaluados permitieron encontrar 7 de 11 poblaciones con ENE elevadas y una población con OFM incrementadas. CONCLUSIONES: Se descarta la resistencia cruzada de tipo fisiológico entre el DDT y lambdacialotrina en las poblaciones de A. aegypti evaluadas. La resistencia fisiológica a lambdacialotrina parece asociarse con el incremento de las ENE. El comportamiento diferencial en los niveles de susceptibilidad y los valores enzimáticos entre poblaciones se asociaron con la variabilidad genética y presión de selección química a nivel local.

OBJECTIVES: To assess the susceptibility status of 13 natural populations of Aedes aegypti (collected from sites in Colombia where dengue is a serious public health problem) to the pyrethroids, deltamethrin and lambda-cyhalothrin, and to the organochlorine, DDT, and to identify any biochemical mechanisms associated with resistance. METHODS: Immature forms of the vector were collected from natural breeding spots at each site and then raised under controlled conditions. Using the F2 generation, bioassays were performed using the World Health Organization's 1981 methodology (impregnated paper) and United States Centers for Disease Control and Prevention's 1998 methodology (impregnated bottles). In populations where mortality rates were consistent with decreased susceptibility, levels of nonspecific esterases (NSE), mixed-function oxidases (MFO), and acetylcholinesterase (AChE) were measured using colorimetric tests. RESULTS: All of the mosquito populations that were tested showed resistance to the organochlorine DDT. In the case of the pyrethroids, widespread resistance to lambda-cyhalothrin was found, but not to deltamethrin. Assessing the biochemical resistance mechanisms showed that 7 of the 11 populations had elevated NSE, and one population, increased MFO. CONCLUSIONS: Physiological cross-resistance between DDT and lambda-cyhalothrin in the A. aegypti populations tested was dismissed. Physiological resistance to lambda-cyhalothrin appears to be associated with increased NSE. The differences in susceptibility levels and enzyme values among the populations were associated with genetic variations and chemicals in use locally.

Acetilcolinesterase, butirilcolinesterase, carboxilesterase e a resistência de peixes neotropicais aos pesticidas organofosforados / Acetylcholinesterase, butyrylcholinesterase, carboxylesterase and the resistance of neotropical fishes to organophosphorus pesticides

Todos os agrupamentos humanos que se organizam para o trabalho usam rios, lagos ou lagoas como depósitos para a decomposição de matéria indesejável. A contaminação do meio aquático por herbicidas e agrotóxicos derivados de práticas agrícolas se tornou, faz tempo, um problema de importância mundial. Precisamos de informações detalhadas sobre a bioquímica da intoxicação de peixes nativos para avaliarmos quais os efeitos de agrotóxicos sobre os processos bioquímicos que mantêm o ciclo de vida dos peixes em águas do Brasil. Organofosfatos, que são agrotóxicos de uso disseminado, podem interargir com as B-esterases butirilcolinesterase (EC 3.1.1.8) e carboxilesterase (EC 3.1.1.1) presentes no fígado e no plasma. Tanto a butirilcolinesterase (BChE) como a carboxilesterase (CarbE), se presentes em concentrações relativamente elevadas, agem como limpadores estequiométricos ("scavengers" moleculares) por ligarem o átomo de fósforo do grupo P=O com a hidroxila de uma serina presente nos seus sítios ativos. Em nossos resultados observamos que curimbatá possui a CarbE plasmática (IC50 74 nM) mais sensível ao organofosfato metilparaoxon quando comparado ao pacu (IC50 691 nM). Isolamos CarbE dos plasmas de curimbatá e pacu. Piavassu não possui uma atividade expressiva de CarbE no sangue, por isso não a isolamos. O tipo e a distribuição das esterases nos tecidos são particulares da espécie. Curimbatá tem alta atividade de CarbE no fígado (237,8 U.g-1) e no sangue (29,85 U.mL-1), pacu é dotado de alta atividade de BChE (134,0 U.g-1) e CarbE (149,6 U.g-1) no fígado, mas o piavussu conta apenas com a BChE do sangue (17,87 U.g-1). Este arsenal enzimático foi suficiente para proteger as AChE de cérebro, músculo e coração das três espécies e evitar a sua intoxicação leve por 0,2 mg metilparation/L. A abordagem cinético-bioquímica para conhecer a inibição das esterases presentes nos tecidos de diferentes espécies de peixes por agrotóxicos é uma ferramenta útil...

Every human group who organizes to work together uses rivers, lakes or ponds as places in which undesirable substances are deposited for decomposition. Contamination of the aquatic environment with herbicides and pesticides derived from agrucultural practices has become a problem of global importance since a long ago. We need detailed information on the biochemistry of the poisoning of native fish to assess the effects of pesticides on the biochemical processes that maintain fishes' life cycle in waters of Brazil. Organophosphates, which are widely used pesticides, can interact with the B-esterases butyrylcholinesterase (EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) in plasma and liver. Butyrylcholinesterase (BChE) and carboxylesterase (CarbE) in relatively high concentrations act as stoichiometric scavengers by linking the phosphorus atom of the P=O group with the serine's hydroxyl they have in their active site. Our results show that the CarbE of curimbata plasma (IC50 74 nm) is more sensitive to the organophosphate metilparaoxon than CarbE of pacu plasma (IC50 691 nm). We isolated CarbE from curimbata and pacu's plasma. Piavussu does not have an expressive activity of CarbE in plasma, so we did not isolate it. The type and distribution of esterases in tissues are peculiar to a species. Curimbata has high CarbE activity in the liver (237.8 U.g-1) and blood (29.85 U.mL-1), pacu is equipped with high activities of BChE (134.0 U.g-1) and CarbE (149.6 U.g-1) in the liver and piavussu relies only on BChE of blood (17.87 U.g-1). This enzymatic arsenal was sufficient to protect AChE from brain, muscle and heart of the three species and protect them against mild intoxication by methilparathion (0.2 mg/L). The biochemical kinetic approach that allows understanding of the inhibition of the esterases in tissues of different fish species is a good tool capable of anticipating the harmful consequences of these drugs.

Bio-synthesis and hydrolysis of ethyl phenylacetate and ethyl 2-phenylpropionate in organic solvent by lyophilized mycelia

To select the best biocatalysts for ethanol acylations with phenylacetic and 2-phenylpropionic acids, lyophilized mycelia of Aspergillus oryzae CBS 10207, A. oryzae MIM, Rhizopus oryzae CBS 11207, R. oryzae CBS 39134, R. oryzae CBS 26028 and R. oryzae CBS 32847 were tested in this study. The carboxylesterase activities of A. oryzae MIM and R. oryzae 11207, which revealed to be the best biocatalysts, were investigated either in 0.1 M phosphate buffer or in n-heptane to catalyze the hydrolysis or the synthesis of ethyl esters of these acids, respectively. A. oryzae proved more effective than R. oryzae, probably due to more favorable microenvironment conditions and thermodynamic scenario. The results in terms of product formation and substrate consumption versus time were used to estimate the maximum conversion yields, the equilibrium constants and the times needed to reach half maximum conversion, thus providing sufficient information about these equilibria.

Micélios liofilizados de Aspergillus oryzae CBS 10207, A. oryzae MIM, Rhizopus oryzae CBS 11207, R. oryzae CBS 39134, R. oryzae CBS 26028 e R. oryzae CBS 32847 foram testados neste estudo com vista à seleção do melhor biocatalisador para efetuar a acilação de etanol com ácidos fenilacético e 2-fenilpropiônico. As atividades carboxilesterásicas de A. oryzae MIM e R. oryzae 11207, que resultaram ser os melhores biocatalisadores, foram investigadas tanto em tampão fosfato 0,1 M como em n-heptano para catalisar a hidrólise ou a síntese dos ésteres etílicos destes ácidos. A. oryzae pareceu ser mais eficaz que R. oryzae, provavelmente devido a condições micro-ambientais e a um cenário termodinâmico mais favoráveis. Os resultados obtidos em termos de formação do produto e consumo dos substratos em função do tempo foram usados para a estimativa dos rendimentos de conversão máximos, as constantes de equilíbrio e os tempos necessários para alcançar metade da conversão máxima, fornecendo desta forma suficientes informações sobre esses equilíbrios.