Multiple resistance to pirimiphos-methyl and bifenthrin in Tribolium castaneum involves the activity of lipases, esterases, and laccase2.

Several recent studies have elucidated the molecular mechanisms that confer insecticide resistance on insect pests. However, little is known about multiple resistance in red flour beetle (Tribolium castaneum) at molecular level. The multiple resistance is characterized as resistance to different classes of insecticides that have different target sites, and is mediated by several enzymatic systems. In this study, we investigated the biochemical and molecular mechanisms involved in multiple resistance of T. castaneum to bifenthrin (pyrethroid [Pyr]) and pirimiphos-methyl (organophosphate [Org]). We used artificial selection, biochemical and in silico approaches including structural computational biology. After five generations of artificial selection in the presence of bifenthrin (F5Pyr) or pirimiphos-methyl (F5Org), we found high levels of multiple resistance. The hierarchical enzymatic cluster revealed a pool of esterases (E), lipases (LIPs) and laccase2 (LAC2) potentially contributing to the resistance in different ways throughout development, after one or more generations in the presence of insecticides. The enzyme-insecticide interaction network indicated that E2, E3, LIP3, and LAC2 are enzymes potentially required for multiple resistance phenotype. Kinetic analysis of esterases from F5Pyr and F5Org showed that pirimiphos-methyl and specially bifenthrin promote enzyme inhibition, indicating that esterases mediate resistance by sequestering bifenthrin and pirimiphos-methyl. Our computational data were in accordance with kinetic results, indicating that bifenthrin has higher affinity at the active site of esterase than pirimiphos-methyl. We also report the capability of these insecticides to modify the development in T. castaneum. Our study provide insights into the biochemical mechanisms employed by T. castaneum to acquire multiple resistance.

Esterase isozymes patterns of grape vine (Vitis vinifera L) are altered in response to fungicide exposure / Padrões de isozimas Esterases de videira (Vitis vinifera L) alterados em resposta à exposição a fungicidas

Current analysis characterizes the effect of different fungicides often applied for pest control on α−and ß-esterase patterns of four economically important table-wine grape cultivars (Italia, Rubi, Benitaka and Brasil) of Vitis vinifera. The α- and ß-esterase patterns in bud leaves of the cultivars were assessed by native PAGE analysis. Cabrio Top® compound inhibited EST-2, EST-5, EST-6, EST-7, EST-8, EST-9 and EST-10 carboxylesterases, whereas EST-4, EST-11, EST-12, EST-13, EST-14 acetylesterases and EST-16 carboxylesterase were detected as weakly stained bands. Carboxylesterases and acetylesterases were also detected as weakly stained bands when exposed to fungicides Orthocide 500®, Positron Duo® and Folicur PM®. No changes in α- and ß-esterase patterns were reported when the vines were exposed to the fungicides Rovral SC®, Kumulus DF®, Curzate M®, Score® or Cuprogarb 500®. The evidence of functional changes in carboxylesterase and acetylesterase levels in current study is a warning to grape producers on the dangers inherent in the indiscriminate use of potent and modern fungicides extensively used in agriculture. The inhibition effect of fungicides on esterase isozyme molecules seems to be independent of the fungicide chemical.

O presente estudo caracterizou o efeito de diferentes fungicidas comumente aplicados como medidas de controle de pragas sobre padrões de α- e ß-esterases de quatro importantes cultivares de uva de mesa (Itália, Rubi, Benitaka e Brasil) de Vitis vinifera. Os padrões de α- e ß-esterases de brotos foliares das cultivares foram avaliados por PAGE. O composto Cabrio Top® inibiu as carboxilesterases EST-2, EST-5, EST-6, EST-7, EST-8, EST-9 e EST-10, enquanto as acetilesterases EST-4, EST-11, EST-12, EST-13, EST-14 e a carboxilesterase EST-16 foram detectadas como bandas fracamente coradas. As carboxilesterases e acetilesterases também foram detectadas como bandas fracamente coradas quando expostas aos fungicidas Orthocide 500®, Positron Duo® e Folicur PM®. Não foram observadas alterações nos padrões de α- e ß-esterases quando as videiras foram expostas aos fungicidas Rovral SC®, Kumulus DF®, Curzate M®, Score® ou Cuprogarb 500®. A evidência de alterações em nível funcional em carboxilesterases e acetilesterases, apresentada neste estudo, pode servir como um alerta aos produtores de uva dos perigos inerentes ao uso indiscriminado de fungicidas potentes e modernos amplamente utilizados hoje na agricultura. O efeito dos fungicidas sobre as enzimas esterases parece ser independente do grupo químico ao qual pertence o fungicida.

Allozyme analysis of the four species of Hypostomus (Teleostei: Loricariidae) from the Ivaí river, upper Paraná river basin, Brazil / Análise aloenzimática de quatro espécies de Hypostomus (Teleostei: Loricariidae) do rio Ivaí, bacia do alto rio Paraná, Brasil

Allozyme electrophoresis analysis were performed in four species of Hypostomus (Loricariidae), H. albopunctatus, H. hermanni, H. regani, and Hypostomus sp. 1/NUP 5612 from the Ivaí river, a tributary of the upper Paraná river. The study of 14 loci revealed diagnostic characters and exclusive alleles in a low frequency. The heterozygosity ranged from 0.000 in H. albopunctatus to 0.199 in H. hermanni, which was higher than the heterozygosity in other samples of Hypostomus in literature, as well as in other fish groups. Hypostomus albopunctatus and H. regani revealed higher similarity (I = 0.804), while H. hermanni and Hypostomus sp. 1/NUP 5612 showed the least genetic identity (I = 0.569). All samples were genetically distinguished, despite there were several shared alleles. The FST value was 0.671, showing a high genetic differentiation among the samples. Hypostomus sp. 1/NUP 5612 was genetically distinguished from the three congeners by the loci Adh-A and G3pdh-B and by present rare exclusive alleles in other six enzymatic systems.

Análises aloenzimáticas foram realizadas em quatro espécies de Hypostomus (Loricariidae), H. albopunctatus, H. hermanni, H. regani e Hypostomus sp. 1/NUP 5612 coletadas no rio Ivaí, um tributário da bacia do alto rio Paraná, através da técnica de eletroforese. O estudo de 14 loci gênicos revelou alelos diagnósticos e alelos exclusivos com uma baixa frequência. A heterozigosidade variou de 0,000 em H. albopunctatus a 0,199 em H. hermanni, a qual foi maior que a média para outras espécies de Hypostomus, como também para outros grupos de peixes já estudadas. Hypostomus albopunctatus e H. regani revelaram maior similaridade (I = 0,804), enquanto que H. hermanni e Hypostomus sp. 1/NUP 5612 mostraram a menor identidade genética (I = 0,569). Todas as populações foram geneticamente distintas apesar de apresentarem muitos alelos em comum. O teste de FST resultou em um valor de 0,671, indicando uma diferenciação significativa entre as populações. Hypostomus sp. 1/NUP 5612 foi geneticamente diferenciada das três congêneres pelos loci Adh-A e G3pdh-B e por apresentar alelos raros exclusivos em outros seis sistemas enzimáticos.

Biochemical and genetic polymorphisms for carboxylesterase and acetylesterase in grape clones of Vitis vinifera L. (Vitaceae) cultivars.

Native polyacrylamide gel electrophoresis (PAGE) was employed to show the highest number of esterase loci and to detect alpha- and beta-esterase polymorphisms in leaf buds of Vitis vinifera cultivars. A total of 16 esterase isozymes were detected in leaf buds from 235 plants including Italia, Rubi, Benitaka, and Brasil cultivars. Biochemical characterization of the grape esterases using ester substrates revealed alpha-, beta-, and alpha/beta-esterases with inhibitor tests distinguishing both carboxylesterases (EST-2, EST-3, EST-5, EST-6, EST-7, EST-8, EST-9, EST-10, and EST-16 isozymes) and acetylesterases (EST-4, EST-11, EST-12, EST-13, EST-14, EST-15 isozymes). No allele variation for alpha-, beta-, and alpha/beta-esterases was detected; however, EST-3 alpha-carboxylesterase was absent in 61.7% of vines, and EST-4 alpha/beta-acetylesterase was absent in one vine of Rubi cv. Null EST-3 carboxylesterase phenotype (61.7%) cannot be explained in this article, but the high genetic polymorphism in four V. vinifera clones is a positive aspect for genetic selection and development of new clones with different characteristics.

Functional classification of esterases from leaves of Aspidosperma polyneuron M. Arg. (Apocynaceae)

Polyacrylamide gel electrophoresis system (PAGE) and inhibition tests for biochemical characterization of alpha- and beta-esterases were used to obtain a functional classification of esterases fromAspidosperma polyneuron. The characterization of alpha- and beta-esterases from young leaves of A. polyneuron by the PAGE system showed fourteen esterase isozymes. The differential staining pattern showed that Est-2 isozyme hydrolyzes beta-naphthyl acetate; Est-6, Est-7 and Est-8 isozymes hydrolyze alpha-naphthyl acetate, and Est-1, Est-3, Est-4, Est-5, Est-9, Est-10, Est-11, Est-12, Est-13, and Est-14 isozymes hydrolyze both alpha- and b-naphthyl acetate. Inhibition pattern of a- and beta-esterases showed that Folidol is a more potent inhibitor that Malathion, while Thiamethoxan (an insecticide with organophosphorus-like action) acts as an Est-4 and Est-6 inhibitor and induces the appearance of Est-5 and Est-7 isozymes as more intensely stained bands. Inhibition tests showed that OPC insecticides inhibit or activate plant esterases. Thus, plant esterases may be used as bioindicators to detect the presence and toxicity of residues of topically applied insecticides in agriculture and may be valuable for monitoring pollutants in the environment

Genetic variability in Hoplias malabaricus (Osteichthyes: Erythrinidae) in fluvial and lacustrine environments in the Upper Paraná river floodplain (Paraná State, Brazil).

Genetic variability in Hoplias malabaricus, from two localities in the upper Paraná Riverfloodplain, was investigated by starch and polyacrylamide gel electrophoresis. A total of 52 specimens were analyzed for 14 enzymatic systems. Twenty-three gene loci of 13 enzymatic systems (AAT, ACP, ADH, GDH, G6PDH, GPI, IDH, LDH, MDH, MEP, PGM, PER, and SOD) were analyzed by starch gel electrophoresis (Penetrose-30). The EST system was analyzed by polyacrylamide gel electrophoresis, and one polymorphic locus was found (EST-1). Twenty-four loci were detected. The proportion of polymorphic loci was 37.5% in the lagoon and 33.3% in the river. Significant differences in allele frequencies of five loci were found between specimens from the two environments. Expected mean heterozygosity (He = 0.14) is the same in the river and lagoon, however, Nei's genetic distance (D) between the population of the two locations was 0.049.