Role of enhanced detoxication in a deltamethrin-resistant population of Triatoma infestans (Hemiptera, Reduviidae) from Argentina

Deltamethrin and other pyrethroids have been extensively used in Argentina since 1980, for the chemical control of Triatoma infestans Klug (Hemiptera: Reduviidae). Recently, resistance to deltamethrin was detected in field populations by the survival of bugs exposed by topical application to the diagnostic dose estimated on the CIPEIN susceptible strain. Results of the current study showed low resistant ratios (RRs) to deltamethrin for the resistant populations (RR ranged from 2.0 for San Luis colony to 7.9 for Salta colony). Biochemical studies were made on the most resistant colony (Salta) and the susceptible strain (CIPEIN), in order to establish the importance of degradative mechanisms as a cause of the detected resistance. Esterase activity was measured on 3 days old first instars through phenylthioacetate and a-naphtyl acetate activities. The results showed a significant difference in no cholinesterase esterase activity from susceptible (7.6 ± 0,7 µM S./i.min.) and Salta resistant colony (9.5 ± 0.8 µM S./i.min.). Cytochrome P450 mono-oxygenase (P450) activity was measured on individual insects through ethoxycoumarine deethylase (ECOD) activity using a fluorescence micro plate reader. The dependence of ECOD activity on age and body region of the nymphs, and pH and time of incubation were studied in order to optimize the measurement. As a result, comparative studies were performed on abdomens of 2 days old first instars at pH 7.2 and 4 h incubation time. ECOD activity of first nymphs was significantly lower in the susceptible colony (61.3 ± 9.08 pg ECOD/ insect) than in the resistant one (108.1± 5.7 pg ECOD/ insect). These results suggest that degradative esterases (no-cholinesterase) and mono-oxygenases cytochrome P450, play an important role in the resistance to deltamethrin in Salta colony from Argentina.

The classification of esterases: an important gene family involved in insecticide resistance - A review

The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.

Evolution of insecticide resistance in non-target black flies (Diptera: Simuliidae) from Argentina

Black flies, a non-target species of the insecticides used in fruit production, represent a severe medical and veterinary problem. Large increases in the level of resistance to the pyrethroids fenvalerate (more than 355-fold) and deltamethrin (162-fold) and a small increase in resistance to the organophosphate azinphos methyl (2-fold) were observed between 1996-2008 in black fly larvae under insecticide pressure. Eventually, no change or a slight variation in insecticide resistance was followed by a subsequent increase in resistance. The evolution of pesticide resistance in a field population is a complex and stepwise process that is influenced by several factors, the most significant of which is the insecticide selection pressure, such as the dose and frequency of application. The variation in insecticide susceptibility within a black fly population in the productive area may be related to changes in fruit-pest control. The frequency of individuals with esterase activities higher than the maximum value determined in the susceptible population increased consistently over the sampling period. However, the insecticide resistance was not attributed to glutathione S-transferase activity. In conclusion, esterase activity in black flies from the productive area is one mechanism underlying the high levels of resistance to pyrethroids, which have been recently used infrequently. These enzymes may be reselected by currently used pesticides and enhance the resistance to these insecticides.

Neuronal in vitro models for the estimation of acute systemic toxicity.

The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

Selective inhibition of CTL activation by a dipalmitoyl-phospholipid that prevents the recruitment of signaling molecules to lipid rafts.

Antigen-specific T-cell activation implicates a redistribution of plasma membrane-bound molecules in lipid rafts, such as the coreceptors CD8 and CD4, the Src kinases Lek and Fyn, and the linker for activation of T cells (LAT), that results in the formation of signaling complexes. These molecules partition in lipid rafts because of palmitoylation of cytoplasmic, membrane proximal cysteines, which is essential for their functional integrity in T-cell activation. Here, we show that exogenous dipalmitoyl-phosphatidylethanolamine (DPPE), but not the related unsaturated dioleoyl-phosphatidylethanolamine (DOPE), partitions in lipid rafts. DPPE inhibits activation of CD8(+) T lymphocytes by sensitized syngeneic antigen-presenting cells or specific major histocompatibility complex (MHC) peptide tetramers, as indicated by esterase release and intracellular calcium mobilization. Cytotoxic, T lymphocyte (CTL)-target cell conjugate formation is not affected by DPPE, indicating that engagement of the T-cell receptor by its cognate ligand is intact in lipid-treated cells. In contrast to other agents known to block raft-dependent signaling, DPPE efficiently inhibits the MHC peptide-induced recruitment of palmitoylated signaling molecules to lipid rafts and CTL activation without affecting cell viability or lipid raft integrity.

Perfil de Suscetibilidade da População de Aedes aegypti (Diptera: Culicidae) da ilha de Santiago, Cabo Verde, a inseticidas

Em Cabo Verde, arquipélago situado na Costa Ocidental Africana, os primeiros casos de dengue ocorreram em 2009, com a notificação de mais de 21.000 casos, a maioria desses registrados na Ilha de Santiago. O mosquito Aedes aegypti foi identificado como vetor, e ações para seu controle, usando os inseticidas temephos (larvicida) e a deltametrina (adulticida), têm sido implementadas. Objetiva-se com esse trabalho avaliar o atual status de suscetibilidade a inseticidas e caracterizar os mecanismos de resistência nessa população. Amostras de A. aegypti da ilha de Santiago foram coletadas através de armadilhas de oviposição, para o estabelecimento de uma população a ser analisada. Foram realizados bioensaios do tipo dose diagnóstica, usando garrafas impregnadas com doses únicas dos adulticidas malathion (organofosforado), deltametrina (piretróide) e cipermetrina (piretróide), e bioensaios do tipo dose resposta, usando múltiplas concentrações dos inseticidas temephos (organofosforado), Bacillus thuringiensis sorovariedade israelensis (bactéria entomopatogênica) e diflubenzuron (inibidor de síntese de quitina). Para a investigação dos mecanismos de resistências, foram realizados testes bioquímicos com substratos específicos para quantificar a atividade das enzimas glutationa S-transferases, esterases (α, β e PNPA) e oxidases de função mista, ligadas a detoxificação de xenobióticos, e a taxa de inibição da acetilcolinesterase ligada a insensibilidade do sítio alvo. Pesquisou-se também a presença de mutaçõeso do tipo kdr (knock-down resistance) associadas à resistência a piretróides, pela análise da sequência dos exons 20 e 21 no gene do canal de sódio. Nos resultados dos bioensaios constatou-se que a população de A. aegypti investigada apresenta resistência aos piretróides deltametrina e cipermetrina (mortalidade <80%) e ao organofosforado temephos (RR90=4), mas é suscetível ao malathion (mortalidade ≥98%), Bacillus thuringiensis sorovariedade israelensis (RR90=0.8) e ao diflubenzuron (RR90=2,2). Em relação a atividade das enzimas ligadas ao processo de detoxificação, foram detectadas alterações nas glutationa S-transferases (25%), oxidases de função mista (18%), esterase-α (19%) e esterase- β (17%). A taxa de inibição da acetilcolinesterase (6%) e a atividade da esterase-PNPA (7%) mostraram que estas estão inalteradas. Nenhuma das mutações do tipo kdr pesquisadas foi detectada. Estes resultados permitem concluir que a população de A. aegypti da ilha de Santiago, Cabo Verde, é suscetível aos inseticidas, excetuando os piretróides testados e o temephos, usados no seu controle; e que ela apresenta alterações em enzimas detoxificadoras que poderão estar implicadas na resistência a esses compostos.

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests. Organophosphate compounds are used in agro-systems, and in programs to control pathogen vectors. Because they are continuously applied, organophosphates often reach water sources and may have an impact on aquatic life. The effects of acute and chronic exposure to the organophosphate insecticide malathion on the midge Chironomus sancticaroli are evaluated. To that end, three biochemical biomarkers, acetylcholinesterase (AChE), alpha (EST-α) and beta (EST-β) esterase were used. Acute bioassays with five concentrations of malathion, and chronic bioassays with two concentrations of malathion were carried out. In the acute exposure test, AChE, EST-α and EST-β activities declined by 66, 40 and 37%, respectively, at 0.251 µg L-1 and more than 80% at 1.37, 1.96 and 2.51 µg L-1. In chronic exposure tests, AChE and EST-α activities declined by 28 and 15% at 0.251 µg L-1. Results of the present study show that low concentrations of malathion can influence larval metabolism, indicating high toxicity for Chironomus sancticaroli and environmental risk associated with the use of organophosphates.

Assessment of soil properties by organic matter and EM-microorganism incorporation

Properties of a claim loam soil, collected in Aranjuez (Madrid) and enriched with organic matter and microorganisms, were evaluated under controlled temperature and moisture conditions, over a period of three months. The following treatments were carried out: soil (control); soil + 50 t ha-1 of animal manure (E50); soil + 50 t ha-1 of animal manure + 30 L ha-1 of effective microorganisms (E50EM); soil + 30 t ha-1 of the combination of various green crop residues and weeds (RC30) and soil + 30 t ha-1 of the combination of various green crop residues and weeds + 30 L ha-1 of effective microorganisms (RC30EM). Soil samples were taken before and after incubation and their physical, chemical, and microbiological parameters analyzed. Significant increase was observed in the production of exopolysaccharides and basic phosphatase and esterase enzyme activities in the treatments E50EM and RC30EM, in correlation with the humification of organic matter, water retention at field capacity, and the cationic exchange capacity (CEC) of the same treatments. The conclusion was drawn that the incorporation of a mixture of effective microorganisms (EM) intensified the biological soil activity and improved physical and chemical soil properties, contributing to a quick humification of fresh organic matter. These findings were illustrated by the microbiological activities of exopolysaccharides and by alkaline phosphatase and esterase enzymes, which can be used as early and integrated soil health indicators.

In rats, oral oleoyl-DHEA is rapidly hydrolysed and converted to DHEA-sulphate

Background: Dehydroepiandrosterone (DHEA) released by adrenal glands may be converted to androgens and estrogens mainly in the gonadal, adipose, mammary, hepatic and nervous tissue. DHEA is also a key neurosteroid and has antiglucocorticoid activity. DHEA has been used for the treatment of a number of diseases, including obesity; its pharmacological effects depend on large oral doses, which effect rapidly wanes in part because of its short half-life in plasma. Since steroid hormone esters circulate for longer periods, we have studied here whether the administration of DHEA oleoyl ester may extend its pharmacologic availability by keeping high circulating levels. Results: Tritium-labelled oleoyl-DHEA was given to Wistar male and female rats by gastric tube. The kinetics of appearance of the label in plasma was unrelated to sex; the pattern being largely coincident with the levels of DHEA-sulfate only in females, and after 2 h undistinguishable from the results obtained using labelled DHEA gavages; in the short term, practically no lipophilic DHEA label was found in plasma. After 24 h only a small fraction of the label remained in the rat organs, with a different sex-related distribution pattern coincident for oleoyl- and free- DHEA gavages. The rapid conversion of oleoyl-DHEA into circulating DHEA-sulfate was investigated using stomach, liver and intestine homogenates; which hydrolysed oleoyl-DHEA optimally near pH 8. Duodenum and ileum contained the highest esterase activities. Pure hog pancreas cholesterol-esterase broke down oleoyl-DHEA at rates similar to those of oleoyl-cholesterol. The intestinal and liver esterases were differently activated by taurocholate and showed different pH-activity patterns than cholesterol esterase, suggesting that oleoyl-DHEA can be hydrolysed by a number of esterases in the lumen (e.g. cholesterol-esterase), in the intestinal wall and the liver. Conclusion: The esterase activities found may condition the pharmacological availability (and depot effect) of orally administered steroid hormone fatty acid esters such as oleoyl-DHEA. The oral administration of oleoyl-DHEA in order to extend DHEA plasma availability has not been proved effective, since the ester is rapidly hydrolysed, probably in the intestine itself, and mainly converted to DHEA-sulfate at least in females.

Degradation of pheromone and plant volatile components by a same Odorant-Degrading Enzyme in the cotton leafworm, Spodoptera littoralis

Background: Odorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant. Methodology: Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis , a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants. Conclusion: SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.

Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.

The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.

Caracterização e diversidade genética do capim-elefante e seus híbridos com milheto mediante padrões isoenzimáticos

Foram avaliadas isoenzimaticamente sete cultivares de capim-elefante (Pennisetum purpureum) e seus híbridos com milheto (P. americanum), selecionados pela Empresa Pernambucana de Pesquisa Agropecuária (IPA), visando à identificação de acessos. Foram estudados, em gel de poliacrilamida, os sistemas peroxidase (POX), esterase (EST), glutamato oxalacetato transaminase (GOT), leucina aminopeptidase (LAP), álcool-desidrogenase (ADH) e fosfatase ácida (ACP), em folhas jovens, aos 28 dias após o corte de uniformização. Não foi observada atividade isoenzimática da ADH e observou-se baixa resolução do sistema LAP, os quais não são indicados para caracterização dos germoplasmas. Os padrões de ACP, GOT, POX e EST permitiram conhecer os fenótipos dos 14 acessos estudados. Foram revelados 9, 3, 13 e 19 diferentes padrões de bandas, respectivamente, sendo possível a identificação da coleção de forma rápida e segura utilizando apenas os padrões de esterase.

Polimorfismo enzimático nos tecidos de pereira

O trabalho foi realizado com o objetivo de avaliar o polimorfismo enzimático em diferentes tecidos de oito cultivares de pereira Pyrus communis L. Os genótipos utilizados fazem parte da coleção de plantas disponíveis na Universidade de Estudos de Bolonha. Para as análises isoenzimáticas foram utilizadas gemas floríferas dormentes no inverno, casca de ramos de um ano obtida de plantas em pleno desenvolvimento, folhas obtidas no início da primavera e folhas de plantas mantidas in vitro. A corrida eletroforética foi realizada em gel de poliacrilamida a gradiente com (5% a 12,5%). Os resultados obtidos com os genótipos utilizados indicaram que o sistema enzimático beta-glucosidase (E.C.3.2.1.21) apresentou atividade apenas nas folhas das plantas in vitro, com uma banda na mesma posição para todas as cultivares, ao passo que os sistemas para as enzimas esterase (E.C.3.1.1.2) e peroxidase (E.C.1.11.1.7) apresentaram elevado polimorfismo. Nas gemas dormentes analisadas, o sistema peroxidase permitiu diferenciar todos os genótipos. As formas isoenzimáticas da esterase permitiram separar todos os genótipos independentemente dos tecidos utilizados.

Alterações dos padrões de isoenzimas em sementes de milho infectadas por fungos

Este trabalho teve como objetivo estudar a interferência dos fungos Aspergillus flavus, Fusarium moniliforme e Penicillium spp. sobre padrões eletroforéticos das sementes de milho. Tais padrões são, normalmente, utilizados na identificação de cultivares e na certificação da pureza genética da espécie em estudo. Sementes da cultivar C-805 foram infectadas artificialmente com os referidos fungos; outra parte delas foi tratada com Benomil e Thiabendazol, e ainda outra parte (controle) não foi tratada. As amostras foram acondicionadas em câmara de crescimento (25°C, 95% de umidade relativa) por um período de 30 dias. Na análise eletroforética foi avaliada também uma amostra de sementes que não permaneceu em câmara de crescimento, visando detectar possíveis interferências das condições do ambiente de crescimento sobre os padrões eletroforéticos. Os resultados obtidos permitiram concluir que a infecção das sementes com os fungos Aspergillus flavus, Fusarium moniliforme e Penicillium spp. promove alterações nos padrões eletroforéticos das isoenzimas malato-desidrogenase, esterase, fosfatase ácida, peroxidase e glutamato-oxalacetato-transaminase. A infecção das sementes com Aspergillus flavus promove alterações tanto na intensidade como no número de bandas dos padrões isoenzimáticos da álcool-desidrogenase e malato-desidrogenase.

Caracterização isoenzimática de genótipos de arroz

Objetivou-se, neste trabalho, caracterizar isoenzimaticamente genótipos de arroz (Oryza sativa L.). A produtividade do arroz irrigado no Rio Grande do Sul é elevada, em virtude da alta tecnologia e sistema de irrigação usados, associados ao potencial alcançado pelas cultivares desenvolvidas através de melhoramento genético. Apenas seis ancestrais contribuem com 86% dos genes das cultivares mais plantadas. Como conseqüência desta estreita base genética, as cultivares apresentam um alto grau de parentesco e de similaridade de suas características morfológicas e agronômicas, o que dificulta a identificação varietal. A concorrência com genótipos, como arroz-vermelho e arroz-preto, de difícil controle por serem da mesma espécie que os cultivados, é considerada como um dos maiores problemas da cultura. Análises de isoenzimas podem ser usadas para o estudo da variabilidade e para estimar as relações genéticas existentes entre estes genótipos. Eletroforese em gel de poliacrilamida foi empregada, portanto, para caracterizar, através de isoenzimas de esterase, 6fosfogluconato desidrogenase, fosfoglucoisomerase e isocitrato desidrogenase em sementes e folhas de plântulas, e de fosfatase ácida e aspartato transaminase em folhas de plântulas, as cultivares BR-IRGA 409, BR-IRGA 410, BRS 6 ('Chuí'), BRS 7 ('Taim'), BRS Agrisul, INIA Taquari, El Paso L 144 e IRGA 417, e ecótipos de arroz-vermelho e arroz-preto. A análise de agrupamento, efetuada por meio do coeficiente de Jaccard e pelo método da média aritmética não ponderada (UPGMA), possibilitou a diferenciação de todos os genótipos, à exceção de BRS 6 ('Chuí') e BRS 7 ('Taim'). Três grupos foram identificados, incluindo-se, em um deles, os ecótipos de arroz-vermelho e arroz-preto, que apresentaram 95% de similaridade.