Unfolding and Aggregation Pathways of Variable Domains from Immunoglobulin Light Chains.

Light chain amyloidosis is the most common form of systemic amyloidosis. This disease is caused by the formation and deposition of amyloid fibers made from immunoglobulin light chains. Environmental conditions such as pH and temperature can affect protein structure and induce the development of these fibers. Several studies have shed light on the native state, stability, dynamics, and final amyloid state of these proteins; however, the initiation process and the fibril formation pathway remain poorly understood structurally and kinetically. To study this, we analyzed the unfolding and aggregation process of the 6aJL2 protein under acidic conditions, with temperature changes, and upon mutation, using biophysical and computational techniques. Our results suggest that the differences in amyloidogenicity displayed by 6aJL2 under these conditions are caused by traversing different aggregation pathways, including unfolded intermediates and the formation of oligomers.

The Common Bean Small Heat Shock Protein Nodulin 22 from Phaseolus vulgaris L. Assembles into Functional High-Molecular-Weight Oligomers.

Small heat shock proteins (sHsps) are present in all domains of life. These proteins are responsible for binding unfolded proteins to prevent their aggregation. sHsps form dynamic oligomers of different sizes and constitute transient reservoirs for folding competent proteins that are subsequently refolded by ATP-dependent chaperone systems. In plants, the sHsp family is rather diverse and has been associated with the ability of plants to survive diverse environmental stresses. Nodulin 22 (PvNod22) is an sHsp of the common bean (Phaseolus vulgaris L.) located in the endoplasmic reticulum. This protein is expressed in response to stress (heat or oxidative) or in plant roots during mycorrhizal and rhizobial symbiosis. In this work, we study its oligomeric state using a combination of in silico and experimental approaches. We found that recombinant PvNod22 was able to protect a target protein from heat unfolding in vitro. We also demonstrated that PvNod22 assembles into high-molecular-weight oligomers with diameters of ~15 nm under stress-free conditions. These oligomers can cluster together to form high-weight polydisperse agglomerates with temperature-dependent interactions; in contrast, the oligomers are stable regarding temperature.

Intrinsically disordered signaling proteins: Essential hub players in the control of stress responses in Saccharomyces cerevisiae.

Cells have developed diverse mechanisms to monitor changes in their surroundings. This allows them to establish effective responses to cope with adverse environments. Some of these mechanisms have been well characterized in the budding yeast Saccharomyces cerevisiae, an excellent experimental model to explore and elucidate some of the strategies selected in eukaryotic organisms to adjust their growth and development in stressful conditions. The relevance of structural disorder in proteins and the impact on their functions has been uncovered for proteins participating in different processes. This is the case of some transcription factors (TFs) and other signaling hub proteins, where intrinsically disordered regions (IDRs) play a critical role in their function. In this work, we present a comprehensive bioinformatic analysis to evaluate the significance of structural disorder in those TFs (170) recognized in S. cerevisiae. Our findings show that 85.2% of these TFs contain at least one IDR, whereas ~30% exhibit a higher disorder level and thus were considered as intrinsically disordered proteins (IDPs). We also found that TFs contain a higher number of IDRs compared to the rest of the yeast proteins, and that intrinsically disordered TFs (IDTFs) have a higher number of protein-protein interactions than those with low structural disorder. The analysis of different stress response pathways showed a high content of structural disorder not only in TFs but also in other signaling proteins. The propensity of yeast proteome to undergo a liquid-liquid phase separation (LLPS) was also analyzed, showing that a significant proportion of IDTFs may undergo this phenomenon. Our analysis is a starting point for future research on the importance of structural disorder in yeast stress responses.

Aggregation pathways of human γ D crystallin induced by metal ions revealed by time dependent methods.

Cataract formation is a slow accumulative process due to protein aggregates promoted by different factors over time. Zinc and copper ions have been reported to induce the formation of aggregates opaque to light in the human gamma D crystallin (HγD) in a concentration and temperature dependent manner. In order to gain insight into the mechanism of metal-induced aggregation of HγD under conditions that mimic more closely the slow, accumulative process of the disease, we have studied the non-equilibrium process with the minimal metal dose that triggers HγD aggregation. Using a wide variety of biophysics techniques such as turbidimetry, dynamic light scattering, fluorescence, nuclear magnetic resonance and computational methods, we obtained information on the molecular mechanisms for the formation of aggregates. Zn(II) ions bind to different regions at the protein, probably with similar affinities. This binding induces a small conformational rearrangement within and between domains and aggregates via the formation of metal bridges without any detectable unfolded intermediates. In contrast, Cu(II)-induced aggregation includes a lag time, in which the N-terminal domain partially unfolds while the C-terminal domain and parts of the N-terminal domain remain in a native-like conformation. This partially unfolded intermediate is prone to form the high-molecular weight aggregates. Our results clearly show that different external factors can promote protein aggregation following different pathways.

Site-Specific Interactions with Copper Promote Amyloid Fibril Formation for λ6aJL2-R24G.

Light-chain amyloidosis (AL) is one of the most common systemic amyloidoses, and it is characterized by the deposition of immunoglobulin light chain (LC) variable domains as insoluble amyloid fibers in vital organs and tissues. The recombinant protein 6aJL2-R24G contains λ6a and JL2 germline genes and also contains the Arg24 by Gly substitution. This mutation is present in 25% of all amyloid-associated λ6 LC cases, reduces protein stability, and increases the propensity to form amyloid fibers. In this study, it was found that the interaction of 6aJL2-R24G with Cu(II) decreases the thermal stability of the protein and accelerates the amyloid fibril formation, as observed by fluorescence spectroscopy. Isothermal calorimetry titration showed that Cu(II) binds to the protein with micromolar affinity. His99 may be one of the main Cu(II) interaction sites, as observed by nuclear magnetic resonance spectroscopy. The binding of Cu(II) to His99 induces larger fluctuations of the CDR1 and loop C″, as shown by molecular dynamics simulations. Thus, Cu(II) binding may be inducing the loss of interactions between CDR3 and CDR1, making the protein less stable and more prone to form amyloid fibers. This study provides insights into the mechanism of metal-induced aggregation of the 6aJL2-R24G protein and sheds light on the bio-inorganic understanding of AL disease.

Different Dynamics in 6aJL2 Proteins Associated with AL Amyloidosis, a Conformational Disease.

Light-chain amyloidosis (AL) is the most common systemic amyloidosis and is caused by the deposition of mainly insoluble immunoglobulin light chain amyloid fibrils in multiple organs, causing organ failure and eventually death. The germ-line λ6a has been implicated in AL, where a single point mutant at amino acid 24 (6aJL2-R24G) has been observed in around 25% of patient samples. Structural analysis has shown only subtle differences between both proteins; nevertheless, 6aJL2-R24G is more prone to form amyloid fibrils. To improve our understanding of the role of protein flexibility in amyloid fibril formation, we have used a combination of solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to complement the structural insight with dynamic knowledge. Fast timescale dynamics (ps-ns) were equivalent for both proteins, but suggested exchange events for some residues. Even though most of the intermediate dynamics (µs-ms) occurred at a similar region for both proteins, the specific characteristics are very different. A minor population detected in the dispersion experiments could be associated with the formation of an off-pathway intermediate that protects from fiber formation more efficiently in the germ-line protein. Moreover, we found that the hydrogen bond patterns for both proteins are similar, but the lifetime for the mutant is significantly reduced; as a consequence, there is a decrease in the stability of the tertiary structure that extends throughout the protein and leads to an increase in the propensity to form amyloid fibers.

Metal-binding polymorphism in late embryogenesis abundant protein AtLEA4-5, an intrinsically disordered protein.

Late embryogenesis abundant (LEA) proteins accumulate in plants during adverse conditions and their main attributed function is to confer tolerance to stress. One of the deleterious effects of the adverse environment is the accumulation of metal ions to levels that generate reactive oxygen species, compromising the survival of cells. AtLEA4-5, a member of group 4 of LEAs in Arabidopsis, is an intrinsically disordered protein. It has been shown that their N-terminal region is able to undergo transitions to partially folded states and prevent the inactivation of enzymes. We have characterized metal ion binding to AtLEA4-5 by circular dichroism, electronic absorbance spectroscopy (UV-vis), electron paramagnetic resonance, dynamic light scattering, and isothermal titration calorimetry. The data shows that AtLEA4-5 contains a single binding site for Ni(II), while Zn(II) and Cu(II) have multiple binding sites and promote oligomerization. The Cu(II) interacts preferentially with histidine residues mostly located in the C-terminal region with moderate affinity and different coordination modes. These results and the lack of a stable secondary structure formation indicate that an ensemble of conformations remains accessible to the metal for binding, suggesting the formation of a fuzzy complex. Our results support the multifunctionality of LEA proteins and suggest that the C-terminal region of AtLEA4-5 could be responsible for antioxidant activity, scavenging metal ions under stress conditions while the N-terminal could function as a chaperone.

The first description of a hormone-sensitive lipase from a basidiomycete: Structural insights and biochemical characterization revealed Bjerkandera adusta BaEstB as a novel esterase.

The heterologous expression and characterization of a Hormone-Sensitive Lipases (HSL) esterase (BaEstB) from the Basidiomycete fungus Bjerkandera adusta is reported for the first time. According to structural analysis, amino acid similarities and conservation of particular motifs, it was established that this enzyme belongs to the (HSL) family. The cDNA sequence consisted of 969 nucleotides, while the gene comprised 1133, including three introns of 57, 50, and 57 nucleotides. Through three-dimensional modeling and phylogenetic analysis, we conclude that BaEstB is an ortholog of the previously described RmEstB-HSL from the phylogenetically distant fungus Rhizomucor miehei. The purified BaEstB was characterized in terms of its specificity for the hydrolysis of different acyl substrates confirming its low lipolytic activity and a noticeable esterase activity. The biochemical characterization of BaEstB, the DLS analysis and the kinetic parameters determination revealed this enzyme as a true esterase, preferentially found in a dimeric state, displaying activity under alkaline conditions and relative low temperature (pH = 10, 20°C). Our data suggest that BaEstB is more active on substrates with short acyl chains and bulky aromatic moieties. Phylogenetic data allow us to suggest that a number of fungal hypothetical proteins could belong to the HSL family.

Melaleuca quinquenervia (Cav.) S.T. Blake (Myrtales: Myrtaceae): Natural alternative for mosquito control.

OBJECTIVE: To evaluate an essential oil with larvicide, adulticide and growth inhibitory activity against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus mosquitoes, of medical importance. METHODS: Standardized methodology by WHO was used to determine the levels of susceptibility of mosquito larvae exposed to the essential oil. To evaluate the adulticide activity with the essential oil at different doses, bottles were impregnated according to the methodology CDC. To determine the development inhibitory activity of Melaleuca quinquenervia (M. quinquenervia) oil in three mosquito species, third instar larvae were exposed to the LC50 and LC90 dose (calculated for each population) of M. quinquenervia oil in glass containers with a capacity of 500 mL. After 24 h exposure, the dead larvae were discarded. The mortality of larvae and pupae were recorded on a daily basis. RESULTS: The calculated LC50 indicates an order of effectiveness of preferential oil for Culex quinquefasciatus (LC50 = 0.0021%), Aedes aegypti (LC50 = 0.0047%) and Aedes albopictus (LC50 = 0.0049%). CONCLUSIONS: The adulticide activity was achieved with impregnated bottles at 40 and 50 mg/mL with the three mosquitoes species. In larvae, a growth inhibition was detected when exposed to sublethal doses. The results indicate that M. quinquenervia is a plant with promising environmentally sustainable source for vector control.

Actividad insecticida de aceite de trementina modificado en Culex quinquefasciatus y Aedes albopictus (Diptera: Culicidae) / Insecticidal activity of modified turpentine oil in Culex quinquefasciatus and Aedes albopictus (Diptera: Culicidae)

Introducción: el estudio de alternativas naturales para el control vectorial es de gran importancia debido a los fenómenos de resistencia a insecticidas sintéticos encontrados en diversas especies de mosquitos. Objetivo: evaluar la actividad insecticida de aceite de trementina modificado en Culex quinquefasciatus y Aedes albopictus. Métodos: se evaluó la actividad larvicida, adulticida e inhibidora del desarrollo de este aceite, según metodologías estandarizadas. Se utilizó para el estudio una cepade Cx. quinquefasciatus (Regla 2013) y una de Ae. albopictus (Fraga 2012). Resultados: el aceite de trementina modificado mostró actividad larvicida para Cx. quinquefasciatus y Ae. albopictus lo que se evidenció por los valores bajos de concentraciones letales que causaron el 90 por ciento de mortalidad (CL90 = 0,0054 por ciento y CL90 = 0,00115 por ciento), en orden. Al evaluar la actividad adulticida se obtuvo el 100 por ciento de derribo a los 30 minutos de los individuos expuestos de ambas cepas a la concentración de 60 mg/mL. Al exponer larvas a sus respectivas CL90 del aceite de trementina modificado, se observó en Cx. quinquefasciatus una mortalidad 2 veces mayor en la fase de larva que en la fase de pupa. Durante los primeros 5 días la mortalidad fue mayor en los estadios pupa y adulto en Ae. Albopictus. Sin embargo, los resultados difieren con Cx. quinquefasciatus en el mismo período. El sexo femenino fue prevaleciente en la mortalidad pupal. La mayor mortalidad en adultos se observó en machos, por lo que ocurrió una desproporción en los sexos de los sobrevivientes. Conclusiones: los resultados encontrados avalan y permiten recomendar el aceite de trementina modificado como un insecticida de origen natural para el control de Ae. albopictus y Cx. quinquefasciatus(AU)

Introduction: the study of natural alternatives for vector control is of great importance due to the phenomena of resistance to synthetic insecticides found in various species of mosquitoes. Objective: to evaluate the insecticidal activity of modified turpentine oil in Culex quinquefasciatus and Aedes albopictus. Methods: the larvicidal, adulticidal and development-inhibitory activity of this oil was evaluated, by standardized methodologies. The study used one strain of Culex quinquefasciatus (Regla, 2013) and another of Aedes albopictus (Fraga, 2012). Results: modified turpentine oil showed larvicidal activity for Culex quinquefasciatus and Aedes albopictus, a fact evidenced by the low values of lethal concentrations that caused 90 percent of mortality (LC90=0.0054 percent and LC90=0.00115 percent, respectively). Evaluating the adulticidal activity, knockdown was obtained in 100 percent and 30 minutes after the individuals of both strains were exposed to the 60 mg/ml concentration. By exposing larvae to their respective modified turpentine oil CL90, Culex quinquefasciatus mortality in the larval stage was observed to be two times higher than in the pupal stage. During the first five days, mortality was higher in the pupa and adult stages in Aedes albopictus. However, these results differ from the Culex quinquefasciatus results for the same period. Female sex was prevalent regarding pupal mortality. Most adult mortality was observed in males, reason why a disproportion occurred concerning the survivors sex. Conclusions: the results found endorse and permit recommending modified turpentine oil as a natural insecticide for controlling Aedes albopictus and Culex quinquefasciatus(AU)

Actividad insecticida de aceite de trementina modificado en Culex quinquefasciatus y Aedes albopictus (Diptera: Culicidae) / Insecticidal activity of modified turpentine oil in Culex quinquefasciatus and Aedes albopictus (Diptera: Culicidae)

Introducción: el estudio de alternativas naturales para el control vectorial es de gran importancia debido a los fenómenos de resistencia a insecticidas sintéticos encontrados en diversas especies de mosquitos. Objetivo: evaluar la actividad insecticida de aceite de trementina modificado en Culex quinquefasciatus y Aedes albopictus. Métodos: se evaluó la actividad larvicida, adulticida e inhibidora del desarrollo de este aceite, según metodologías estandarizadas. Se utilizó para el estudio una cepa de Cx. quinque fasciatus (Regla 2013) y una de Ae. albopictus (Fraga 2012). Resultados: el aceite de trementina modificado mostró actividad larvicida para Cx. quinquefasciatus y Ae. albopictus lo que se evidenció por los valores bajos de concentraciones letales que causaron el 90 por ciento de mortalidad (CL90 = 0,0054 por ciento y CL90 = 0,00115 por ciento), en orden. Al evaluar la actividad adulticida se obtuvo el 100 por ciento de derribo a los 30 minutos de los individuos expuestos de ambas cepas a la concentración de 60 mg/mL. Al exponer larvas a sus respectivas CL90 del aceite de trementina modificado, se observó en Cx. quinque fasciatus una mortalidad 2 veces mayor en la fase de larva que en la fase de pupa. Durante los primeros 5 días la mortalidad fue mayor en los estadios pupa y adulto en Ae. Albopictus.Sin embargo, los resultados difieren con Cx. quinque fasciatus en el mismo período. El sexo femenino fue prevaleciente en la mortalidad pupal. La mayor mortalidad en adultos se observó en machos, por lo que ocurrió una desproporción en los sexos de los sobrevivientes. Conclusiones: los resultados encontrados avalan y permiten recomendar el aceite de trementina modificado como un insecticida de origen natural para el control de Ae. albopictus y Cx. quinque fasciatus(AU)

Inhibition of Light Chain 6aJL2-R24G Amyloid Fiber Formation Associated with Light Chain Amyloidosis.

Light chain amyloidosis (AL) is a deadly disease characterized by the deposition of monoclonal immunoglobulin light chains as insoluble amyloid fibrils in different organs and tissues. Germ line λ VI has been closely related to this condition; moreover, the R24G mutation is present in 25% of the proteins of this germ line in AL patients. In this work, five small molecules were tested as inhibitors of the formation of amyloid fibrils from the 6aJL2-R24G protein. We have found by thioflavin T fluorescence and transmission electron microscopy that EGCG inhibits 6aJL2-R24G fibrillogenesis. Furthermore, using nuclear magnetic resonance spectroscopy, dynamic light scattering, and isothermal titration calorimetry, we have determined that the inhibition is due to binding to the protein in its native state, interacting mainly with aromatic residues.

Vigilancia entomológica con énfasis en Aedes aegypti (Díptera: Culicidae) en campos de desplazados en Haití, 2010-2011 / Entomological surveillance with an emphasis on Aedes aegypti (Díptera: Culicidae) in displaced persons camps of Haiti, 2010-2011

Introducción: el terremoto en Haití en enero del 2010, condujo al agravamiento de las condiciones higiénicas, sanitarias y ambientales de un gran número de personas sin viviendas. Objetivo: evaluar la vigilancia vectorial con énfasis sobre Aedes aegypti, en un campo de desplazados post terremoto en la comuna de Carrefour en Haití, Junio 2010-Junio 2011. Métodos: se determinaron la presencia y la densidad de Ae. aegypti, Anopheles albimanus y Culex quinquefasciatus por medio de encuestas larvales. Se emplearon también medidas de control adulticidas y uso de larvicidas para Ae. aegypti. Resultados: se identificaron 7 especies de mosquitos. Los valores obtenidos de los índices casa, depósito y Breteau evidenció el mantenimiento de las densidades de Ae. aegypti con un incremento en la segunda mitad del año. Los depósitos misceláneos constituyeron el mayor por ciento de criaderos con 56 por ciento del total, seguido por los neumáticos con 27 por ciento. An. albimanus y Cx. quinquefasciatus mostraron escasa presencia en el área estudiada. Los tratamientos adulticidas contribuyeron a controlar las densidades de mosquitos en las instituciones con presencia cubana y en las áreas del campo de desplazados. Conclusiones: durante el período estudiado se mantuvo una vigilancia y control constante sobre los mosquitos contribuyendo a que no se reportaran enfermedades transmitidas por estos vectores en el campo de desplazados(AU)

Introduction: as a result of the earthquake occurring in Haiti in 2010, health, sanitation and environmental conditions worsened and a large number of persons lost their homes. Objective: evaluate vector surveillance with an emphasis on Aedes Aegypti in a post-earthquake displaced persons camp in the commune of Carrefour, Haiti, from June 2010 to June 2011. Methods: determination was made of the presence and density of Ae. aegypti, Anopheles albimanus and Culex quinquefasciatus by means of larval surveys. Adult control measures and larvicides against Ae. Aegypti were also used. Results: seven mosquito species were identified.The values obtained for the indices household, container and Breteau showed that Ae. aegypti densities remained stable, with an increase in the second half of the year. Most breeding sites were miscellaneous containers (56 percent), followed by pneumatic tires (27 percent). An. albimanus and Cx. quinquefasciatus showed scant presence in the study area. Adulticide treatments contributed to the control of mosquito densities at institutions with the presence of Cuban cooperation personnel and in the displaced persons camp. Conclusions: mosquito surveillance and control were permanent during the study period, as a result of which there were no reports of diseases transmitted by this vector in the displaced persons camp(AU)

Vigilancia entomológica con énfasis en aedes aegypti (Díptera: Culicidae) en campos de desplazados en Haití, 2010-2011 / Entomological surveillance with an emphasis on aedes aegypti (Díptera: Culicidae) in displaced persons camps of Haiti, 2010-2011

INTRODUCCIÓN: el terremoto en Haití en enero del 2010, condujo al agravamiento de las condiciones higiénicas, sanitarias y ambientales de un gran número de personas sin viviendas. OBJETIVO: evaluar la vigilancia vectorial con énfasis sobre Aedes aegypti, en un campo de desplazados post terremoto en la comuna de Carrefour en Haití, Junio 2010-Junio 2011. MÉTODOS: se determinaron la presencia y la densidad de Ae. aegypti, Anopheles albimanus y Culex quinquefasciatus por medio de encuestas larvales. Se emplearon también medidas de control adulticidas y uso de larvicidas para Ae. aegypti. RESULTADOS: se identificaron 7 especies de mosquitos. Los valores obtenidos de los índices casa, depósito y Breteau evidenció el mantenimiento de las densidades de Ae. aegypti con un incremento en la segunda mitad del año. Los depósitos misceláneos constituyeron el mayor por ciento de criaderos con 56 % del total, seguido por los neumáticos con 27 %. An. albimanus y Cx. quinquefasciatus mostraron escasa presencia en el área estudiada. Los tratamientos adulticidas contribuyeron a controlar las densidades de mosquitos en las instituciones con presencia cubana y en las áreas del campo de desplazados. CONCLUSIONES: durante el período estudiado se mantuvo una vigilancia y control constante sobre los mosquitos contribuyendo a que no se reportaran enfermedades transmitidas por estos vectores en el campo de desplazados.

INTRODUCTION: as a result of the earthquake occurring in Haiti in 2010, health, sanitation and environmental conditions worsened and a large number of persons lost their homes. OBJECTIVE: evaluate vector surveillance with an emphasis on Aedes Aegypti in a post-earthquake displaced persons camp in the commune of Carrefour, Haiti, from June 2010 to June 2011. METHODS: determination was made of the presence and density of Ae. aegypti, Anopheles albimanus and Culex quinquefasciatus by means of larval surveys. Adult control measures and larvicides against Ae. Aegypti were also used. RESULTS: seven mosquito species were identified.The values obtained for the indices household, container and Breteau showed that Ae. aegypti densities remained stable, with an increase in the second half of the year. Most breeding sites were miscellaneous containers (56 %), followed by pneumatic tires (27 %). An. albimanus and Cx. quinquefasciatus showed scant presence in the study area. Adulticide treatments contributed to the control of mosquito densities at institutions with the presence of Cuban cooperation personnel and in the displaced persons camp. CONCLUSIONS: mosquito surveillance and control were permanent during the study period, as a result of which there were no reports of diseases transmitted by this vector in the displaced persons camp.

Eficacia del pyriproxyfeno en cepas de referencia de Aedes aegypti susceptible y resistente a temefos / Efficacy of pyriproxyfen in reference strains of Aedes aegypti susceptible and resistant to temephos

Introducción: el uso intensivo del larvicida químico temefos (abate) en Cuba, ha generado el desarrollo de mecanismos de resistencia en Aedes (Stegomyia) aegypti (Linnaeus, 1762). Por esta razón, apremia la necesidad de evaluar otras alternativas de control, como el pyriproxyfeno, inhibidor de crecimiento. Objetivos: determinar la eficacia de pyriproxyfeno como larvicida, pupicida y adulticida en cepas de referencia de Aedes aegypti y establecer su efecto sobre la reproducción. Métodos: para el estudio se utilizó una cepa de Aedes aegypti de referencia susceptible (Rockefeller) y otra resistente (SANtemF13) a temefos. La evaluación de pyriproxyfeno se realizó a través de bioensayos recomendados por la Organización Mundial de la Salud. Se determinó el efecto en la reproducción, midiendo la fecundidad y viabilidad de los huevos (fertilidad) en larvas de la cepa Rockefeller, sobrevivientes a dosis subletales de pyriproxyfeno. Resultados: en Rockefeller, pyriproxyfeno manifestó actividad larvicida en 32 por ciento, pupicida en 21 por ciento y adulticida solo 1 por ciento. En SANtem F13, resultó larvicida en 31 por ciento, pupicida 12 por ciento y adulticida 1 por ciento. El análisis de varianza entre rangos de dosis letales para larvas, pupas y adultos de las cepas referenciales, Rockefeller y SANtem F13, no mostró diferencias significativas (p= 0,000001). El pyriproxyfeno resultó ser un excelente inhibidor de la emergencia de adultos en las cepas de Aedes aegypti estudiadas a concentraciones bajas, inferiores a las recomendadas por la Organización Mundial de la Salud en mosquitos del género Aedes. También tuvo fuerte efecto subletal y afectó la fertilidad de los adultos, en condiciones de laboratorio. Conclusión: el inhibidor de crecimiento pyriproxyfeno representa una buena alternativa a incluir dentro de las estrategias de control integrado de Aedes aegypti en Cuba, sin afectarse su eficacia por la resistencia que esta especie ha...(AU)

Introduction: intensive use of chemical larvicide temephos (abate) in Cuba has resulted in the development of resistance mechanisms in Aedes (Stegomyia) aegypti (Linnaeus, 1762). Therefore, there is an urgent need to consider other control alternatives, such as pyriproxyfen, a growth inhibitor. Objectives: determine the efficacy of pyriproxyfen as larvicide, pupicide and adulticide in reference strains of Aedes aegypti and establish its effect on reproduction. Methods: the study was based on a strain of reference Aedes aegypti susceptible to temephos (Rockefeller) and a strain resistant to temephos (SANtemF13). Pyriproxyfen was evaluated with bioassays recommended by the World Health Organization. Determination was made of the effect of pyriproxyfen on reproduction by measuring fecundity and egg viability (fertility) in larvae of the Rockefeller strain surviving sublethal doses of pyriproxyfen. Results: in the Rockefeller strain, pyriproxyfen was larvicidal in 32 percent, pupicidal in 21 percent and adulticidal in a mere 1 percent. In the SANtem F13 strain, it was larvicidal in 31 percent, pupicidal in 12 percent and adulticidal in 1 percent. Analysis of variance between ranges of lethal doses for larvae, pupas and adults of the reference strains Rockefeller and SANtem F13 did not show any significant differences (p= 0.000001). Pyriproxyfen was found to be an excellent inhibitor of adult emergence in the strains of Aedes aegypti studied when used at low concentrations, below those recommended by the World Health Organization for mosquitoes of the genus Aedes. It also showed a strong sublethal effect and affected the fertility of adults in laboratory conditions. Conclusion: growth inhibitor pyriproxyfen is a good alternative to be included among integrated control strategies against Aedes aegypti in Cuba without affecting its efficacy, due to the resistance that this species has developed to larvicide temephos(AU)

Actividad incrementada de las enzimas citocromo P450 monooxigenasas en cepas cubanas de Aedes aegypti de referencia, resistentes a insecticidas / Increased activity of cytochrome P450 monooxygenase enzymes in reference insecticide-resistant Aedes aegypti strains from Cuba

Introducción: las enzimas desintoxicadoras citocromo P450 monooxigenasas (MFO) constituyen uno de los principales mecanismos de resistencia de Aedes aegypti a insecticidas. En Cuba, aunque la presencia de estas enzimas se ha estudiado in vivo mediante el uso de sinergistas, su actividad no se ha determinado cuantitativamente in vitro, elemento que resulta imprescindible para abordar estudios de resistencia metabólica en los insectos. Objetivo: estandarizar un método para detectar la actividad de las citocromo P450 monooxigenasas in vitro, y determinarla entonces, en larvas y adultos de cepas de referencia de Aedes aegypti. Métodos: se utilizaron 3 cepas de laboratorio de Aedes aegypti seleccionadas por 14 o 15 generaciones con temefos, deltametrina o propoxur, respectivamente, y una cepa susceptible a los insecticidas. Resultados: se establecieron las condiciones para los ensayos de actividad enzimática (concentración de proteínas y de sustrato, 0,4 mg/mL y 12 mmol/L, respectivamente; y tiempo de reacción de 10 min). Hubo un incremento significativo de la actividad de las citocromo P450 monooxigenasas en las cepas resistentes, con una mayor frecuencia fenotípica de este carácter en el estadio larva. Conclusiones: las modificaciones a la técnica para la determinación de la actividad enzimática permitieron discriminar entre mosquitos de cepas susceptibles y resistentes en los estadios larva y adulto, por lo que se cuenta con otra herramienta para la detección de la resistencia metabólica en Cuba(AU)

Introduction: cytochrome P450 monooxygenase detoxifying enzymes (MFO) are one of the main resistance mechanisms of Aedes aegypti to insecticides. In vivo studies of the presence of these enzymes have been conducted in Cuba with the use of synergists. However, their activity has not been quantitatively determined in vitro, an indispensable step in studies about metabolic resistance in insects. Objective: standardize a method to detect the activity of cytochrome P450 monooxygenase in vitro, and then determine such activity in larvae and adults of Aedes aegypti reference strains. Methods: the study was based on three laboratory strains of Aedes aegypti selected for 14 or 15 generations with temephos, deltamethrin or propoxur, respectively, and a strain susceptible to insecticides. Results: the conditions for enzyme activity assays were established (protein and substrate concentration: 0.4 mg/mL and 12 mmol/L, respectively, and reaction time: 10 min). There was a significant increase in cytochrome P450 monooxygenase activity in resistant strains, with a higher phenotypic frequency in the larval stage. Conclusions: modifications to the technique used for determination of enzymatic activity made it possible to distinguish between mosquitoes from susceptible and resistant strains in larval and adult stages, providing a new tool for the detection of metabolic resistance in Cuba(AU)

Eficacia del pyriproxyfeno en cepas de referencia de Aedes aegypti susceptible y resistente a temefos / Efficacy of pyriproxyfen in reference strains of Aedes aegypti susceptible and resistant to temephos

Introducción: el uso intensivo del larvicida químico temefos (abate) en Cuba, ha generado el desarrollo de mecanismos de resistencia en Aedes (Stegomyia) aegypti (Linnaeus, 1762). Por esta razón, apremia la necesidad de evaluar otras alternativas de control, como el pyriproxyfeno, inhibidor de crecimiento. Objetivos: determinar la eficacia de pyriproxyfeno como larvicida, pupicida y adulticida en cepas de referencia de Aedes aegypti y establecer su efecto sobre la reproducción. Métodos: para el estudio se utilizó una cepa de Aedes aegypti de referencia susceptible (Rockefeller) y otra resistente (SANtemF13) a temefos. La evaluación de pyriproxyfeno se realizó a través de bioensayos recomendados por la Organización Mundial de la Salud. Se determinó el efecto en la reproducción, midiendo la fecundidad y viabilidad de los huevos (fertilidad) en larvas de la cepa Rockefeller, sobrevivientes a dosis subletales de pyriproxyfeno. Resultados: en Rockefeller, pyriproxyfeno manifestó actividad larvicida en 32 por ciento, pupicida en 21 por ciento y adulticida solo 1 por ciento. En SANtem F13, resultó larvicida en 31 por ciento, pupicida 12 por ciento y adulticida 1 por ciento. El análisis de varianza entre rangos de dosis letales para larvas, pupas y adultos de las cepas referenciales, Rockefeller y SANtem F13, no mostró diferencias significativas (p= 0,000001). El pyriproxyfeno resultó ser un excelente inhibidor de la emergencia de adultos en las cepas de Aedes aegypti estudiadas a concentraciones bajas, inferiores a las recomendadas por la Organización Mundial de la Salud en mosquitos del género Aedes. También tuvo fuerte efecto subletal y afectó la fertilidad de los adultos, en condiciones de laboratorio. Conclusión: el inhibidor de crecimiento pyriproxyfeno representa una buena alternativa a incluir dentro de las estrategias de control integrado de Aedes aegypti en Cuba, sin afectarse su eficacia por la resistencia que esta especie ha...

Introduction: intensive use of chemical larvicide temephos (abate) in Cuba has resulted in the development of resistance mechanisms in Aedes (Stegomyia) aegypti (Linnaeus, 1762). Therefore, there is an urgent need to consider other control alternatives, such as pyriproxyfen, a growth inhibitor. Objectives: determine the efficacy of pyriproxyfen as larvicide, pupicide and adulticide in reference strains of Aedes aegypti and establish its effect on reproduction. Methods: the study was based on a strain of reference Aedes aegypti susceptible to temephos (Rockefeller) and a strain resistant to temephos (SANtemF13). Pyriproxyfen was evaluated with bioassays recommended by the World Health Organization. Determination was made of the effect of pyriproxyfen on reproduction by measuring fecundity and egg viability (fertility) in larvae of the Rockefeller strain surviving sublethal doses of pyriproxyfen. Results: in the Rockefeller strain, pyriproxyfen was larvicidal in 32 percent, pupicidal in 21 percent and adulticidal in a mere 1 percent. In the SANtem F13 strain, it was larvicidal in 31 percent, pupicidal in 12 percent and adulticidal in 1 percent. Analysis of variance between ranges of lethal doses for larvae, pupas and adults of the reference strains Rockefeller and SANtem F13 did not show any significant differences (p= 0.000001). Pyriproxyfen was found to be an excellent inhibitor of adult emergence in the strains of Aedes aegypti studied when used at low concentrations, below those recommended by the World Health Organization for mosquitoes of the genus Aedes. It also showed a strong sublethal effect and affected the fertility of adults in laboratory conditions. Conclusion: growth inhibitor pyriproxyfen is a good alternative to be included among integrated control strategies against Aedes aegypti in Cuba without affecting its efficacy, due to the resistance that this species has developed to larvicide temephos

Actividad incrementada de las enzimas citocromo P450 monooxigenasas en cepas cubanas de Aedes aegypti de referencia, resistentes a insecticidas / Increased activity of cytochrome P450 monooxygenase enzymes in reference insecticide-resistant Aedes aegypti strains from Cuba

Introducción: las enzimas desintoxicadoras citocromo P450 monooxigenasas (MFO) constituyen uno de los principales mecanismos de resistencia de Aedes aegypti a insecticidas. En Cuba, aunque la presencia de estas enzimas se ha estudiado in vivo mediante el uso de sinergistas, su actividad no se ha determinado cuantitativamente in vitro, elemento que resulta imprescindible para abordar estudios de resistencia metabólica en los insectos. Objetivo: estandarizar un método para detectar la actividad de las citocromo P450 monooxigenasas in vitro, y determinarla entonces, en larvas y adultos de cepas de referencia de Aedes aegypti. Métodos: se utilizaron 3 cepas de laboratorio de Aedes aegypti seleccionadas por 14 o 15 generaciones con temefos, deltametrina o propoxur, respectivamente, y una cepa susceptible a los insecticidas. Resultados: se establecieron las condiciones para los ensayos de actividad enzimática (concentración de proteínas y de sustrato, 0,4 mg/mL y 12 mmol/L, respectivamente; y tiempo de reacción de 10 min). Hubo un incremento significativo de la actividad de las citocromo P450 monooxigenasas en las cepas resistentes, con una mayor frecuencia fenotípica de este carácter en el estadio larva. Conclusiones: las modificaciones a la técnica para la determinación de la actividad enzimática permitieron discriminar entre mosquitos de cepas susceptibles y resistentes en los estadios larva y adulto, por lo que se cuenta con otra herramienta para la detección de la resistencia metabólica en Cuba

Introduction: cytochrome P450 monooxygenase detoxifying enzymes (MFO) are one of the main resistance mechanisms of Aedes aegypti to insecticides. In vivo studies of the presence of these enzymes have been conducted in Cuba with the use of synergists. However, their activity has not been quantitatively determined in vitro, an indispensable step in studies about metabolic resistance in insects. Objective: standardize a method to detect the activity of cytochrome P450 monooxygenase in vitro, and then determine such activity in larvae and adults of Aedes aegypti reference strains. Methods: the study was based on three laboratory strains of Aedes aegypti selected for 14 or 15 generations with temephos, deltamethrin or propoxur, respectively, and a strain susceptible to insecticides. Results: the conditions for enzyme activity assays were established (protein and substrate concentration: 0.4 mg/mL and 12 mmol/L, respectively, and reaction time: 10 min). There was a significant increase in cytochrome P450 monooxygenase activity in resistant strains, with a higher phenotypic frequency in the larval stage. Conclusions: modifications to the technique used for determination of enzymatic activity made it possible to distinguish between mosquitoes from susceptible and resistant strains in larval and adult stages, providing a new tool for the detection of metabolic resistance in Cuba