RESUMEN
We have recently discovered that a GDSL lipase/esterase (TcGLIP) in Tanacetum cinerariifolium catalyzed acyltransferase activity to form an ester bond in the natural insecticide, pyrethrin. TcGLIP contained Ser40 in Block I, Gly64 in Block II, Asn168 in Block III and Asp318 and His321 in Block V, suggesting underlying hydrolase activity, although little is known about their role in acyltransferase activity. We expressed TcGLIP here in Esherichia coli as a fusion with maltose-binding protein (MBP), part of the fusion being cleaved with a protease to obtain MBP-free TcGLIP. A kinetic analysis revealed that the MBP moiety scarcely influenced the kinetic parameters. The effects on acyltransferase activity of mutations of Gly64, Asn168, Asp318 and His321 were investigated by using MBP-fused TcGLIP. Mutations of these amino acids markedly reduced the acyltransferase activity, suggesting their critical role in the production of pyrethrins.
Asunto(s)
Aciltransferasas/metabolismo , Biocatálisis , Chrysanthemum cinerariifolium/enzimología , Lipasa/química , Lipasa/metabolismo , Piretrinas/metabolismo , Secuencia de Aminoácidos , Esterificación , Lipasa/genética , Proteínas de Unión a Maltosa/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium.
Asunto(s)
Aciltransferasas/metabolismo , Chrysanthemum cinerariifolium/enzimología , Insecticidas/metabolismo , Lipasa/metabolismo , Piretrinas/metabolismo , Aciltransferasas/genética , Aciltransferasas/aislamiento & purificación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Chrysanthemum cinerariifolium/química , Chrysanthemum cinerariifolium/citología , Chrysanthemum cinerariifolium/genética , Esterasas/genética , Esterasas/aislamiento & purificación , Esterasas/metabolismo , Ésteres , Flores/enzimología , Flores/genética , Expresión Génica/genética , Insecticidas/análisis , Insecticidas/química , Cinética , Lipasa/genética , Lipasa/aislamiento & purificación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Tallos de la Planta/enzimología , Tallos de la Planta/genética , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Piretrinas/análisis , Piretrinas/química , Proteínas Recombinantes de Fusión , Plantones/enzimología , Plantones/genética , Especificidad por SustratoRESUMEN
After the chemical structure of "natural pyrethrins," the insecticidal ingredient of pyrethrum flowers, was elucidated, useful synthetic pyrethroids provided with various characteristics have been developed by organic chemists throughout the world, leading to the advancement of pyrethroid chemistry. Even in pyrethroids with high selective toxicity, a chemical design placing too much importance on efficacy improvements may invite loss of the safety margin. It is strongly hoped that the development of household pyrethroids and their preparations for use in living environments around humans and pets will be achieved in the future by retaining the characteristics of natural pyrethrins.
Asunto(s)
Insecticidas/farmacología , Piretrinas/química , Piretrinas/farmacología , Chrysanthemum cinerariifolium/crecimiento & desarrollo , Resistencia a los Insecticidas , Relación Estructura-ActividadRESUMEN
Plants emit specific blends of volatile organic compounds (VOCs) in response to mechanical wounding. Such induced VOCs have been shown to mediate in plant and interplant communication, yet little is known about the time- and dose-response relationships in VOC-mediated communications. Here, we employed young seedlings of Chrysanthemum cinerariaefolium to examine the effects of volatiles emitted by artificially damaged seedlings on the biosynthesis of the natural insecticides pyrethrins in intact conspecific plants. Wounded leaves emitted (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate and (E)-ß-farnesene as dominant wound-induced VOCs. Exposing intact seedlings to a mixture of these VOCs at concentrations mimicking those emitted from wounded seedlings, as well as placing the intact seedlings next to the wounded seedlings, resulted in enhanced pyrethrin contents in the intact seedlings. Thus we quantified mRNA transcripts of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), chrysanthemyl diphosphate synthase (CPPase), 13-lipoxygenase (13-LOX) and allene oxide synthase (AOS) genes in intact seedlings exposed to the VOC mixture to show that DXS and 13-LOX gene expression reached a maximum at 3 h, whereas CPPase and AOS reached it at 6 h. Interestingly, both increasing and decreasing the VOC mixture concentrations from those observed on injury reduced the expression of DXS, CPPase and AOS genes to the control level. Also, separating the VOC mixture into individual components eliminated the ability to enhance the expression of all the biosynthetic genes examined. This is the first study showing that the wound-induced VOCs function as a blend to control the biosynthesis of second metabolites at specific concentrations.
Asunto(s)
Chrysanthemum/efectos de los fármacos , Chrysanthemum/metabolismo , Piretrinas/metabolismo , Compuestos Orgánicos Volátiles/farmacología , Vías Biosintéticas/efectos de los fármacos , Vías Biosintéticas/genética , Chrysanthemum/genética , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Piretrinas/química , Plantones/efectos de los fármacos , Plantones/metabolismo , Factores de TiempoRESUMEN
With the advancement from natural pyrethrins to synthetic pyrethroids, their applications have expanded from household insecticides for indoor use against sanitary pests to outdoor use for agriculture, forestry, animal health, termite control, and many other pest situations. However, high fish toxicity and development of pyrethroid resistance in some pests have been cited as common shortcomings of pyrethroids. To overcome these pyrethroid problems such as high fish toxicity, Katsuda and fellow scientists invented silafluofen by introducing a silicone atom into the pyrethroidal chemical structure in 1984. In addition to the high insecticidal activity and low mammalian toxicity, this compound features low fish toxicity, chemical stability under sunlight, in the soil and under alkaline environments. These features make silafluofen unique among pyrethroids. In Japan, silafluofen has been used as an agricultural insecticide for 15 years since 1995 for various plants, especially useful for paddy rice protection because of its low fish toxicity. Over the last 20 years, silafluofen-based termiticides including emulsifiable concentrate (EC) and oil formulations have been widely used in Japan for soil treatment and timber treatments. Additional silafluofen product lines include anti-termitic plastic sheets which are laid under buildings. In this paper, literature on the development of silafluofen and its use in Japan are reviewed. On the other hand, in Thailand, we proceeded with development works of silafluofen-based termiticides from 2005 by starting laboratory efficacy tests and field efficacy tests in Phuket. Both laboratory and field tests showed good efficacy as a soil termiticide, suggesting that the material will perform well for commercial use in high biological hazard environments such as Thailand and can be used in environments close to water where fish toxicity might be a concern with other pyrethroids.
RESUMEN
We collected 11 groups of Aedes aegypti mosquitoes from various locations of Thailand. After rearing in the laboratory, the colonies were tested for KT50 values to dl,d-T80-allethrin 0.5% mosquito coils in a 25 m3 room semi-field test and KD50 and LD50 values to dl,d-T80-allethrin by a topical application method. Two groups of mosquitoes were susceptible to allethrin similar to a SS (known allethrin sensitive) group, and other 9 groups showed various levels of lower susceptibility to allethrin; of these 6 had susceptibilities similar to a BS (known allethrin resistant) group with extremely low susceptibility, while the remaining 3 groups had susceptibilities to allethrin between the SS and BS groups. The KD50 values with the topical application were found to correlate highly with the KT50 values in the 25 m3 room semi-field test, providing a useful test method for insect susceptibility evaluation. The allethrin mosquito coils, even at higher concentrations, had no activity against the 6 decreased susceptibility groups, similar to the BS group. With the 25 m3 room semi-field test, mosquito coils with d,d-T-prallethrin at concentrations of 0.1 to 0.15% plus a synergist and those with methoxymethyl-tetrafluorobenzyl tetramethylcyclopropane carboxylate (K-3050) at a concentration of 0.1% plus a synergist were found to be highly effective against these mosquito groups. These two pyrethroids had smaller KD50 and LD50 values for topical application, and were more effective than dl,d-T80-allethrin, having the potential to control Ae. aegypti mosquitoes with low allethrin susceptibility.
Asunto(s)
Aedes , Insectos Vectores , Insecticidas , Control de Mosquitos/métodos , Piretrinas , Aletrinas , Animales , TailandiaRESUMEN
The bioefficacy of mosquito coils containing several pyrethroids were tested in a 25 m3 room against Culex pipiens quinquefasciatus, Aedes aegypti and Anopheles dirus. The test results were compared with tests against Culex pipiens pallens in Japan. Based on the KT50 values (the 50% knockdown time) of mosquito coils containing dl, d-T80-allethrin, d, d-T-prallethrin and methoxymethyl-tetrafluorobenzyl tetramethyl-cyclopropanecarboxylate (K-3050) at doses of 0.05-0.5% (w/w) with or without a synergist, the pyrethroid susceptibility of the four mosquito species was as follows: Cx. p. quinquefasciatus was several times more tolerant to pyrethroids than Cx. p. pallens, Ae. aegypti was a further several times more tolerant than Cx. p. quinquefasciatus, and An. dirus was more susceptible than Cx. p. pallens (KT50 value: about half of Cx. p. pallens). The order of their susceptibilities is common for pyrethroids. Mosquito coils containing d, d-T-prallethrin and K-3050 at doses of 0.05-0.2% (w/w) and N-(2-ethylhexyl)bicycle-[2,2,1]-hept-5-ene-2,3-dicarboxyimide as a synergist at a ratio of 2 times the active ingredient were highly effective against Ae. aegypti, the most important mosquito vector for dengue fever.
Asunto(s)
Enfermedades Transmisibles/parasitología , Culicidae/efectos de los fármacos , Control de Mosquitos/métodos , Piretrinas/farmacología , Clima Tropical , Animales , Humanos , Japón , TailandiaRESUMEN
Four strains (SS, BS, A and B) of Aedes aegypti collected from different sites in Bangkok and at different times were examined for their pyrethroid susceptibility. Mosquito coils containing dl, d-T80-allethrin, d, d-T-prallethrin and methoxymethyl-tetrafluorobenzyl tetramethyl-cyclopropanecarboxylate (K-3050) with or without a synergist were tested by the 25 m3 semi-field test method. One strain (SS) was the most susceptible with KT50 values of about < 30 minutes for all mosquito coils, while the other three strains (BS, A and B) were found to be around 10 to 20 times more tolerant to pyrethroids than the SS strain. A similar tendency for the pyrethroid susceptibility of the four strains was obtained with tests by topical application method. In field efficacy tests, mosquito coils with d, d-T-prallethrin 0.20% plus N-(2-ethylhexyl)bicycle-[2,2,1]- hept-5- ene-2,3-dicarboxyimide as a synergist exhibited a repellent effect of about 85%, while those with K-3050 0.10% plus the synergist exhibited a greater repellent effect of about 90%. In contrast, the repellent effect of commercial dl, d-T80-allethrin 0.20% coils was as low as about 50%. The d, d-T-prallethrin and K-3050 coils with the synergist were confirmed to be highly effective in repelling Ae. aegypti.
Asunto(s)
Aedes/efectos de los fármacos , Repelentes de Insectos/farmacología , Insectos Vectores/efectos de los fármacos , Piretrinas/farmacología , Aletrinas/farmacología , Animales , Resistencia a Medicamentos , Sinergistas de Plaguicidas/farmacología , Piretrinas/química , TailandiaRESUMEN
The biosynthetic pathway to natural pyrethrins in Chrysanthemum cinerariaefolium seedlings was studied using [1-13C]d-glucose as a precursor, with pyrethrin I isolated using HPLC from a leaf extract. The 13C NMR spectrum of pyrethrin I from the precursor-administered seedlings indicated that the acid moiety was biosynthesized from d-glucose via 2-C-methyl-d-erythritol 4-phosphate, whereas the alcohol moiety was possibly biosynthesized from linolenic acid.