Requirement of Catalytic-Triad and Related Amino Acids for the Acyltransferase Activity of Tanacetum cinerariifolium GDSL Lipase/Esterase TcGLIP for Ester-Bond Formation in Pyrethrin Biosynthesis

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.     

Evaluation of two methods for quantifying passeriform lice

Two methods commonly used to quantify ectoparasites on live birds are visual examination and dust‐ruffling. Visual examination provides an estimate of ectoparasite abundance based on an observer's timed inspection of various body regions on a bird. Dust‐ruffling involves application of insecticidal powder to feathers that are then ruffled to dislodge ectoparasites onto a collection surface where they can then be counted. Despite the common use of these methods in the field, the proportion of actual ectoparasites they account for has only been tested with Rock Pigeons (Columba livia), a relatively large‐bodied species (238–302 g) with dense plumage. We tested the accuracy of the two methods using European Starlings (Sturnus vulgaris; ～75 g). We first quantified the number of lice (Brueelia nebulosa) on starlings using visual examination, followed immediately by dust‐ruffling. Birds were then euthanized and the proportion of lice accounted for by each method was compared to the total number of lice on each bird as determined with a body‐washing method. Visual examination and dust‐ruffling each accounted for a relatively small proportion of total lice (14% and 16%, respectively), but both were still significant predictors of abundance. The number of lice observed by visual examination accounted for 68% of the variation in total abundance. Similarly, the number of lice recovered by dust‐ruffling accounted for 72% of the variation in total abundance. Our results show that both methods can be used to reliably quantify the abundance of lice on European Starlings and other similar‐sized passerines.     

Engineering of tomato type VI glandular trichomes for trans-chrysanthemic acid biosynthesis,the acid moiety of natural pyrethrin insecticides

Glandular trichomes, known as metabolic cell factories, have been proposed as highly suitable for metabolically engineering the production of plant high-value specialized metabolites. Natural pyrethrins, found only in Dalmatian pyrethrum (Tanacetum cinerariifolium), are insecticides with low mammalian toxicity and short environmental persistence. Type I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of the three rethrolone-type alcohols. To test if glandular trichomes can be made to synthesize trans-chrysanthemic acid, we reconstructed its biosynthetic pathway in tomato type VI glandular trichomes, which produce large amounts of terpenoids that share the precursor dimethylallyl diphosphate (DMAPP) with this acid. This was achieved by coexpressing the trans-chrysanthemic acid pathway related genes including TcCDS encoding chrysanthemyl diphosphate synthase and the fusion gene of TcADH2 encoding the alcohol dehydrogenase 2 linked with TcALDH1 encoding the aldehyde dehydrogenase 1 under the control of a newly identified type VI glandular trichome-specific metallocarboxypeptidase inhibitor promoter. Whole tomato leaves harboring type VI glandular trichomes expressing all three aformentioned genes had a concentration of total trans-chrysanthemic acid that was about 1.5-fold higher (by mole number) than the levels of β-phellandrene, the dominant monoterpene present in non-transgenic leaves, while the levels of β-phellandrene and the representative sesquiterpene β-caryophyllene in transgenic leaves were reduced by 96% and 81%, respectively. These results suggest that the tomato type VI glandular trichome is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.     

Identification and characterization of a GDSL lipase-like protein that catalyzes the ester-forming reaction for pyrethrin biosynthesis in Tanacetum cinerariifolium- a new target for plant protection

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.     

Aerosol distribution and efficacy in a commercial food warehouse

A series of field trials were conducted in a commercial food storage facility to evaluate exposure of stored‐product insects to aerosol formulations of synergized pyrethrins and the insect growth regulator methoprene. When adults of Tribolium castaneum (Herbst), the red flour beetle, and Tribolium confusum (Jacqueline DuVal), the confused flour beetle were exposed with and without a food source to synergized pyrethrin aerosol, there was no difference in adult mortality with respect to availability of food at either 7 or 14 days after exposure (P≥ 0.05). However, mortality was lower in T. confusum (40.4% and 79.3% with flour at 7 and 14 days, 38.9% and 84.8% without flour at 7 and 14 days) compared to T. castaneum (96.5% and 99.8% with flour at 7 and 14 days, 91.0% and 98.7% without flour at 7 and 14 days). Few late‐stage larvae and pupae of either species exposed to the pyrethrin aerosol emerged as adults. In tests with methoprene aerosol, adult emergence of exposed 3‐and 4‐week‐old larvae of T. confusum was less than 2%. Only 0.3% of 4‐week‐old larvae of T. castaneum exposed in open and obstructed areas emerged as adults. Emergence of adults from eggs of Plodia interpunctella (Hübner), the Indianmeal moth, embedded in culture media and exposed to the methoprene aerosol was 13.2%± 3.5%. Results show that the aerosols evaluated in our study could give effective control of some of the major stored‐product insect pests in commercial food storage facilities, and may offer an alternative to fumigation.     

缬氨酸杀虫菊酯的合成

缬氨酸杀虫菊酯是一类很有发展前景的杀虫杀螨剂,它的菊酸部分为Ｒ－构型,菊醇部分为Ｓ－构型,其合成是由相应的缬氨酸或者它的活性衍生物与相应的醇经酯化而制得。具体过程是Ｄ－缬氨酸经６ＮＨＢｒ／ＮａＮＯ２溴化制成（Ｒ）－α－溴代异戊酸,再与对三氟甲基苯胺反应合成（Ｒ）－Ｎ－［（４－三氟甲基）苯基］α－缬氨酸,后被Ｎ－氯代琥珀酰亚胺氯化制成（Ｒ）－Ｎ－［２－氯－４－（三氟甲基）苯基］α－缬氨酸,最后与（Ｓ）－α－氰基－３－苯氧苯基甲醇用ＤＣＣ脱水酯化合成缬氨酸杀虫菊酯Ｉ（Ｒ１＝Ｆ３Ｃ,Ｒ２＝Ｈ,ＣＬ,Ｒ３＝Ｈ）。还给出了两种菊酯２×４种异构体杀虫（烟草夜蛾和家蝇）活性数据。     

Effects of autumn kaolin and pyrethrin treatments on the spring population of Dysaphis plantaginea in apple orchards

The impact of treatments with the pyrethrin insecticide Pyrethrum FS and the repellent kaolin product Surround^® WP to control the autumn forms of Dysaphis plantaginea Pass. (Hom., Aphididae), a major insect pest of apple, was assessed in 1‐year field experiments in Switzerland. Single and multiple applications of pyrethrin and kaolin were tested at different dates after apple harvesting in autumn 2002 when sexuales of the rosy apple aphid were present. Repeated applications of Surround^® WP in autumn significantly reduced the number of females in autumn and, consequently, the number of hatched fundatrices in spring. Single kaolin treatments were less effective. Unexpectedly, neither single nor multiple applications of the contact insecticide Pyrethrum FS had a knock‐down effect on females in autumn. However, pyrethrin significantly reduced the number of hatched fundatrices in spring. Neither pesticide completely controlled the rosy apple aphid but with a more detailed analysis of factors influencing the efficacy of autumn treatments a new approach to control this serious pest could be achieved.     

Light effects on pyrethrum shoot cultures

Light consistently increased the pyrethrin content of pyrethrum shoot organ cultures. Under the experimental conditions used, the pyrethrin increase was not dependent upon a constant light supply.