Insecticide resistance in Chinese populations of the Culex pipiens complex through esterase overproduction

In most parts of China, mosquitoes have been subjected to organophosphate (OP) insecticide treatments since the mid-1960s, and resistance gene monitoring in the Culex pipiens complex (Diptera: Culicidae) started in only a few locations from the end of the 1980s. Many resistant alleles at the Ester locus have been found in field populations, including those commonly found around the world (Ester(B1) and Ester(2)), and those endemic to China (Ester(B6), Ester(B7), Ester(8), and Ester(9)). This situation is atypical, and may represent a complex situation for the evolution of insecticide resistance genes in China. To increase our understanding of the Chinese situation and our ability to manage resistance in the C. pipiens complex, a large study was performed. Twenty field populations were sampled from Beijing to Guangzhou. Bioassays with five insecticides (dichlorvos, parathion, chlorpyrifos, 2-sec-butylphenyl methyl carbamate, and propoxur) disclosed resistance levels variable according to the geographic origin, and up to 85-fold for dichlorvos. Six overproduced esterases were identified, including two that have not been previously described. Most of them were found in all samples, although at variable frequencies, suggesting variable selection or a transient situation, e.g., each one was recently restricted to a particular geographic area. The results are discussed in the context of recent alterations to insecticide campaigns, and of the evolution of resistance genes in Chinese C. pipiens populations.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

Xenobiotics removal from polluted water by a multifunctional constructed wetland

Removal efficiencies on xenobiotics from polluted water in a twin-shaped constructed wetland consisting of a vertical flow chamber with the crop plant Colocasia esculenta L. Schott and a reverse vertical flow one with Ischaemum aristatum var. glaucum Honda, were assessed by chemical analysis and bioassays. After a four-month period of application, removal efficiencies of the applied pesticides parathion and omethoate were 100%, with no detectable parathion and omethoate in the effluent. For the applied herbicides, the decontamination was less efficient with removal efficiencies of 36% and 0% for 4-chloro-2-methyl-phenoxyacetic acid and dicamba, respectively. As shown by toxicity assay with duckweed Lemna minor L., growth retardation may occur if the water treated for herbicide removal is used in irrigation of sensitive cultivars in agriculture or horticulture. In contrast to I. aristatum var. glaucum Honda, the crop C esculenta L. Schott has a high yield in biomass production as a valuable source of renewable energy. (C) 2002 Elsevier Science Ltd. All rights reserved.

KINETICS OF SIZE-FRACTIONATED AND DISSOLVED ALKALINE-PHOSPHATASE IN A FARM POND

Nutrient addition bioassays were conducted in 10 L carboys with water from a eutrophic farm pond. The four bioassay treatments each conducted in triplicate were control (no nutrients added), +N (160 mu mol L(-1) NH4Cl), +P (10 mu mol L(-1) KH2PO4), and N+P (160 mu mol L(-1) NH4Cl and 10 mu mol L(-1) KH2PO4). The size fractionated (0.2-0.8, 0.8-3, > 3 mu m) contents of the carboys were analyzed after 7 d for alkaline phosphatase activity (APA) and chlorophyll-a content. Chlorophyll data suggested P deficiency in ammonium and control mesocosms and no P deficiency with phosphate additions. Pond water also was collected in June, August, October, and March for measurement of APA. In water from the pond, the greatest V-max of APA usually was associated with microorganisms in the size classes between 0.8-3 mu m. In mesocosm experiments, the N+P treatment increased V-max of dissolved and particulate associated APA in the 0.2-0.8 mu m size range and in dissolved form. The V-max of APA in the largest size-fraction (> 3 mu m) increased markedly with P deficiency (+N treatment) and decreased in the P-enrichment treatment. The patterns of APA and chlorophyll associated with different size fractions often varied independently among different treatments and seasons and not always as a function of P deficiency, indicating the difficulty of attempting to normalize APA to phytoplankton biomass or chlorophyll. The Michaelis half saturation constant of APA in the pond water showed no strong trends with varied seasons or size fraction.

Calibration of a solid-state copper ion-selective electrode in cupric ion buffers containing chloride

It is shown that near-Nernstian calibration slopes can be obtained with a Cu1.8Se electrode in a range of cupric ion buffers in spite of a high chloride content. Best results are obtained with the ligands ethylenediamine, glycine and histidine. The onset of cupric ion toxicity towards marine organisms falls within the pCu calibration range obtained with glycine, and the Cu1.8Se electrode could, therefore, be useful for monitoring cupric ion activity in bioassays in sea-water media.

A General Route to Construct Diverse Multifunctional Fe3O4/Metal Hybrid Nanostructures

We have developed a simple, efficient, economical, and general approach to construct diverse multifunctional Fe3O4/metal hybrid nanostructures displaying magnetization using 3-aminopropyltrimethoxysilane (APTMS) as a linker. High-density Au nanoparticles (NPs) could be supported on the surface of superparamagnetic Fe3O4 spheres and used as seeds to construct Au shell-coated magnetic spheres displaying near-infrared (NIR) absorption., which may make them promising in biosensor and biomedicine applications. High-density flower-like Au/Pt hybrid NPs could be supported on the surface of Fe3O4 spheres to construct multifunctional hybrid spheres with high catalytic activity towards the electron-transfer reaction between potassium ferricyanide and sodium thiosulfate. High-density Ag or Au/Ag core/shell NPs could also be supported on the surface of Fe3O4 spheres and exhibited pronounced surface-enhanced Raman scattering (SERS), which may possibly be used as an optical probe with magnetic function for application in high-sensitivity bioassays.

7-Amino actinomycin D bound to single-stranded hairpin DNA enhanced by loop sequence-dependent luminescent Eu3+ and Tb3+ binding

Lanthanide Eu3+ and Tb3+ ions have been widely used in luminescent resonance energy transfer (LRET) for bioassays to study metal binding microenvironments. We report here that Eu3+ or Tb3+ can increase the binding affinity of antitumor antibiotic drug agent, 7-amino actinomycin D (7AACTD), binding to 5'-GT/TG-5' or 5'-GA/AG-5' mismatched stem region of the single-stranded hairpin DNA. Further studies indicate that the effect of Eu3+ or Tb3+ on 7AACTD binding is related to DNA loop sequence. Our results will provide new insights into how metal ions can enhance antitumor agents binding to their targets.

Comparison of two marine sponge-associated Penicillium strains DQ25 and SC10: differences in secondary metabolites and their bioactivities

Two strains of Penicillium, DQ25 and SC10, isolated from marine sponge Haliclona angulata (Bowerbank) and Hymeniacidon sp. respectively, were subjected to stationary cultivation under GYP medium for 30 days. The fermentation extracts were undergone bioactivities assays against human pathogens, phytopathogenic fungi and brine shrimp (Artemia salina). Bioassays-guided compounds isolation was performed by Silica gel columns and Sephadex LH-20 chromatography. Spectroscopic methods were used to structures elucidation of the compounds. Results showed the activities of secondary metabolites of strain DQ25 were generally stronger than that of strain SC10. Major bioactive molecules isolated from strain DQ25 were a 1,4-naphthoquinone derivative and an unidentified alkaloid. The two components were not isolated from the extract of strain SC10. ITS sequences revealed that these two species have the greatest similarity with Penicillium vinaceum and Penicillium granulatum respectively.

Toxicity of lead, cadmium and mercury on embryogenesis, survival, growth and metamorphosis of Meretrix meretrix larvae

In order to assess the toxicity of heavy metals on the early development of Meretrix meretrix, the effects of mercury (Hg), cadmium (Cd) and lead (Pb) on embryogenesis, survival, growth and metamorphosis of larvae were investigated. The EC50 for embryogenesis was 5.4 mu g l(-1) for Hg, 1014 mu g l(-1) for Cd and 297 mu g l(-1) for Pb, respectively. The 96 h LC50 for D-shaped larvae was 14.0 mu g l(-1) for Hg, 68 mu g l(-1) for Cd and 353 mu g l(-1) for Pb, respectively. Growth was significantly retarded at 18.5 mu g l(-1) (0.1 mu M) for Hg, 104 mu g l(-1) (1 mu M) for Cd and 197 mu g l(-1) (1 mu M) for Pb, respectively. The EC50 for metamorphosis, similar to 48 h LC50, was higher than 96 h LC50. Our results indicate that the early development of M. meretrix is highly sensitive to heavy metals and can be used as a test organism for ecotoxicology bioassays in temperate and subtropical regions.

Synthesis, structure and biological activities of 2-(4-methoxybenzoyl)-N-phenyl-2-(1,2,4-triazol-1-yl)thioacetamide

The title compound, 2-(methoxybenzoyl)-N-phenyt-2-(1,2,4-triazol-1-yl)thioacetamide was synthesized by several reactions from 4-methoxyacetophenone, triazole and phenyl isothiocyanate. The structure was identified by elemental analysis, H-1 NMR, MS and IR. The single crystal structure of 2-(methoxybenzoyl)-N-phenyl-2-(1,2,4-triazol-1-yl)thioacetamide was determined with X-ray diffraction. The preliminary bioassays show that the title compound exhibits weak antifungal activities and plant-growth regulatory activity.

Studies on synthesis, structure and biological activities of novel triazole compounds containing thioamide

Four novel triazole compounds containing thioamide group were designed and synthesized by using triazole, phenyl isothiocyanate and aryl ethyl ketone as raw material. Their structures were conformed by elemental analysis, H-1 NMR, IR and MS spectra. The crystal structure of 1-[1-anilinothiocarbonyl-1-(4-fluorobenzoyl)methyl]-1,2,4-trizole has been determined by X-ray diffraction analysis. The preliminary bioassays have shown that the title compounds exhibit certain antifungal activity.

Selección de un aislado del nucleopoliedrovirus de Helicoverpa armigera (HearNPV) para su uso como base de un bioinsecticida

Helicoverpa armigera (Lepidoptera; Noctuidae), conocida como el taladro del tomate, es una especie polífaga y de amplia distribución, responsable de grandes pérdidas económicas en más de 60 cultivos a lo largo de las regiones tropicales y subtropicales del mundo. Estas plagas se controlan mayoritariamente con plaguicidas químicos, aunque existe un gran interés por desarrollar otros agentes de control biológico. Entre estos, se encuentra el nucleopoliedrovirus de Helicoverpa armigera (HearNPV, Baculoviridae), que por sus características de seguridad y eficacia, sería útil para impulsar los programas de gestión integrada de plagas que se fomentan desde la Directiva 2009/128/CEE. El objetivo de este trabajo fue realizar una caracterización bioquímica y biológica de varios aislados de HearNPV : un aislado silvestre español (Badajoz) HearNPV-SP1, un aislado chino HearSNPV-G4, tres aislados sudafricanos (HearNPV-Whl, HearNPV-Kzn, HearNPV-Alb) y la materia activa de un producto comercial en uso en Europa (HearNPV-Hx). El análisis con las enzimas de restricción determinó que la enzima BglII generaba perfiles similares pero con fragmentos característicos en todos los casos a excepción de los aislados HearNPV-Kzn y HearNPVAlb, que no pudieron ser diferenciados entre sí con ninguna de las enzimas probadas. El análisis filogenético, basado en las secuencias parciales de los genes poliedrina (polh), lef-8 y lef-9, donde se incluyeron las secuencias correspondientes a 18 genomas mostró que el aislado HearNPV-Whl es filogenéticamente próximo a las cepas de origen ibérico, mientras que los aislados HearNPV-Hx y HearNPV-Alb comparten la misma rama que los aislados asiáticos y australiano. La caracterización insecticida de los aislados HearNPV-SP1, HearNPV-Hx y HearNPV-G4 reveló que la virulencia (TMM) del aislado HearNPV-SP1 (104 h) fue significativamente menor que la de los aislados HearNPV-G4 (109 h) y HearNPV-Hx (111 h). En este trabajo, se determinó que el tiempo de acción del HearNPV-SP1 es menor al de otros bioinsecticidas en uso en Europa, por lo que se confirma la posibilidad de mejorar los productos activos en uno de los aspectos más sensibles de cara a su comercialización como es su tiempo de actuación.

Mix it and fix it: functions of composite olfactory signals in ring-tailed lemurs.

Animals communicating via scent often deposit composite signals that incorporate odorants from multiple sources; however, the function of mixing chemical signals remains understudied. We tested both a 'multiple-messages' and a 'fixative' hypothesis of composite olfactory signalling, which, respectively, posit that mixing scents functions to increase information content or prolong signal longevity. Our subjects-adult, male ring-tailed lemurs (Lemur catta)-have a complex scent-marking repertoire, involving volatile antebrachial (A) secretions, deposited pure or after being mixed with a squalene-rich paste exuded from brachial (B) glands. Using behavioural bioassays, we examined recipient responses to odorants collected from conspecific strangers. We concurrently presented pure A, pure B and mixed A + B secretions, in fresh or decayed conditions. Lemurs preferentially responded to mixed over pure secretions, their interest increasing and shifting over time, from sniffing and countermarking fresh mixtures, to licking and countermarking decayed mixtures. Substituting synthetic squalene (S)-a well-known fixative-for B secretions did not replicate prior results: B secretions, which contain additional chemicals that probably encode salient information, were preferred over pure S. Whereas support for the 'multiple-messages' hypothesis underscores the unique contribution from each of an animal's various secretions, support for the 'fixative' hypothesis highlights the synergistic benefits of composite signals.

Multi-function oxidases are responsible for the synergistic interactions occurring between repellents and insecticides in mosquitoes

Background: With the spread of pyrethroid resistance in mosquitoes, the combination of an insecticide (carbamate or organophosphate) with a repellent (DEET) is considered as a promising alternative strategy for the treatment of mosquito nets and other relevant materials. The efficacy of these mixtures comes from the fact that they reproduce pyrethroid features and that positive interactions occur between insecticides and repellent. To better understand the mechanisms involved and assess the impact of detoxifying enzymes (oxidases and esterases) in these interactions, bioassays were carried out in the laboratory against the main dengue vector Aedes aegypti. Methods: Topical applications of DEET and propoxur (carbamate), used alone or as a mixture, were carried out on female mosquitoes, using inhibitors of the two main detoxification pathways in the insect. PBO, an inhibitor of multi-function oxidases, and DEF, an inhibitor of esterases, were applied one hour prior to the main treatment. Results: Results showed that synergism between DEET and propoxur disappeared in the presence of PBO but not with DEF. This suggests that oxidases, contrary to esterases, play a key role in the interactions occurring between DEET and cholinesterase inhibitors in mosquitoes. Conclusion: These findings are of great interest for the implementation of "combination nets" in the field. They support the need to combine insecticide with repellent to overcome insecticide resistance in mosquitoes of public health importance.

Recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season

Two media were developed which specifically allow the cultivation of Bacillus thuringiensis while it is in the vegetative as opposed to the spore form. Using these media B. thuringiensis was shown conclusively for the first time to exist in an active form on the phylloplane. The profile of its appearance in vegetative and spore form was followed over a growing season on clover (Trifolium hybridum) in the field. Three simultaneous and sudden rises and declines of both spore and vegetative cell densities were observed. The most common other spore-former on these leaves was Bacillus cereus but the fluctuations in appearance of these two very closely related species were not co-incident. Using specific PCR primers a considerable diversity of cry toxin gene types was found in isolates that had been recovered in vegetative form ([`]vegetative isolates') with the majority possessing multiple [delta]-endotoxin genes while some had only one of those tested. Bioassays against a lepidopteran insect of purified [delta]-endotoxins showed that they were no more potent than those from a laboratory-adapted strain. PCR primers for an internal region of the vip3A gene produced amplification in 70% of the vegetative isolates compared to 25% of the laboratory-adapted strains tested.