Construction and Single-Crystal Structures of N-Isoxazolin-5-ylcarbonylindole Derivatives, and Their Pesticidal Activities and Toxicology Study.

To explore natural product-based pesticide candidates, a series of indole derivatives containing the isoxazoline skeleton at the N-1 position were synthesized by 1,3-dipolar [2 + 3] cycloaddition reaction. Their structures were characterized by melting points (mp), infrared (IR) spectra, proton nuclear magnetic resonance spectra (1H NMR), carbon-13 nuclear magnetic resonance spectra (13C NMR), and high resolution mass spectrometry (HRMS). The single-crystal structures of five compounds were presented. Against Tetranychus cinnabarinus Boisduval, compound 3b showed greater than 3.8-fold acaricidal activity of indole and good control effects under glasshouse conditions. Against Aphis citricola Van der Goot, compounds 3b and 3q exhibited 48.3- and 36.8-fold aphicidal activity of indole and 6-methylindole, respectively. Particularly, compound 3b showed good bioactivities against T. cinnabarinus and A. citricola. Against Eriosoma lanigerum Hausmann, compound 3h and 3i showed 2.1 and 1.9 times higher aphicidal activity compared to indole. Furthermore, the construction of the epidermal cuticle layer of 3b-treated carmine spider mites was distinctly damaged, which ultimately led to their death.

Optimization of Osthole as a Pesticide Candidate: Synthesis, Crystal Structures, and Agrochemical Properties of Acrylate Derivatives of Isopropenyl 2,3-Dihydrobenzofurans.

For high-value-added application of osthole derivatives as a pesticide candidate in crop protection, by the use of osthole as a lead compound, a series of novel acrylate derivatives of isopropenyl 2,3-dihydrobenzofurans were prepared by the successive bromination, rearrangement, and esterization reactions. Three-dimensional structures of four compounds were determined by single-crystal X-ray diffraction. The possible mechanism for construction of this new isopropenyl 2,3-dihydrobenzofuran skeleton from the osthole was presented. Against Plutella xylostella Linnaeus, compound 32 (R = PhCH2CH2) displayed 3.5-fold potent insecticidal activity of osthole. Against Tetranychus cinnabarinus Boisduval, compound 40 (LC50: 0.165 mg/mL; R = (CH2)13CH3) showed 8.3-fold pronounced acaricidal activity of osthole (LC50: 1.367 mg/mL); notably, its control effect can be comparable to that of the commercial acaricide spirodiclofen. Additionally, the scanning electron microscopy imaging method demonstrated that compound 40 can destroy the stratum corneum of T. cinnabarinus. Compound 40 can be further explored as a lead acaricidal agent.

GLB-13 is associated with oxidative stress resistance in Caenorhabditis elegans.

Globins constitute a superfamily of heme-binding proteins that is widely present in many species. There are 33 putative globins in the genome of Caenorhabditis elegans, where glb-13 is a homolog of neuroglobin (Ngb) based on sequence analysis and specific expression in neurons. Here we examined whether glb-13 as well as Ngb is also associated with resistance to reactive oxygen species (ROS) induced by paraquat. Our results showed that the mRNA level of glb-13 was significantly upregulated after paraquat exposure. Expression of a green fluorescent protein (GFP) reporter gene directed by the glb-13 promoter was increased by paraquat exposure. The mutant C. elegans strain glb-13(tm2825) was sensitive to paraquat-induced oxidative stress. Overexpression of human Ngb (hNgb) in C. elegans neuronal cells can rescue the paraquat sensitive phenotype of the mutant strain. glb-13 mutation or hNgb overexpression did not affect the expression of antioxidant enzymes such as superoxide dismutase (SOD). To examine the ROS-scavenging capabilities of hNgb and glb-13, we further examined the level of ROS in glb-13 mutant and hNgb transgenic (hNgb-Tg) worms. There was no statistical difference in ROS levels in the untreated controls; however in paraquat-treated worms, the ROS level was statistically repressed in the hNgb-Tg relative to enhanced green fluorescent protein (EGFP)-Tg worms or wildtype animals. Additionally, the ROS level of glb-13 mutant was statistically higher than the wildtype animals. Furthermore, hNgb overexpression diminished the ROS level of glb-13 mutant. In conclusion, hNgb can rescue the ROS sensitive phenotype of the glb-13 mutant strain. The protein GLB-13 seems to have an hNgb-like function, suggesting the importance of the globin protein family in maintaining the homeostasis of ROS signals. Our data provided evidence for the first time that glb-13 is associated with the resistance against oxidative stress-induced toxicity.

[Development and application of the physical hypoxic models of C. elegans].

OBJECTIVE: To develop a suitable hypoxic injury model, which is important for revealing pathological molecular mechanism of hypoxia. METHODS: We focused on C. elegans by treatment with different hypoxic times and systematically observed mortality, movement, Cellular morphology and the related-protein expression of the animals. RESULTS: We demonstrated that hypoxia (0.2% partial pressure of oxygen) induced morphological cell defects, and then leading to death of C. elegans. The mortality of C. elegans increased along with hypoxic time, while hypoxia-inducible factor (HIF-1) was significantly up-regulated. In addition, by using neuron-specific transgenic wonns with green fluorescent protein--we observed the neuron-specffic injury caused by hypoxic stress. CONCLUSION: We successfully established an effective, convenient physical hypoxic model of C. elegans, which will facilitate the studies of hypoxic pathology and molecular mechanisms of hypoxic response in the future.

Autosomal interactions and mechanisms of pyrethroid resistance in house flies, Musca domestica.

Five BC1 lines and 16 house fly mass-cross homozygous lines were generated from crosses of the pyrethroid resistant ALHF (wild-type) and susceptible aabys (bearing recessive morphological markers on each of five autosomes) strains. Each of the resulting homozygous lines had different combinations of autosomes from the resistant ALHF strain. Levels of resistance to permethrin were measured for each line to determine the autosomal linkage, interaction and, possibly, regulation in pyrethroid resistance of house flies. Results indicated that factors on autosome 4 are not involved in the development of resistance in house flies, while factors on autosomes 1, 2, 3 and 5 play important roles in pyrethroid resistance. The sodium channel gene has been mapped on autosome 3 and multiple cytochrome P450 genes overexpressed in resistant ALHF house flies have been genetically mapped on autosome 5, suggesting that P450 mediated detoxification and sodium channel-mediated target site insensitivity located on autosomes 3 and 5, respectively, are major factors related to resistance development in house flies. However, neither the factors on autosome 3 or 5 alone, nor the factors from both autosomes 3 and 5 combined could confer high levels of resistance to pyrethroid. In addition, strong synergistic effects on resistance was obtained when autosomes 1 and 2 interact with autosome 3 and/or 5, suggesting that the trans factors on autosomes 1 and 2 may interact with factors on autosomes 3 and 5, therefore, playing regulatory roles in the development of sodium channel insensitivity- and P450 detoxification-mediated resistance.

Sodium sulfite is a potential hypoxia inducer that mimics hypoxic stress in Caenorhabditis elegans.

Physical and chemical hypoxia have been widely used in the study of hypoxic injury; however, both of these hypoxia models have their own limitations. Physical hypoxia is usually difficult to control and maintain. Chemical hypoxia, which is usually induced by chemical hypoxia-mimicking agents, such as CoCl(2), may result in heavy metal toxicity or impose security threats. To develop a more suitable hypoxia model, we focused on sodium sulfite (Na(2)SO(3)) and evaluated its ability to remove dissolved oxygen in aqueous solutions. Our results showed that sodium sulfite successfully induced hypoxic conditions. The degree of hypoxia and the guarantee period of the sodium sulfite solution could be easily controlled by the concentration of soluble sodium sulfite. In addition, we used sodium sulfite to create a hypoxia model in Caenorhabditis elegans. Similar to physical hypoxia, the sodium sulfite solutions induced hypoxia-related death in the worms and led to morphologic cell defects and C. elegans hypoxia inducible factor 1 stabilization. Taken together, our data show that sodium sulfite is a potential hypoxia inducer that mimics hypoxic stress in C. elegans.

Differential expression of genes in pyrethroid resistant and susceptible mosquitoes, Culex quinquefasciatus (S.).

The transcriptional regulation of gene expression is a primary means by which insects adapt to a changing environment. The evolution of insecticide resistance is conferred through mechanisms, typically requiring the interaction of multiple genes. Consequently, the characterization of gene regulation and interactions in resistance is fundamental for achieving an understanding of the complex processes responsible for resistance. cDNA macroarray technology offers a promising new approach for investigating the complicated processes responsible for resistance development by revealing the interrelations of all of the elements in a resistant system simultaneously. In the current study, we compared the gene expression profiles of resistant and susceptible Culex quinquefasciatus mosquitoes, using a combination of subtractive PCR hybridization and cDNA microarray technique. By screening of 1500 cDNA clones from a resistant-susceptible mosquito subtractive library, we identified a set of genes with up-regulated expression in insecticide resistant Culex mosquitoes through transcriptional profiling compared to those in susceptible mosquitoes. These genes are vital for cellular and molecular metabolism, signal transduction, vesicular and molecular transport, protein biosynthesis, ubiquitination, and neuronal survival, but most have not previously been implicated in insecticide resistance. Functional studies of resistance-associated up-regulated genes should shed new light on both the molecular basis of resistance and the regulatory pathways that lead to it.

Chlorpyrifos resistance in mosquito Culex quinquefasciatus.

Two mosquito strains of Culex quinquefasciatus Say, MAmCq and HAmCq, were collected from Mobile and Huntsville, AL, respectively, after the control of mosquitoes with insecticides proved difficult. A synergism study showed that resistance to chlorpyrifos in MAmCq and HAmCq was not suppressed by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF), suggesting that P450 monooxygenase- and hydrolase-mediated detoxication does not contribute to chlorpyrifos resistance in either strain. Diethyl maleate (DEM) did not cause any significant change in the level of chlorpyrifos toxicity to HAmCq. However, DEM enhanced toxicity of chlorpyrifos to MAmCq 2.5-fold, indicating that glutathione S-transferase (GST)-mediated detoxication may play a minor role in the resistance of MAmCq. An inhibition study of acetylcholinesterase (AChE) by chlorpyrifos showed that bimolecular rate constants (Ki) of chlorpyrifos for the inhibition of AChE in adults and larvae of the susceptible S-Lab strain were 2.2- and 1.9-fold higher, respectively, than in the HAmCq strain and 3.4- and 3.8-fold higher than in the MAmCq strain. The single mutation, G119S, resulting from a single nucleotide polymorphism (SNP), G to A, in ace-1 acetylcholinesterase gene was present in HAmCq and MAmCq mosquitoes. The frequency of the heterozygote for the G119S mutant allele in the HAmCq and MAmCq mosquito populations was 0.25 and 0.45, respectively, and no individuals in either of these mosquito strains were homozygous for the A allele. It thus seems likely that the presence of heterozygous individuals for the G119S allele in HAmCq and MAmCq populations may be a response to the insensitivity of AChE observed in these two mosquito strains.

Resistance in the mosquito, Culex quinquefasciatus, and possible mechanisms for resistance.

Two mosquito strains of Culex quinquefasciatus (Say), MAmCq(G0) and HAmCq(G0), were collected from Mobile and Huntsville, Alabama, respectively. MAmCq(G0) and HAmCq(G0) were further selected in the laboratory with permethrin for one and three generations, respectively. The levels of resistance to permethrin in MAmCq(G1) (after one-generation selection) and HAmCq(G3) (after three-generation selection) increased rapidly. Resistance to permethrin in MAmCq(G1) and HAmCq(G3) was partially suppressed by piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF) and diethyl maleate (DEM), inhibitors of cytochrome P450 monooxygenases, hydrolases and glutathione S-transferases (GST), respectively, suggesting these three enzyme families are important in conferring permethrin resistance in both strains. A substitution of leucine to phenylalanine (L to F) resulting from a single nucleotide polymorphism (SNP), termed the kdr mutation, in the para-homologous sodium channel gene has been reported as a very common mutation associated with pyrethroid resistance of insects. A 341-bp sodium channel gene fragment, where the kdr mutation resides, was generated by PCR from genomic DNAs of Cx. quinquefasciatus strains. We found that the kdr mutation was present in both permethrin-selected and unselected HAmCq and MAmCq mosquito populations, suggesting that the kdr mutation plays the role in permethrin resistance. There was no significant change in the frequency and heterozygosity of the A to T SNP for the kdr allele between permethrin-selected and unselected MAmCq and HAmCq mosquitoes, indicating that other mechanisms are involved in the evolution of resistance in mosquitoes selected by permethrin in the laboratory.

Insecticide resistance in Alabama and Florida mosquito strains of Aedes albopictus.

The susceptibility of four strains of Aedes albopictus (Skuse) to permethrin, deltamethrin, resmethrin, chlorpyrifos, malathion, propoxur, fipronil, imidacloprid, spinosad, and Bacillus thuringiensis variety israelensis (Bti) was determined. The HAmAal and MAmAal strains were collected in 2002 and 2003, respectively, from Huntsville and Mobile, AL, and the VBFmAal and SFmAal strains were collected in 1998 from Vero Beach and southern Florida, respectively. The HAmAal strain showed a 22-fold elevated level of resistance to deltamethrin compared with the susceptible Ikaken laboratory strain, whereas the VBFmAal strain showed a six-fold lower sensitivity to deltamethrin compared with Ikaken. However, comparison of resistance ratios for deltamethrin at LC50 and LC90 (21-fold) and the gradual slopes of dose-response curves indicated that the field population of this mosquito strain was heterogenous in response to deltamethrin. All four mosquito strains showed elevated levels of resistance to chlorpyrifos, with resistance ratios from 10 to 33. Nevertheless, except for the relatively low resistance to deltamethrin and chlorpyrifos, all mosquito strains showed a similar susceptibility or lower tolerance to the remaining insecticides tested compared with the susceptible Ikaken strain, even though some, such as permethrin, resmethrin, malathion, and Bti, have been used in the field for a long time, especially in Alabama. These results indicate that the development of resistance to insecticides in Ae. albopictus is slow and conventional insecticides, such as permethrin, resmethrin, malathion, and Bti, and relatively new insecticides, such as fipronil, imidacloprid, and spinosad, may all be valuable for the management of this important mosquito.

Insecticide resistance and cross-resistance in Alabama and Florida strains of Culex quinquefasciatus [correction].

Insecticide resistance and cross-resistance was determined for three strains of Culex quinquefasciatus Say in the southeastern United States. HAmCq and MAmCq strains were collected in 2002 from Huntsville and Mobile, AL. The VBFmCq strain was collected in 1998 from Vero Beach, FL. VBFmCq, HAmCq, and MAmCq larvae showed resistance to permethrin with resistance ratios of 13, 100, and 940, respectively, compared with the susceptible S-Lab strain. Levels of resistance in HAmCq and MAmCq larvae were 200- and 830-fold to resmethrin and 4- and 70-fold to malathion, respectively. VBFmCq, HAmCq, and MAmCq strains all demonstrated a great ability to develop tolerance and/or cross-resistance to different insecticides, including deltamethrin (50-, 100-, and 300-fold), chlorpyrifos (150-, 33-, and 720-fold), fipronil (10-, 5-, and 15-fold), and imidacloprid (7.5-, 5- and 10-fold, respectively). Comparison of resistance ratios for pyrethroids, organophosphates, and imidacloprid at LC50 and LC90 and gradual slopes of dose-response curves indicated that VBFmCq, HAmCq, and MAmCq were heterozygous in response to these insecticides. All three strains showed high levels of susceptibility to Bacillus thuringiensis variety israelensis (Bti) and spinosad, although these mosquitoes had been extensively exposed to Bti. Thus, we conclude that Bti and spinosad may be valuable for the management of Cx. quinquefasciatus, especially in situations where local strains are highly resistant to other insecticides.