Inhibition kinetics on carboxylesterase and acetylcholinesterase of Liposcelis bostrychophila and Liposcelis entomophila (Psocop., Liposcelididae) of two insecticides

Abstract: The inhibition kinetics on carboxylesterase (CarE) and acetylcholinesterase (AChE) of Liposcelis bostrychophila and Liposcelis entomophila of paraoxon and carbosulfan were compared. The results showed that L. entomophila exhibited significantly greater specific activity of CarE than L. bostrychophila [0.045 vs. 0.012 μmol of α‐naphthyl acetate (α‐NA) hydrolysed/mg protein/min]. Moreover, L. entomophila showed higher affinity (i.e. lower K_m value) to the substrate α‐NA than L. bostrychophila (0.29 vs. 0.67 mm ). For AChE‐specific activity and affinity, no significant differences between the two species were observed. Based on the I₅₀ values, AChE of L. bostrychophila was more sensitive to paraoxon and carbosulfan than that of L. entomophila. According to inhibition kinetics, the results revealed that AChE of L. bostrychophila was 3.8‐fold more sensitive to inhibition by paraoxon than that of L. entomophila, but L. entomophila was 1.5‐fold more susceptible to carbosulfan.     

Insect nicotinic acetylcholine receptor gene families: from genetic model organism to vector,pest and beneficial species

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their mammalian counterparts. Thus, Drosophila melanogaster and Anopheles gambiae each possess 10 nAChR genes while Apis mellifera has 11. Although these are among the smallest nAChR gene families known, receptor diversity can be considerably increased by alternative splicing and mRNA A-to-I editing, thereby generating species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that act on particular pests while sparing beneficial insects. Electrophysiological studies on cultured Drosophila cholinergic neurons show partial agonist actions of the neonicotinoid imidacloprid and super-agonist actions of another neonicotinoid, clothianidin, on native nAChRs. Recombinant hybrid heteromeric nAChRs comprising Drosophila Dα2 and a vertebrate β2 subunit have been instructive in mimicking such actions of imidacloprid and clothianidin. Unitary conductance measurements on native nAChRs indicate that more frequent openings of the largest conductance state may offer an explanation for the superagonist actions of clothianidin.     

小菜蛾烟碱型乙酰胆碱受体α亚基cDNA的克隆、序列分析与不同发育阶段表达分析

烟碱型乙酰胆碱受体(nAChR)介导昆虫中枢神经系统中胆碱能突触兴奋性神经递质的快速传递,也是新烟碱类杀虫剂和多杀菌素的作用靶标。本研究利用RT-PCR和RACE技术,克隆了小菜蛾Plutella xylostella nAChR α亚基的一个新基因(Pxα8)的全长cDNA(GenBank登录号为EU914853)。Pxα8的cDNA序列全长1 744 bp,开放阅读框为1 602 bp,编码534个氨基酸,具有nAChR α亚基的典型特征,与其他昆虫nAChR α8亚基具有77%～96%的相似性,与果蝇nAChR β2亚基具有76%的相似性。Pxα8的开放阅读框存在单核苷酸多态性位点,导致多个位点氨基酸的替换。雌性4龄幼虫的多态性位点多于雄性4龄幼虫,而且雌、雄4龄幼虫的多态性位点均不相同。半定量RT-PCR研究结果表明,Pxα8 mRNA在成虫期表达量高于蛹期和4龄幼虫期。本研究结果为进一步研究小菜蛾nAChR 亚基的多样性和对多杀菌素的靶标抗性机制提供重要基础。     

Comparative Proteomics Studies of Insect Cuticle by Tandem Mass Spectrometry: Application of a Novel Proteomics Approach to the Pea Aphid Cuticular Proteins

The cuticle is a biological composite material consisting principally of N‐acetylglucosamine polymer embedded in cuticular proteins (CPs). CPs have been studied and characterized by mass spectrometry in several cuticular structures and in many arthropods. Such analyses were carried out by protein extraction using SDS followed by electrophoresis, allowing detection and identification of numerous CPs. To build a repertoire of cuticular structures from Bombyx mori, Apis mellifera and Anopheles gambiae the use of SDS and electrophoresis was avoided. Using the combination of hexafluoroisopropanol and of a surfactant compatible with MS, a high number of CPs was identified in An. gambiae wings, legs and antennae, and in the thoracic integument cuticle of Ap. mellifera pupae. The exoskeleton analysis of B. mori larvae allowed to identify 85 CPs from a single larva. Finally, the novel proteomics approach was tested on cuticles left behind after the molt from the fourth instar of Acyrthosiphon pisum. Analysis of these cast cuticles allowed to identify 100 Ac. pisum CPs as authentic cuticle constituents. These correspond to 68% of the total putative CPs previously annotated for this pea aphid. While this paper analyzes only the recovered cuticular proteins, peptides from many other proteins were also detected.     

Expressed sequence tag analysis of the diapausing queen of the bumblebee Bombus ignitus

We constructed a full‐length cDNA library from diapausing queens of the bumblebee Bombus ignitus. A total of 480 randomly selected clones was sequenced by single‐run 5′‐end sequencing. Of these, there were 437 high quality clones, 23 poor quality clones and 20 read‐fail clones. Each high quality clone sequence was searched against a public protein database. The most frequently found matching genes were ribosomal proteins (12.5%), p10 (3.58%), cytochrome P450 monooxygenase (3.13%) and sensory appendage protein (2.9%). Sequence similarity analysis between bumblebees and other insect species showed that 72 out of 437 (16.5%) bumblebee expressed sequence tags (EST) matched sequences of Apis mellifera, with matches to Drosophila melanogaster (6.6%), Caenorhabditis briggsae (6.2%), Lysiphlebus testaceipes (4.8%), Periplaneta americana (3.7%) and Anopheles gambiae (3.4%) following, suggesting that sequence similarity of bumblebee EST is closest to that of A. mellifera. Functional classification of EST based on Gene Ontology showed that most genes found by sequencing are associated with physiological processes in the bumblebee. The results of sequencing and analysis of our 437 cDNA demonstrated that high‐throughput EST sequencing and data analysis are powerful means for identifying novel genes and for expression profiling. Our bumblebee EST collection could be a useful platform for further studies of gene expression in diapausing bumblebees.     

Selective actions of Lynx proteins on different nicotinic acetylcholine receptors in the locust,Locusta migratoria manilensis

Nicotinic acetylcholine receptors (nAChRs) are major neurotransmitter receptors and targets of neonicotinoid insecticides in the insect nervous system. The full function of nAChRs is often dependent on associated proteins, such as chaperones, regulators and modulators. Here, three Lynx (Ly‐6/neurotoxin) proteins, Loc‐lynx1, Loc‐lynx2 and Loc‐lynx3, were identified in the locust, Locusta migratoria manilensis. Co‐expression with Lynx resulted in a dramatic increase in agonist‐evoked macroscopic currents on nAChRs Locα1/β2 and Locα2/β2 in Xenopus oocytes, but no changes in agonist sensitivity. Loc‐lynx1 and Loc‐lynx3 only modulated nAChRs Locα1/β2 while Loc‐lynx2 modulated Locα2/β2 specifically. Meanwhile, Loc‐lynx1 induced a more significant increase in currents evoked by imidacloprid and epibatidine than Loc‐lynx3, and the effects of Loc‐lynx1 on imidacloprid and epibatidine were significantly higher than those on acetylcholine. Among three lynx proteins, only Loc‐lynx1 significantly increased [³H]epibatidine binding on Locα1/β2. The results indicated that Loc‐lynx1 had different modulation patterns in nAChRs compared to Loc‐lynx2 and Loc‐lynx3. Taken together, these findings indicated that three Lynx proteins were nAChR modulators and had selective activities in different nAChRs. Lynx proteins might display their selectivities from three aspects: nAChR subtypes, various agonists and different modulation patterns.

     

Rediocides A and G as Potential Antitoxins Against Cobra Venom

Rediocides A and G, the principle components of Trigonostemon reidioides (Kurz ) Craib , which is known as Lotthanong in Thai, were investigated for a detoxification mechanism against Naja kaouthia venom by in silico, in vitro, and in vivo methods. Molecular dockings of α‐cobratoxin with rediocides A and G were performed, and the binding energies were found to be ?14.17 and ?14.14 kcal/mol, respectively. Rediocides bind to α‐cobratoxin at the same location as α‐cobratoxin binds to the nicotinic acetylcholine receptor (nAChR), i.e., at the Asp27, Phe29, Arg33, Gly34, Lys35, and Val37 residues. α‐Cobratoxin cannot bind to nAChR, because some of its binding sites are occupied with rediocides. From in vitro SDS‐PAGE, it was found that rediocides can diminish the bands of α‐cobratoxin. In the presence of acetylcholine‐binding protein (AChBP), it was apparent that rediocides can bind both α‐cobratoxin and AChBP. From an in vivo test, it was found that injection of rediocides at 0.5 mg/kg immediately after an α‐cobratoxin dose of three times LD₅₀ cannot prolong the survival time of mice. However, rediocide can prolong the survival time, if it is injected 30 min before the injection of α‐cobratoxin. The in vitro SDS‐PAGE and the in vivo results support the in silico detoxification mechanism of rediocides against cobra venom at a molecular level.     

Subcutaneous Fat Shows Higher Thyroid Hormone Receptor‐α1 Gene Expression Than Omental Fat

The aims of this work were to evaluate thyroid hormone receptor‐α (TRα), TRα1, and TRα2 mRNA gene expression and TRα1:TRα2 ratio, identified as candidate factors for explaining regional differences between human adipose tissue depots. TRα, TRα1, and TRα2 mRNA levels, and the gene expressions of arginine–serine‐rich, splicing factor 2 (SF2), heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), and Spot 14 (S14) were evaluated in 76 paired adipose tissue samples obtained from a population of 38 women who varied widely in terms of obesity and body fat distribution. Gene expression for these factors was also studied in stromal‐vascular cells (SVCs) and mature adipocytes (MAs) from eight paired fat depots. TRα gene and TRα1 mRNA expression were increased 1.46‐fold (P = 0.006) and 1.80‐fold (P < 0.0001), respectively, in subcutaneous (SC) vs. visceral fat. These differences in gene expression levels were most significant in the obese group, in which the TRα1:TRα2 ratio was 2.24‐fold (P < 0.0001) higher in SC vs. visceral fat. S14 gene expression was also increased by 2.42‐fold (P < 0.0001) and correlated significantly with TRα and TRα1 gene expression and with the TRα1:TRα2 ratio. In agreement with these findings, hnRNP A1:SF2 ratio was decreased by 1.39‐fold (P = 0.001). TRα and S14 levels were 2.1‐fold (P < 0.0001) and 112.4‐fold (P < 0.0001), respectively, higher in MAs than in SVCs from both fat depots. In summary, genes for TR‐α, their upstream regulators, and downstream effectors were differentially expressed in SC vs. omental (OM) adipose tissue. Our findings suggest that TRα1 could contribute to SC adipose tissue expandability in obese subjects.     

Regulation of c‐Ret,GFRα1, and GFRα2 in the substantia nigra Pars compacta in a rat model of Parkinson's disease

Glial cell line‐derived neurotrophic factor (GDNF) family members have been proposed as candidates for the treatment of Parkinson's disease because they protect nigral dopaminergic neurons against various types of insult. However, the efficiency of these factors depends on the availability of their receptors after damage. We evaluated the changes in the expression of c‐Ret, GFRα1, and GFRα2 in the substantia nigra pars compacta in a rat model of Parkinson's disease by in situ hybridization. Intrastriatal injection of 6‐hydroxydopamine (6‐OHDA) transiently increased c‐Ret and GFRα1 mRNA levels in the substantia nigra pars compacta at 1 day postlesion. At later time points, 3 and 6 days, the expression of c‐Ret and GFRα1 was downregulated. GFRα2 expression was differentially regulated, as it decreased only 6 days after 6‐OHDA injection. Triple‐labeling studies, using in situ hybridization for the GDNF family receptors and immunohistochemistry for neuronal or glial cell markers, showed that changes in the expression of c‐Ret, GFRα1, and GFRα2 in the substantia nigra pars compacta were localized to neurons. In conclusion, our results show that nigral neurons differentially regulate the expression of GDNF family receptors as a transient and compensatory response to 6‐OHDA lesion. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 343–351, 2002     

Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are members of the Cys‐loop ligand‐gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two α, and one each of β, γ, and δ subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular. The transmembrane domain consists of four α‐helical segments (M1–M4). Pioneering structural information was obtained using electronmicroscopy of Torpedo nAChR. The recently solved X‐ray structure of the first eukaryotic Cys‐loop receptor, a truncated (intracellular domain missing) glutamate‐gated chloride channel α (GluClα) showed the same overall architecture. However, a significant difference with regard to the vertical alignment between the channel‐lining segment M2 and segment M3 was observed. Here, we used functional studies utilizing disulfide trapping experiments in muscle nAChR to determine the spatial orientation between M2 and M3. Our results are in agreement with the vertical alignment as obtained when using the GluClα structure as a template to homology model muscle nAChR, however, they cannot be reconciled with the current Torpedo nAChR model. The vertical M2–M3 alignments as observed in X‐ray structures of prokaryotic Gloeobacter violaceus ligand‐gated ion channel and GluClα are in agreement. Our results further confirm that this alignment in Cys‐loop receptors is conserved between prokaryotes and eukaryotes.     

Chemotaxonomic Screening of Seed Oils from the Family Saxifragaceae and Comparison with Data on Seed Oils from Grossulariaceae Obtained from Literature

Seeds of 25 members of the family Saxifragaceae, 1 × Astilbe, 1 × Darmera, 1 × Leptarrhena, 1 × Tellima, 3 × Mitella, and 18 × Saxifraga were investigated regarding oil content, as well as composition and content of fatty acids and vitamin E active compounds. The results were compared with results obtained from literature for members of the genus Ribes belonging to the closely related family Grossulariaceae to find chemometric differences between the different genera and between members of the family Saxifragaceae and Grossulariaceae, respectively. Members of the family Saxifragaceae are dominated by high amounts of linoleic and α‐linolenic acid which together account for about 80% of the total fatty acids. While α‐linolenic acid is characteristic for members of the genus Saxifraga, in other genera, linoleic acid is predominant. In comparison to members of the family Saxifragaceae members of the family Grossulariaceae also contain γ‐linolenic acid and stearidonic acid which allow a significant differentiation between both families. By principle component analysis, members of both families were divided into three distinct groups, i) species with a high content of α‐linolenic acid (genus Saxifraga), ii) species with high amounts of γ‐linolenic acid and stearidonic acid (genus Ribes), and iii) species with higher amounts of linoleic acid (other members of the family Saxifragaceae). The composition of the vitamin E active compounds was characterized by a high content of γ‐tocopherol in most members of the family Saxifragaceae, but no chemotaxonomic relevance.     

Oplopane Sesquiterpenes from Ligularia knorringiana and Their Anti‐Complementary Activity

Three new oplopane sesquiterpenes, knorringianalarins D – F ( 1 – 3 , respectively), and five known analogues ( 4 – 8 , respectively), were isolated from the roots and rhizomes of Ligularia knorringiana. The structures of three new compounds were identified as 4‐acetoxy‐11α,12‐epoxy‐2β‐hydroxy‐3β‐(2‐methylbutyryloxy)‐9α‐(4‐methylsenecioyloxy)oplop‐10(14)‐ene ( 1 ), 3β,4‐diacetoxy‐9α‐(4‐acetoxy‐4‐methylsenecioyloxy)‐11α,12‐epoxy‐8α‐(2‐methylbutyryloxy)oplop‐10(14)‐ene ( 2 ), and (1R,5R,6R,7R,9R)‐5,9,11‐trihydroxy‐4,15‐dinoroplop‐10(14)‐en‐3‐one ( 3 ) based on spectroscopic methods including 1D‐ and 2D‐NMR, mass spectrometry, and CD spectroscopy techniques. All compounds were evaluated for their anti‐complementary activity on the classical pathway of the complement system in vitro. Among which, three oplopane sesquiterpenes ( 3 , 7 , and 8 ) exhibited better anti‐complementary effects with CH₅₀ values ranging from 0.33 to 0.89 mm , which are plausible candidates for developing potent anti‐complementary agents.     

Sgβ1, a novel locust (Schistocerca gregaria) non-α nicotinic acetylcholine receptor-like subunit with homology to the Drosophila melanogaster Dβ1 subunit

The cloning, sequencing and functional expression of Sgβ1, a novel locust (Schistocerca gregaria) non-α nicotinic acetylcholine receptor (nAChR) subunit is described. This subunit shows 80% identity with the Drosophila melanogaster Dβ1 and 92% identity with the Locusta migratoria β1, non-α subunits but only 38% identity to Sgα1 (also referred to as αL1), a previously cloned S. gregaria nAChR α-subunit. When expressed in Xenopus laevis oocytes, Sgβ1 does not respond to nicotine. Responses to nicotine are observed, however, in oocytes co-expressing Sgα1 and Sgβ1, but the pharmacology is indistinguishable from that of currents produced by expressing Sgα1 alone. We conclude that either Sgβ1 does not co-assemble with Sgα1, or that it is unable to contribute to the functional properties of the receptor, in the Xenopus oocyte expression system.     

Chemical Constituents and Insecticidal Activities of Ajania fruticulosa Essential Oil

The insecticidal activity and chemical constituents of the essential oil from Ajania fruticulosa were investigated. Twelve constituents representing 91.0% of the essential oil were identified, and the main constituents were 1,8‐cineole ( 41.40% ), (+)‐camphor ( 32.10% ), and myrtenol (8.15%). The essential oil exhibited contact toxicity against Tribolium castaneum and Liposcelis bostrychophila adults with LD₅₀ values of 105.67 μg/adult and 89.85 μg/cm², respectively. The essential oil also showed fumigant toxicity against two species of insect with LC₅₀ values of 11.52 and 0.65 mg/l, respectively. 1,8‐Cineole exhibited excellent fumigant toxicity (LC₅₀ = 5.47 mg/l) against T. castaneum. (+)‐Camphor showed obvious fumigant toxicity (LC₅₀ = 0.43 mg/l) against L. bostrychophila. Myrtenol showed contact toxicity (LD₅₀ = 29.40 μg/cm²) and fumigant toxicity (LC₅₀ = 0.50 mg/l) against L. bostrychophila. 1,8‐Cineole and (+)‐camphor showed strong insecticidal activity to some important insects, and they are main constituents of A. fruticulosa essential oil. The two compounds may be related to insecticidal activity of A. fruticulosa essential oil against T. castaneum and L. bostrychophila.     

Studies on the resting behaviour and host choice of Anopheles gambiae and An. arabiensis from Muleba,Tanzania

The relative efficacy of a mechanical (Prokopack) collection method vs. manual aspiration in the collection of resting mosquitoes was evaluated in northern Tanzania before and after an intervention using indoor residual spraying and longlasting insecticide‐treated nets. In smoke‐free houses mosquitoes were collected from the roof and walls, but in smoky houses mosquitoes were found predominantly on the walls. Anopheles gambiae (Diptera: Culicidae) constituted 97.7% of the 312 An. gambiae complex specimens identified before but only 19.3% of the 183 identified after the intervention. A single sampling with the Prokopack collected a third of the available insects. Anopheles gambiae completed its gonotrophic development indoors, whereas Anopheles arabiensis did so outdoors. In both species gonotrophic development took 2 days. Most unfed resting An. arabiensis collected outdoors were virgins, whereas the majority of engorged insects were parous (with well‐contracted sacs). Daily survival was estimated to be 80.0%. Only 9.4% of the engorged An. arabiensis collected outdoors and 47.1% of those collected indoors had fed on humans. Using the Prokopack sampler is more efficient than manual methods for the collection of resting mosquitoes. Malaria transmission may have been affected by a change in vector composition resulting from a change in feeding, rather than reduced survival. Monitoring the proportions of members of the An. gambiae complex may provide signals of an impending breakdown in control.     

Alternative splicing of the Anopheles gambiae nicotinic acetylcholine receptor, Agamαβ9, generates both alpha and beta subunits

Nicotinic acetylcholine receptors (nAChRs) are the members of the cys-loop ligand-gated ion channel superfamily and are formed by five subunits arranged around a central ion channel. Each subunit is encoded by a separate gene and is classified as either α or non-α depending on the presence or absence, respectively, of two adjacent cysteine residues which are important for acetylcholine binding. Here, we report for the first time a single nAChR gene encoding both α and non-α subunits. Specifically, alternative splicing of the Anopheles gambiae nAChR subunit, previously called Agamα9 and renamed here Agamαβ9, generates two variants, one possessing the two cysteines (denoted Agamαβ9^α) and the other lacking the cysteine doublet (Agamαβ9^β). Attempts to heterologously express functional nAChRs consisting of the Agamαβ9 splice variants in Xenopus laevis oocytes were unsuccessful. Our findings further characterise a potential target to control the malaria mosquito as well as provide insights into the diversification of nAChRs.     

A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis

High levels of resistance to spinosad, a macrocyclic lactone insecticide, have been reported previously in western flower thrips, Frankliniella occidentalis, an economically important insect pest of vegetables, fruit and ornamental crops. We have cloned the nicotinic acetylcholine receptor (nAChR) α6 subunit from F. occidentalis (Foα6) and compared the nucleotide sequence of Foα6 from susceptible and spinosad‐resistant insect populations (MLFOM and R1S respectively). A single nucleotide change has been identified in Foα6, resulting in the replacement of a glycine (G) residue in susceptible insects with a glutamic acid (E) in resistant insects. The resistance‐associated mutation (G275E) is predicted to lie at the top of the third α‐helical transmembrane domain of Foα6. Although there is no direct evidence identifying the location of the spinosad binding site, the analogous amino acid in the C. elegans glutamate‐gated chloride channel lies in close proximity (4.4 Å) to the known binding site of ivermectin, another macrocyclic lactone pesticide. The functional consequences of the resistance‐associated mutation have been examined in the human nAChR α7 subunit. Introduction of an analogous (A272E) mutation in α7 abolishes the modulatory effects of spinosad whilst having no significant effect upon activation by acetylcholine, consistent with spinosad having an allosteric mechanism of action.     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.