Consumption of a toxic food by caterpillars increases with dietary exposure: support for a role of induced detoxification enzymes

We examined whether there is a link between feeding behavior and detoxification enzyme activity, which increases over time in proportion to the ingested dose of toxic allelochemicals. We hypothesized that, for insects consuming a toxic but nondeterrent food, consumption would initially decline but then increase after continuous exposure in conjunction with the induction of detoxification enzyme activity. We tested this hypothesis by observing the feeding behavior of fall armyworms (Spodoptera frugiperda) when offered diets containing indole 3-carbinol (I3C), a well-known inducer of detoxification enzymes in this species. The I3C concentrations used were toxic but nondeterrent, thereby avoiding any confounding influence of preingestive responses. The feeding pattern of caterpillars previously unexposed to dietary I3C (i.e. with basal detoxification enzyme activity) was substantially altered when offered I3C-containing diets. Compared with control caterpillars fed I3C-free diets, they exhibited fewer feeding bouts and had a greater proportion of long (> 60 min) non-feeding pauses between bouts. In contrast, the feeding pattern of caterpillars pre-exposed to an I3C diet (i.e. with induced detoxification enzyme activity) did not differ from that of control caterpillars. We conclude that there is a relationship between feeding behavior and detoxification activity in these caterpillars, and propose two postingestive mechanisms that could link these processes.     

飞蝗解毒酶系活力测定方法

飞蝗Locusta migratoria是重要的农业害虫,代谢抗性是飞蝗主要的农药抗性机制之一。与代谢抗性相关的解毒酶系主要有:非专一性酯酶系(Non-specficesterases,ESTs)、谷胱甘肽S-转移酶系(Glutathione S-transferases,GSTs)和细胞色素P450单加氧酶系(Cytochrome P450 monooxygenases,P450s),解毒酶系活力的测定是研究飞蝗农药代谢机制的重要途径。本文详细介绍了飞蝗解毒酶系的测定方法,为蝗虫及其他昆虫解毒酶系的测定提供参考。     

A gene from the fungal plant pathogen Nectria haematococca that encodes the phytoalexin-detoxifying enzyme pisatin demethylase defines a new cytochrome P450 family

The gene PDAT9 from the fungus Nectria haematococca encodes pisatin demethylase, an enzyme that detoxifies the phytoalexin pisatin, an antimicrobial compound produced by pea in response to infection by this plant pathogen. PDAT9 was found to contain an open reading frame (ORF) encoding 515 amino acids and four introns of 52–58 nucleotides each within its coding region. The amino acid sequence F-G-A-G-S-R-S-C-I-G, indicative of the fifth ligand binding site present in all cytochrome P454s, occurs as residues 446 to 455, confirming that PDAT9 is a cytochrome P450. The deduced amino acid sequence is distinct from all other reported cytochrome P-450s, and PDAT9 has been assigned to a new cytochrome P450 family, CYP57. A 1.3 kb SacI fragment of the PDAT9 ORF that lacked the fifth ligand binding site, hybridized to unique DNA fragments in N. haematococca isolates known to possess PDA genes that encode different whole cell phenotypes for pisatin demethylating activity. These genes were also tentatively identified as cytochrome P450s by the hybridization of the same fragments to separate subclones of PDAT9, one of which contained the fifth ligand sequence. That probe also hybridized to DNA other than that attributed to pisatin demethylase genes; these other DNAs are presumed to represent other cytochrome P450s.     

柑橘全爪螨田间种群敏感性测定及三种主要解毒酶活性比较

为明确重庆地区柑橘全爪螨Panonychus citri(McGregor)对常用杀螨剂的抗性水平,本研究采用阿维菌素、哒螨灵、三唑锡、螺螨酯4种不同类型杀螨剂对柑橘全爪螨重庆北碚种群、璧山种群、武隆种群和忠县种群进行了田间敏感性测定。结果表明,柑橘全爪螨4个种群对三唑锡表现最不敏感,致死中浓度LC50在209.9～370.9mg/L之间。璧山种群对阿维菌素敏感性最高,武隆种群和忠县种群对阿维菌素的相对抗性分别达12倍和11倍。哒螨灵监测结果表明,北碚种群的抗性水平显著高于其他3个种群。而北碚种群对螺螨酯的LC50仅为1.2mg/L,显著低于其他种群。柑橘全爪螨4个种群解毒酶活性研究发现,解毒酶活性的高低与不同种群抗性水平之间并没有明显相关性,这可能同各地区施药背景不同、综合防治措施不同、各杀螨剂作用机理不同、不同种群体内代谢抗性及靶标抗性水平差异有关。     

6种植物次生物质对斜纹夜蛾解毒酶活性的影响

草食性昆虫取食植物时遇到宿主植物中大量次生物质的化学防御,研究昆虫适应植物毒素的反防御策略具有重要的科学意义。分别添加0.01%肉桂酸、0.01%水杨酸、0.01%花椒毒素、0.02%槲皮素、0.05%黄酮和0.1%香豆素等6种植物次生物质的人工饲料饲养斜纹夜蛾(Spodoptera litura)五龄幼虫48 h后,测定斜纹夜蛾幼虫中肠和脂肪体中谷胱甘肽S-转移酶(GSTs)、羧酸酯酶(CarE)、P450的酶含量及头部乙酰胆碱酯酶(AChE)的活性,利用半定量RT-PCR检测中肠和脂肪体中CYP4M14和CYP4S9的基因表达水平。结果表明:取食肉桂酸和香豆素后,斜纹夜蛾中肠中CarE的酶活性分别提高了1.67和1.37倍,取食6种次生物质均能显著提高斜纹夜蛾脂肪体中GSTs酶活性。取食肉桂酸和香豆素48 h后,脂肪体中P450酶含量比对照增加2.93和14.50倍。取食肉桂酸、花椒毒素、槲皮素和香豆素后,斜纹夜蛾头部AchE酶活性与对照相比提高了1.53、1.80、2.36和1.56倍。6种次生物质均可诱导脂肪体中CYP4M14基因表达,槲皮素、肉桂酸和香豆素强烈诱导CYP4S9在脂肪体中表达。表明,斜纹夜蛾具有利用植物次生物质诱导其解毒酶的能力,进而提高其对毒素的抗性。     

亚致死浓度氰氟虫腙对小菜蛾三种解毒酶及P450 mRNA表达量的影响

【目的】为探讨亚致死浓度氰氟虫腙对小菜蛾Plutella xylostella(L.)3种解毒酶和细胞色素P450基因表达量的影响。【方法】采用叶片浸渍法,测定用氰氟虫腙LC10和LC25处理72 h后小菜蛾羧酸酯酶、谷胱甘肽S-转移酶和多功能氧化酶3种解毒酶的活性;应用实时荧光定量PCR(Real-time PCR)方法测定了其在24、48和72 h时对P450基因(Px CYP4、Px CYP6和Px CYP9家族)表达的影响。【结果】结果表明,氰氟虫腙LC10和LC25处理对羧酸酯酶活性影响不显著;对谷胱甘肽S-转移酶活性存在显著抑制作用(P<0.05);LC25处理后多功能氧化酶活性显著增加(P<0.05),为对照的1.39倍。同时,LC25氰氟虫腙处理组小菜蛾P450基因Px CYP4M19、Px CYP6BF1V2、Px CYP6CN1和Px CYP6CV2 m RNA的相对表达量分别为对照组的2.33~8.5倍、1.32~3.48倍、1.18~2.53倍、2.02~7.22倍。【结论】结果显示,氰氟虫腙可能通过诱导这4种P450基因m RNA的上调表达而增强了小菜蛾多功能氧化酶酶活性。     

Drug metabolizing enzyme induction by benzoquinolines,acridine, and quinacrine; Tricyclic aromatic molecules containing a single heterocyclic nitrogen

Rats were treated with nitrogen-containing phenanthrene (3,4-, 5,6-, or 7,8-benzoquinoline) or anthracene (acridine or quinacrine) derivatives at a dose of 75 mg/kg, daily for 3 days. The hepatic drug metabolizing enzyme response ranged from no induction (quinacrine) through low (5,6-benzoquinoline), intermediate (acridine), and high (3,4-benzoquinoline) magnitude increases of only phase II enzymes, to induction of both phase I and phase II enzymes (7,8-benzoquinoline). The phase I enzyme response of 7,8-benzoquinoline was an induction of CYP1A. All three benzoquinolines, but neither anthracene derivative, elevated NAD(P)H quinone oxidoreductase activity. A similar pattern but of lesser magnitude was seen with glutathione S-transferase activity. 3,4-Benzoquinoline was the only agent to significantly increase microsomal epoxide hydrolase activity (2.3-fold). Both 3,4- and 7,8-benzoquinoline increased UDP-glucuronosyltransferase activity toward 4-nitrophenol (40% and 70%, respectively), but only the 3,4-isomer increased activity toward morphine (75%), diclofenac (75%), and testosterone (23%), and only the 7,8-isomer increased activity toward chloramphenicol (105%). 3,4-Benzoquinoline elevated the hepatic mRNA concentration of UGT2B1 but not UGT1*6. Acridine treatment increased UDP-glucuronosyltransferase activity toward morphine (47%), 1-naphthol (28%), testosterone (19%), and estrone (19%). Quinacrine failed to elevate any UDP-glucuronosyltransferase activity and depressed activities toward testosterone and estrone by 20%. This study shows that some tricyclic aromatic compounds containing a single heterocyclic nitrogen atom have the potential for use as chemoprotective agents based upon their ability to selectively induce only phase II enzymes. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 297–303, 1997.     

Food consumption, utilization and detoxification enzyme activity of larvae of three polyphagous noctuid moth species when fed the botanical insecticide rotenone

We assessed the effects of the isoflavonoid rotenone, an insecticidal allelochemical occurring in various legumes, on larval performance of three polyphagous noctuid species: the corn earworm (CEW), the fall armyworm (FAW) and the southern armyworm (SAW). As rotenone concentration was increased up to 1% fresh mass in an artificial diet, neither mortality (for all three species) nor food consumption (for SAW and FAW) was significantly affected, but developmental time of the latter two species was prolonged. In contrast, for CEW developmental time was shortened and food consumption declined, especially at the two highest rotenone concentrations. Final biomass of all three species declined as dietary rotenone increased.Analysis of covariance (ANCOVA) was used to evaluate larval food utilization analogous to, but without the potential statistical problems of, the traditional ratio-based indices approximate digestibility (AD) and efficiency of conversion of digested food (ECD). Frass output, statistically adjusted to account for differences in consumption, increased with rotenone concentration for all three species, suggesting that, when ingested, this allelochemical interfered with the digestion and/or absorption of ingested food, especially at the higher concentrations tested.Significant statistical interactions in the ANCOVA necessitated the use of utilization plots to examine the linear regressions between biomass gain and absorption at each rotenone concentration. At the two highest concentrations, rotenone tended to reduce the amount of food absorbed by the larvae, as well as their ability to convert the absorbed food to biomass.When piperonyl butoxide, an inhibitor of the polysubstrate monooxygenase (PSMO) system known to metabolize rotenone, was added to the diet along with 0.5 or 1% rotenone, mortality increased significantly only for FAW, approaching or exceeding 50%. The only apparent effects of dietary rotenone on PSMO (aldrin epoxidase) activity (adjusted by ANCOVA for the covariate, crude homogenate protein) were for CEW, where activity decreased in all rotenone treatments compared to the control, and for SAW, where activity at the 3 lowest rotenone concentrations (0.001–0.1% fm) declined about 50% below that of larvae on the control diet, whereas at 0.5 and 1% rotenone, it increased approximately 2-fold over that on the control diet.     

Temperature-independent increase in the detoxifying enzyme activity of insecticide-resistant small brown planthoppers and Drosophila

The populations of migrated small brown planthoppers (SBPHs) collected in Korea were reported to have higher insecticide resistance and percentages of viruliferous individuals than the populations of overwintering SBPHs. Therefore, the migrated SBPHs might survive after insecticide treatments and cause severe damage to rice plants. In this study, the changes in the biochemical properties of resistant SBPHs with temperature changes were investigated. The activities of detoxifying enzymes known to be involved in insecticide resistance showed no correlation with temperature, but the resistant strains had consistently higher detoxifying enzyme activities than the susceptible strains. Interestingly, the amount of reactive oxygen species (ROS) increased with temperature in all strains.Chlorantraniliprole-resistant strains of Drosophila melanogaster were examined to investigate whether the phenomena observed in the resistant SBPHs were conserved in other insects with resistance to an insecticide with different modes of action. Similar to the resistant SBPHs, the resistant Drosophila also exhibited increased amounts of ROS and detoxifying enzyme activity compared to the control Drosophila. Also in the resistant Drosophila, only the ROS showed a temperature-dependent increase.Taken together, in addition to the involvement of increased activities of detoxifying enzymes, the resistant insects also had a temperature-dependent significant increase in ROS. Thus, the development of tools to induce ROS toxicity could lead to the development of new control methods to eliminate resistant insect strains.     

Identification and classification of detoxification enzymes from Culex quinquefasciatus (Diptera: Culicidae)

Molecular characterization of the insecticide resistance has become a hot research topic ever since the first disease transmitting arthropod (Anopheles gambiae) genome sequence has unveiled in 2002. A recent publication of the Culex quinquefasciatus genome sequence has opened up new opportunities for molecular and comparative genomic analysis of multiple mosquito genomes to characterize the insecticide resistance. Here, we utilized a whole genome sequence of Cx. quinquefasciatus to identify putatively active members of the detoxification supergene families, namely cytochrome P450s (P450s), glutathione-S-transferases (GSTs), and choline/carboxylesterases (CCEs). The Culex genome analysis revealed 166 P450s, 40 GSTs, and 62 CCEs. Further, the comparative genomic analysis shows that these numbers are considerably higher than the other dipteran mosquitoes. These observed speciesspecific expansions of the detoxification super gene family members endorse the popular understanding of the involvement of these gene families in protecting the organism against multitudinous classes of toxic substances during its complex (aquatic and terrestrial) life cycle. Thus, the generated data set may provide an initial point to start with to characterize the insecticide resistance at a molecular level which could then lead the development of an easy to use molecular marker to monitor the incipient insecticide resistance in field environs.     

人工湿地污水处理系统中的植物效应与基质酶活性

为了解植物在湿地污水净化系统中的作用与机理,构建了A-B-C三级串连垂直流人工湿地系统,并选择水葱(Scirpus validus)风车草(Cyperus alternifolius)、美人蕉(Canna indica)、和芦苇(Phragmites australis)作为测试植物,进行了生活污水处理试验.结果表明,系统中有植物单元单位面积污染物的去除量均高于无植物对照单元.与对照相比,在A、B、C三级系统中由植物效应产生的CODCr去除增量分别为43.52 g/(m2·d),20.38 g/(m2·d),30.94 g/(m2·d);TN去除增量为13.14 g/(m2·d),28.61 g/(m2·d),6.97 g/(m2·d);TP去除增量为1.2 g/(m2·d),0.66 g/(m2·d),0.06 g/(m2·d).从A到C级,污水中污质浓度递减,呈现根系活力显著增强,而植物生长量、过氧化物酶含量、生长量与氮磷积累量等显著下降趋势.基质酶活性在对照单元中较低,在有植物单元中,基质酶活性明显增大并与根系活力成正相关.     

复合微生态制剂对幼刺参营养成分和 酶活力的影响

目的研究复合微生态制剂对幼刺参体壁营养成分、几种消化酶和免疫酶活力的影响。方法在封闭式循环系统中投喂不同的微生态制剂进行30 d刺参养殖实验。结果投喂液态复合微生态制剂组和粉状复合微生态制剂组的刺参体壁的粗脂肪、总糖、粗蛋白含量最高,两组与对照组相比差异有统计学意义(Ps0.05)。添加微生态制剂的3个实验组刺参肠道蛋白酶、淀粉酶活力均比对照组高,且差异有统计学意义(Ps0.05)。实验组刺参的肠道、体壁、体液过氧化氢酶活力均比对照组高,其中投喂液态复合微生态制剂实验组活力最高。实验组刺参组织的酸性磷酸酶和碱性磷酸酶活力也明显高于对照组(与对照组相比差异有统计学意义,Ps0.05)。结论微生态制剂可以有效改善幼刺参体壁营养成分,促进机体消化活力,提高刺参免疫力。     

Tannic acid as a natural antioxidant compound: Discovery of a potent metabolic enzyme inhibitor for a new therapeutic approach in diabetes and Alzheimer's disease

Multiple studies have been recorded on the synthesis and design of multi‐aim anti‐Alzheimer molecules. Using dual butyrylcholinesterase/acetylcholinesterase inhibitor molecules has attracted more interest in the therapy for Alzheimer's disease. In this study, a tannic acid compound showed excellent inhibitory effects against acetylcholine esterase (AChE), α‐glycosidase, α‐amylase, and butyrylcholinesterase (BChE). IC₅₀ values of tannic acid obtained 11.9 nM against α‐glycosidase and 3.3 nM against α‐amylase, respectively. In contrast, K_i values were found of 50.96 ± 2.18 µM against AChE and 53.17 ± 4.47 µM against BChE. α‐Glycosidase inhibitor compounds can be utilized as a novel group of antidiabetic drugs. By competitively decreasing glycosidase activity, these inhibitor molecules help to hamper the fast breakdown of sugar molecules and thereby control the blood sugar level.     

不同虫态烟蚜对杀虫剂敏感性及其与解毒酶活力关系

为更好地指导生产,本文研究了不同虫态烟蚜Myzus persicae对吡虫啉和丁硫克百威敏感性及其与羧酸酯酶、多功能氧化酶、谷胱甘肽S-转移酶比活力相关性。结果表明,3龄若虫对吡虫啉敏感性水平最低,4龄若虫对丁硫克百威敏感性水平最低。不同虫态烟蚜解毒酶活力随龄期逐渐增强,之后逐渐减弱,3龄若虫高于其他任何虫态,且多功能氧化酶和羧酸酯酶比活力与杀虫剂敏感性间存在强的负相关性。田间最佳防治虫态为1龄若虫、2龄若虫和成虫。     

Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides

The effect of exposure of Aedes aegypti larvae to sub-lethal doses of the pyrethroid insecticide permethrin, the organophosphate temephos, the herbicide atrazine, the polycyclic aromatic hydrocarbon fluoranthene and the heavy metal copper on their subsequent tolerance to insecticides, detoxification enzyme activities and expression of detoxification genes was investigated. Bioassays revealed a moderate increase in larval tolerance to permethrin following exposure to fluoranthene and copper while larval tolerance to temephos increased moderately after exposure to atrazine, copper and permethrin. Cytochrome P450 monooxygenases activities were induced in larvae exposed to permethrin, fluoranthene and copper while glutathione S-transferase activities were induced after exposure to fluoranthene and repressed after exposure to copper. Microarray screening of the expression patterns of all detoxification genes following exposure to each xenobiotic with the Aedes Detox Chip identified multiple genes induced by xenobiotics and insecticides. Further expression studies using real-time quantitative PCR confirmed the induction of multiple CYP genes and one carboxylesterase gene by insecticides and xenobiotics. Overall, this study reveals the potential of xenobiotics found in polluted mosquito breeding sites to affect their tolerance to insecticides, possibly through the cross-induction of particular detoxification genes. Molecular mechanisms involved and impact on mosquito control strategies are discussed.     

Cross-induction of systemic induced resistance between an insect and a fungal pathogen in Austrian pine over a fertility gradient

Evidence for cross-induction of systemic resistance or susceptibility in plant–fungus–herbivore interactions is mostly derived from herbaceous model systems and not perennial woody plants. Furthermore, the effects of environmental variables such as soil fertility on these tripartite interactions are generally unknown. This study examined cross-induction of systemic resistance in Pinus nigra (Austrian pine) to infection by Sphaeropsis sapinea (a fungal pathogen), or feeding by Neodiprion sertifer (European pine sawfly), by prior induction with either S. sapinea or N. sertifer, over a fertility gradient. In a replicated 3-year study, cross-induction of systemic induced resistance (SIR) was found to be both asymmetric within a single year and variable between years. Prior induction with insect defoliation induced SIR to subsequent fungal challenge in 2006 but not in 2005. In 2005, a fertility-independent negative systemic effect of the fungal infection on herbivore growth was detected while herbivore survival was affected by a significant interaction between induction treatment and fertility level in 2006. Prior infection by the fungus induced SIR against the same fungus in both years regardless of fertility levels. This is the first report of whole-plant SIR against a defoliating insect induced by a fungal pathogen and vice versa, under variable nutrient availability, in a conifer or any other tree. Alieta Eyles and Rodrigo Chorbadjian contributed equally to the paper.     

The influence of cytochrome P450 enzyme activity on the composition and quantity of volatile organics in expired breath

Abstract

We have previously described a method to capture, identify and quantify volatile components in expired breath. The purpose of this research is to provide a non-invasive means to measure biomarkers of metabolism in vivo. In the present studies, the effect of 1-aminobenzotriazole (ABT), an inhibitor of diverse cytochrome P450 (P450) enzymes, on the composition of volatile organic chemicals (VOCs) expired in the breath of male F-344 rats was determined in parallel with the catalytic activities and total content of hepatic P450. lntraperitoneal administration of ABT (100 mg kg-¹) to rats resulted in markedly diminished hepatic microsomal P450 content and activities. The extent of inhibition was near maximal at 4 h, at which time approximately 50% of the total P450 content, about 65% of the CYPlA2 activity, 55% of the CYP2E1 activity, and about 80% of CYP2B activity were lost. Inhibition was maintained to 48 h post-dosing, but P450 content and activities had largely been restored by day 7. Concomitant with the inhibition of P450 were corresponding increases (up to several hundred-fold) in the molar amount of volatiles appearing in the breath of ABT-treated animals, and the rebound of P450 levels was attended by corresponding decreases in the appearance of breath volatiles. These studies indicate that P450 plays a major role in the metabolism of VOCs appearing in breath, and that these chemicals can serve as markers on P450 activity in vivo.     

The influence of cytochrome P450 enzyme activity on the composition and quantity of volatile organics in expired breath

We have previously described a method to capture, identify and quantify volatile components in expired breath. The purpose of this research is to provide a non-invasive means to measure biomarkers of metabolism in vivo. In the present studies, the effect of 1-aminobenzotriazole (ABT), an inhibitor of diverse cytochrome P450 (P450) enzymes, on the composition of volatile organic chemicals (VOCs) expired in the breath of male F-344 rats was determined in parallel with the catalytic activities and total content of hepatic P450. lntraperitoneal administration of ABT (100 mg kg-¹) to rats resulted in markedly diminished hepatic microsomal P450 content and activities. The extent of inhibition was near maximal at 4 h, at which time approximately 50% of the total P450 content, about 65% of the CYPlA2 activity, 55% of the CYP2E1 activity, and about 80% of CYP2B activity were lost. Inhibition was maintained to 48 h post-dosing, but P450 content and activities had largely been restored by day 7. Concomitant with the inhibition of P450 were corresponding increases (up to several hundred-fold) in the molar amount of volatiles appearing in the breath of ABT-treated animals, and the rebound of P450 levels was attended by corresponding decreases in the appearance of breath volatiles. These studies indicate that P450 plays a major role in the metabolism of VOCs appearing in breath, and that these chemicals can serve as markers on P450 activity in vivo.