磷化氢、二氧化碳混合气体对腐食酪螨成螨的生物学效应

本文研究结果表明：腐食酪螨Tyrophagus putretcentiae(Schrank)成螨在0%、8%、16%(容积比)CO₂气体中耗氧量随CO₂浓度的增加而增加,当在32%、64% CO₂：气体中,该成螨的耗氧量反倒低于其在正常大气中的耗氧量。在0%、8%、16% 32% 64%CO₂与0.05mg/LPH₃混合气体中该戍螨对PH₃的吸收量分别为1.11±0.92、1.79±0.56 、5.14±1.13、7.60±1.80、8.08±0.85μg/hr'g,在同一CO₂浓度条件下试螨对PH₃的吸收量在高浓度PH₃(0.45mg/L)中明显大于在低浓度PH₃,(0.05mg/L)中,但PH₃,吸收量的增加倍数远远低于PH₃浓度的增加倍数。PH₃,对该螨过氧化氢酶的抑制体内酶高于离体酶,细胞色素c氧化酶受PH₃抑制则相反。被PH₃抑制的过氧化氢酶和细胞色素c氧化酶活性恢复时间分别为二周和一周。本文还对PH_{3的可能杀螨机理及CO2在此过程中的作用进行了讨论。}     

A comparison of some effects of phosphine, hydrogen cyanide and anoxia in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae)

The effects of phosphine, hydrogen cyanide and anoxia on levels of ATP, pyruvate and lactate in Rhyzopertha dominica are compared. The effect of phosphine on anaerobiosis is not directly comparable either with HCN or anoxia. Reduction of catalase by feeding 3 amino 1,2,4 triazole does not enhance the toxicity of phosphine in treated insects.     

Biochemistry of phosphine uptake in susceptible and resistant strains of two species of stored product beetles

1. Uptake of phosphine by resistant strains of Rhyzopertha dominica and Tribolium castaneum was lower than by susceptible ones.2. There was no active uptake of phosphine in vitro by insect fractions and gel filtration of R. dominica cytosol showed no evidence of the presence of a phosphine-binding factor.3. Phosphine appeared to be converted mainly to hypophosphite in insects and the difference in accumulation by resistant and susceptible insects is postulated to be due to differences in the rate of oxidation of phosphine in vivo.     

Are mitochondrial lineages,mitochondrial lysis and respiration rate associated with phosphine susceptibility in the maize weevil Sitophilus zeamais?

Phosphine is the most widely used fumigant with ever‐growing problems of phosphine resistance among insect pests of stored products. One such insect is the maize weevil Sitophilus zeamais, a key pest of stored cereals. Despite its importance as a fumigant, the mechanisms of phosphine toxicity and resistance remain unclear, although the mitochondrion is broadly recognised as its site of action. Here we explored the phosphine susceptibility of maize weevil populations and its association with insect respiration rate, and we tested the association of phosphine susceptibility with the mitochondrial lineages from the field populations studied. We also assessed the action of phosphine in the degradation of mitochondria from muscle cells. Survival under phosphine treatment varied among weevil populations and was negatively correlated with the respiration rate and body mass of the insect. Phosphine produced little lysis of mitochondria and the more phosphine‐resistant population exhibited a slightly higher mitochondria fluorescence intensity under confocal imaging. Therefore, reduced respiration rate is correlated with reduced phosphine activity, but its association with high mitochondria fluorescence intensity in muscle cells seems marginal. There was no association between mitochondrial lineages and phosphine susceptibility, which evolved independently, and the mitochondrial gene fragments of cytochrome oxidase I and II were not useful molecular markers of phosphine susceptibility.     

赤拟谷盗的磷化氢敏感和抗性品系体内羧酸酯酶的活性比较

本文比较测定了赤拟谷盗Tribolium castaneum(Herbst)的磷化氢抗性(Rf=327)和敏感品系害虫的羧酸酯酶活性,研究了该害虫同一品系不同个体间羧酸酯酶的活性差异,比较了两品系害虫在系列磷化氢浓度下熏蒸24h和6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的羧酸酯酶活性。主要结果为:未熏蒸的抗性害虫幼虫和蛹体内的羧酸酯酶活性分别高于敏感品系的1.37和1.16倍;敏感和抗性害虫同品系内不同个体间羧酸酯酶活性分布频率都存在明显差异,抗性害虫中酶活性大的个体数量占的比例较大;磷化氢浓度分别为0.69×10-2、2.78×10-2、5.56×10-2、8.33×10-2和11.11×10-2mg/L时都可导致敏感害虫羧酸酯酶的活性降低,但活性受抑制的程度不因浓度高低呈相应的增减。浓度分别为5.56×10-2、11.11×10-2、13.89×10-2、20.83×10-2和27.78×10-2mg/L的熏蒸中抗性害虫体内酶活性增加,且活性增高的程度与浓度增减也不呈对应变化。在6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的结果中,敏感害虫的酶活性随时间延长而下降,抗性害虫的活性则随时间延长而增大。研究表明赤拟谷盗对磷化氢的抗性可能与羧酸酯酶的活性增加有关。     

Effects of phosphine on the neural regulation of gas exchange in Periplaneta americana

Phosphine is used for fumigating stored commodities, however an understanding of the physiological response to phosphine in insects is limited. Here we show how the central pattern generator for ventilation in the central nervous system (CNS) responds to phosphine and influences normal resting gas exchange. Using the American cockroach, Periplaneta americana, that perform discontinuous gas exchange (DGE) at rest, we simultaneously measure ventilatory nervous output from the intact CNS, VCO(2) and water loss from live specimens. Exposure to 800 ppm phosphine at 25 degrees C for 2 h (n=13) during recording did not cause any mortality or obvious sub-lethal effects. Within 60 s of introducing phosphine into the air flow, all animals showed a distinct CNS response accompanied by a burst release of CO(2). The initial ventilatory response to phosphine displaced DGE and was typically followed by low, stable and continuous CO(2) output. CNS output was highest and most orderly under normoxic conditions during DGE. Phosphine caused a series of ventilatory CNS spikes preceding almost complete cessation of CNS output. Minimal CNS output was maintained during the 2 h normoxic recovery period and DGE was not reinstated. VCO(2) was slightly reduced and water loss significantly lower during the recovery period compared with those rates prior to phosphine exposure. A phosphine narcosis effect is rejected based on animals remaining alert at all times during exposure.     

Studies in biochemical genetics in Drosophila

The metabolism of the imaginal discs of wild type, miniature, vestigial, and four-jointed varieties of Drosophila was investigated using the Cartesian diver ultramicrorespirometer. Wild type and vestigial wing disc respiration is inhibited by cyanide and azide and thus is mediated by an iron or copper porphyrin system, presumable cytochrome-cytochrome oxidase. Respiration is also inhibited by certain hydroxynaphthoquinones, believed to inactivate some enzyme between cytochromes b and c. The respiration of the vestigial and miniature wing discs is increased to normal by the addition of ascorbic acid and to a lesser extent by p-phenylenediamine and hydroquinone, hence the cytochrome oxidase and cytochrome c systems of vestigial and miniature wing discs are normal and the effects of these genes are on enzymes below cytochrome c in the respiratory chain. The respiratory enzymes of the developing imaginal discs of insects are similar to those of a wide variety of cells from bacteria to mammals. The correlation of these biochemical findings with embryological studies of the discs is discussed.     

Properties of copper-dependent o-diphenol oxidase activity in the potato aphid Macrosiphum euphorbiae (thomas)

Potato aphid Macrosiphum euphorbiae (Thomas) was found to contain high amounts of o-diphenol oxidase activity. Enzyme activity was largely distributed into the postmitochondrial supernatant from Brij-35 extracted aphids and occurs in a latent form that was activated up to 45-fold by pretreatment with isopropanol. The aphid enzyme has a broad pH optimum near 6, and utilized L-dopa (K_m = 1.4 mM, V_max = 348 nmol/min-mg protein), dopamine, and 4-methylcatechol the best out of the twelve substrates tested. In addition, this activity is a typical copper-dependent oxidase in that it is potently inhibited by phenylthiourea (50% inhibition at 30nM) and other copper chelators, including salicylhydroxamic acid. The above properties are common to most insect tyrosinases. However, the aphid enzyme lacked the o-hydroxylase and laccase components and the optimal activity at higher temperatures that are typical of cuticular tyrosinases of other insects. The high levels of o-diphenol oxidase in aphids compared to other insects is surprising, since the major function associated with these enzymes, that of melanization and sclerotization of cuticle, is of much less importance to aphids. The possibility that aphids use this enzyme to metabolize dietary phenolics is discussed.     

Use of nitrocellulose membrane to activate and measure insect prophenol oxidase

A method for the activation and measurement of insect prophenol oxidase using nitrocellulose membrane is presented. Using this method we were able to conveniently activate both crude and purified prophenol oxidase from insects belonging to three different orders. This rapid method allows for prophenol oxidase activation, in the absence of a prophenol oxidase-activating system, and in the presence of high ionic strength, protease inhibitors, or chelator.     

灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2积累

植物对昆虫取食活动进行成功防御的关键,取决于对昆虫口腔反吐物的激发子的快速识别。实验利用无损伤微测系统及激光共聚焦显微镜,研究了沙冬青细胞经灰斑古毒蛾口腔反吐物诱导后Ca2+流及H2O2的变化。结果发现:灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2的积累,表明Ca2+内流及H2O2的积累是沙冬青细胞对口腔反吐物产生应答的早期响应事件;Ca2+钙通道阻断剂仅部分抑制Ca2+内流,说明Ca2+内流除经过质膜上的Ca2+通道进入细胞外,尚存在其他的内流途径;灰斑古毒蛾口腔反吐物中的某些成分与沙冬青细胞的质膜结合后,诱导质膜上形成允许Ca2+通过的孔道,而GdCl3不能抑制这类孔道的活性。胞外Ca2+螯合剂EGTA完全抑制H2O2的积累,GdCl3预处理仅部分抑制了H2O2的积累,说明灰斑古毒蛾诱导的沙冬青细胞内H2O2的积累依赖于Ca2+内流;抑制剂实验表明,H2O2的积累主要来源于质膜上NADPH氧化酶的作用。     

Influence of phosphine resistance genes on flight propensity and resource location in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae): the landscape for selection

Phosphine resistance in Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) has evolved through changes to enzymes involved in basic metabolic pathways. These changes impose metabolic stress and could affect energy‐demanding behaviours. We therefore tested whether phosphine resistance alleles impact the movement of these insects in their quest for new resources. We measured walking and flight parameters of four T. castaneum genotypes: (1) a field‐derived population, (2) a laboratory cultured, phosphine‐susceptible reference strain, (3) a laboratory cultured, phosphine‐resistant reference strain, and (4) a resistant introgressed strain that is almost identical genetically to the susceptible population. The temporal pattern of flight was identical across all populations, but resistant beetles took flight significantly less, walked more slowly, and located resources less successfully than did susceptible beetles. Also, the field‐derived beetles (proved not to be carrying resistance genes) walked significantly faster and more directly towards food resources, and had a higher propensity for flight when compared to the susceptible laboratory beetles. These negative effects suggest survival of beetles with the resistance alleles will be compromised should they leave phosphine application sites. The field for selection therefore extends beyond the site at which phosphine fumigant imposed its effect, and other mutations are also likely to be affected in this way.     

The locus of inhibition of NADH oxidation by benzothiadiazoles in beef heart submitochondrial particles

6-Chloro-1,2,3-benzothiadiazole (6-Cl-BTD) is an effective inhibitor of NADH oxidase (site I) but not of succinate oxidase in beef heart submitochondrial particles. For NADH oxidase activity maximal inhibition (80-85%) was achieved at 0.75mM 6-Cl-BTD. A similar level of inhibition was also observed (half maximal inhibitory concentration 0.5mM) towards NADH-duroquinone reductase; NADH-juglone reductase was slightly inhibited (23%) at 0.5mM 6-Cl-BTD while NADH-ferricyanide reductase was unaffected. The data suggest that 6-Cl-BTD interacts with an electron transport site on the oxygen side of NADH dehydrogenase and inhibitory studies with 6-Cl-BTD and rotenone indicate that it might correspond with one of the two sites affected by rotenone. The substituted 1,2,3-benzothiadiazoles (BTDs) are perhaps best known for their activity as inhibitors of cytochrome P-450-mediated mixed-function oxidation (MFO). In vitro, the BTDs are potent inhibitors of MFO activities in microsomes from mammalian liver and insect tissues and they have been demonstrated to inhibit aminopyrine metabolism in perfused rat liver. In vivo, they reportedly prolong hexobarbital sleeping time in mice, inhibit the irreversible binding of labeled trichloro-ethylene to microsomal protein and effectively enhance the toxicity (synergize) of pyrethrin, organophosphorus-containing and carbamate insecticides to insects.     

Insect melanogenesis. II. Inability of Manduca phenoloxidase to act on 5,6-dihydroxyindole-2-carboxylic acid

Eumelanins in animals are biosynthesized by the combined action of tyrosinase, 3,4-dihydroxyphenylalanine (DOPA)chrome isomerase, and other factors. Two kinds of eumelanins were characterized from mammalian systems; these are 5,6-dihydroxyindole (DHI)-melanin and 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin. In insects, melanin biosynthesis is initiated by phenoloxidase and supported by DOPAchrome isomerase (decarboxylating). Based on the facts that DOPA is a poor substrate for insect phenoloxidases and DHI is the sole product of insect DOPAchrome isomerase reaction, it is proposed that insects lack DHICA-melanin. Accordingly, the phenoloxidase isolated from the hemolymph of Manduca sexta failed to oxidize DHICA. Control experiments reveal that mushroom tyrosinase, as well as laccase, which is a contaminant in the commercial preparations of mushroom tyrosinase, are capable of oxidizing DHICA. Neither the whole hemolymph nor the cuticular extracts of M. sexta possessed any detectable oxidase activity towards this substrate. Thus, insects do not seem to produce DHICA-eumelanin. A useful staining procedure to localize DHICA oxidase activity on gels is also presented.     

COⅡ基因在昆虫分子系统学研究中的作用和地位

细胞色素氧化酶Ⅱ(cytochromeoxidaseⅡ,COⅡ)基因位于线粒体DNA(mtDNA)上,编码细胞色素氧化酶亚基Ⅱ,该亚基为细胞色素c提供重要的结合位点。COⅡ基因进化速率较快,是昆虫分子系统学研究中理想的分子标记。目前,已经利用该基因从各个分类水平对昆虫系统发育关系、物种形成与分化、种群遗传与变异及生物地理等方面做了广泛的研究。研究表明,利用该基因可以很好地解决昆虫属、种及种下分类单元的系统发育问题,但是在解决科、亚科等高级阶元的系统发育关系时仍存在一些局限,COⅡ基因与其他mtDNA及核基因的联合分析能够更好地解决昆虫的系统发育问题。     

唾液成分在刺吸式昆虫与植物关系中的作用

近年来,人们对刺吸式昆虫唾液成分的研究,揭示出其在刺吸式昆虫与植物关系中的重要作用。对多数刺吸式昆虫而言,他们取食时会分泌胶状和水状两种唾液,其中胶状唾液会在取食早期分泌形成唾液鞘来围绕并保护口针,通过直接和间接的作用来帮助取食;而水状唾液中则包含了果胶酶、纤维素酶、多酚氧化酶、过氧化物酶、碱性磷酸酯酶、蔗糖酶等组分,来帮助刺吸式昆虫对植物穿刺、消化食物、解毒次生物质并破坏植物的防御反应。有趣的是,唾液成分同时还可以诱导植物的防御反应,包括诱导植物的伤信号引起直接防御反应和诱导植物产生挥发物吸引植食者的天敌引起间接防御反应。并且,许多刺吸式昆虫取 食能够特异性地引发植物的病理反应,有研究推测刺吸式昆虫唾液中多聚半乳糖醛酸酶、碱性磷酸酯酶、蔗糖酶、多酚氧化酶等成分可能是某些植物特定病理反应的激发子,但是目前还没有定论,同时许多刺吸式昆虫唾液中的氨基酸和蛋白酶还是引起植物虫瘿的原因之一。 迄今的研究表明,刺吸式昆虫会根据不同的寄主植物和不同的生理需要,通过唾液组分的改变,来达到取食和发育的目的。对刺吸式昆虫唾液成分和作用机理的研究,可以为揭示刺吸式昆虫致害机理特别是传毒机理、指导害虫有效治理、阐明其与植物的协同进化等提供一定的思路。     

Inheritance and Characterization of Strong Resistance to Phosphine in Sitophilus oryzae (L.)

Sitophilus oryzae (Linnaeus) is a major pest of stored grain across Southeast Asia and is of increasing concern in other regions due to the advent of strong resistance to phosphine, the fumigant used to protect stored grain from pest insects. We investigated the inheritance of genes controlling resistance to phosphine in a strongly resistant S. oryzae strain (NNSO7525) collected in Australia and find that the trait is autosomally inherited and incompletely recessive with a degree of dominance of -0.66. The strongly resistant strain has an LC50 52 times greater than a susceptible reference strain (LS2) and 9 times greater than a weakly resistant strain (QSO335). Analysis of F2 and backcross progeny indicates that two or more genes are responsible for strong resistance, and that one of these genes, designated So_rph1, not only contributes to strong resistance, but is also responsible for the weak resistance phenotype of strain QSO335. These results demonstrate that the genetic mechanism of phosphine resistance in S. oryzae is similar to that of other stored product insect pests. A unique observation is that a subset of the progeny of an F1 backcross generation are more strongly resistant to phosphine than the parental strongly resistant strain, which may be caused by multiple alleles of one of the resistance genes.     

烟粉虱MEAM1隐种漆酶-1基因全长cDNA克隆、序列分析与组织表达

多酚氧化酶是多种刺吸式昆虫唾液中的关键酶类,可以通过干扰寄主植物正常的氧化还原反应来调控食物来源使其更有利于昆虫本身。昆虫体内的漆酶-1属于多酚氧化酶家族,被认为参与昆虫取食过程中的金属离子代谢、降解植物次生有毒物质以及免疫防御等生理活动。为研究漆酶-1基因在烟粉虱MEAM1隐种Bemisia tabaci Middle East-Asia Minor 1组织中的功能,利用RACE技术首次获得了烟粉虱MEAMI隐种漆酶-1（laccase-1,lac-1）基因的cDNA全长序列,命名为Btlac-1 ( GenBank 登录号： JQ966215）。结果表明,该基因含有一个2 733 bp的开放阅读框,编码910个氨基酸,其编码产物含有3个Cu-oxidase功能域, 属于蓝多铜氧化酶家族成员。同源性比较分析表明,Btlac-1与黑尾叶蝉Nephotettix cincticeps和豌豆蚜Acyrthosiphon pisum的lac-1编码的氨基酸序列一致性达58%。Btlac-1在不同组织和发育时期的转录表达分析表明, Btlac-1在烟粉虱MEAM1隐种中肠中的转录水平最高,显著高于头胸部和腹部组织中,且在各个发育时期均有表达,在成虫期的表达水平较高。该结果为进一步明确lac-1在烟粉虱取食过程的生理作用奠定基础,也为探讨刺吸式昆虫与植物相互作用机制提供理论依据。     

Steady-state oxygen kinetics of terminal oxidases in Trypanosoma mega

Steady-state oxygen kinetics of Trypanosoma mega reveal the presence of 3 oxidases. These include an oxidase which is sensitive to salicylhydroxamic acid (SHAM) but insensitive to sodium azide. This oxidase could be the L-alpha glycerophosphate oxidase present in bloodstream trypanosomes. In addition, and oxidase is present wthich is azide-sensitive but SHAM-insensitive. This oxidase is inhibited by CO and is probably cytochrome aa3. A 3rd oxidase is insensitive to both azide and SHAM but is inhibited by CO and is possibly cytochrome o. Reciprocal plots of T. mega reveal the presence of 2 oxidases that are inhibited by CO. These results are discussed in the light of previous evidence suggesting the presence of several oxidases and a branched electron transport system in T. mega.