An improvement of cucumber cotyledon greening bioassay for cytokinins

The cucumber cotyledon greening bioassay for cytokinins was modified by using 95% acetone-ethanol instead of 80% acetone as extraction solvent. The cotyledons were extracted directly with a 2:1 (v/v) acetone-ethanol solution in dark for 24 hours, omitting the homogenization and centrifugation operations of the previous bioassay. The modified bioassay is more convenient and especially useful in screening cytokinin-active substance from a large number of samples.     

Isolation and primary culture of Necturus maculosus (Amphibia: urodela) hepatocytes

Summary In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochemical analyses. A typical perfusion yielded 1.7×10⁵ viable hepatocytes per gram body weight with an average viability of 86±5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8°C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.     

绿盲蝽雄虫体外挥发物研究

采用气质联用和嗅觉仪对分部解剖的绿盲蝽Apolygus lucorum(Meyer-Dür)雄虫体外挥发物进行鉴定和生测。结果表明:绿盲蝽雄虫浸提物中的主要组分有13种,包括醇类、酸类和酯类。相对含量较高的依次有丁酸己酯、反-2-丁酸己烯酯和己醇。绿盲蝽雄虫从交配不活跃期进入活跃期,其体内的丁酸己酯、反-2-丁酸己烯酯和己醇的含量都明显变化,其中丁酸己酯和反-2-丁酸己烯酯明显增加,而己醇的含量减少,表明绿盲蝽雄虫在交配活跃期,可能有大量的丁酸己酯和反-2-丁酸己烯酯释放到体外。比较绿盲蝽雄虫的不同部位的浸提物的含量,发现丁酸己酯、反-2-丁酸己烯酯和己醇主要存在于虫体胸部。嗅觉反应测试中绿盲蝽雌虫对丁酸己酯和反-2-丁酸己烯酯有明显的趋性,同时含量很少的丁酸庚酯对雌虫有明显引诱作用。因此推测丁酸己酯、反-2-丁酸己烯酯和丁酸庚酯可能是绿盲蝽雄虫释放到体外的挥发性引诱成分,并主要由胸部内的腺体分泌。     

橘小实蝇植物源引诱活性物质的生物测定

植物源引诱物质可显著提高橘小实蝇Bactrocera dorsalis(Hendel)蛋白饵剂的应用效果,本文测试了柑橘、番木瓜、芒果、番石榴、杨桃的叶片浸提物、果实以及已知植物次生物质对橘小实蝇的引诱效果。植物浸提物的生物测定结果表明,杨桃的二氯甲烷浸提物的引诱效果最佳,平均引诱率为20.83%,对于不同的萃取剂而言,二氯甲烷、乙醇浸提物效果明显优于石油醚,三者浸提的平均引诱率分别为15.67%、15.17%和10.50%;对于不同寄主植物而言,柑橘浸提物的效果最佳,3种有机溶剂浸提物的平均引诱率为18.33%,其中石油醚、二氯甲烷和乙醇浸提物的引诱率分别为20.00%、15.83%和19.17%,其他植物浸提物的引诱效果为杨桃>番木瓜>芒果>番石榴,3种有机溶剂浸提物的平均引诱率分别为17.22%、11.67%、11.11%和10.56%。寄主果实的生物测定结果表明,柑橘的平均引诱率为65.83%,其中雌虫和雄虫的引诱率分别为61.67%和70.00%,明显高于杨桃、番石榴和番木瓜,三者的引诱率分别为31.67%、31.67%和21.67%,与柑橘引诱率差异显著(P<0.05)。已知植物次生物质的生物测定结果表明,甲基丁香酚的引诱效果最佳,平均引诱率为45.00%,其中雄虫和雌虫的引诱率分别为86.67%和3.33%;乙酸乙酯和番石榴香精的引诱效果次之,平均引诱率分别为32.50%和28.33%,其中雌虫的引诱率均为36.67%;杨桃香精和柠檬酸的引诱效果较差,平均引诱率分别为25.00%和26.67%。     

一种改进的粉虱成虫生物测定方法

生物测定是检测害虫抗药性的一项重要技术。利用100mL插口圆底聚丙烯离心管对现在应用较多的粉虱成虫生物测定方法——琼脂保湿浸叶法进行了改进。改进后的方法不影响粉虱成虫的持续取食,具有操作简单、结果重复性好及无需对成虫麻醉等优点。同时发现茄子叶片对于B型烟粉虱Bemisia tabaci(Gennadius)B-biotype和温室白粉虱Trialeurodes vaporariorum(Westwood)成虫均是一种非常适合的生测材料。利用该方法分3次独立测定了烯啶虫胺对B型烟粉虱和温室白粉虱混合日龄成虫的毒力,结果具有很好的重复性。     

茚虫威对不同抗药性品系小菜蛾呼吸代谢的影响

以小菜蛾Plutella xylostella(L.)敏感品系(S)、田间品系(F)及茚虫威汰选抗性品系(T17)幼虫为供试虫源,采用便携式光合作用测定仪测定小菜蛾3～4龄幼虫在受药前后或不同受药剂量等条件下呼吸速率的变化,从能量代谢的角度研究抗药性机制。结果表明,汰选抗性品系、田间品系与敏感品系同龄期小菜蛾幼虫在未受药条件下的呼吸速率差异不显著,表明抗药性的产生并未影响小菜蛾本底的呼吸速率。以各个品系的茚虫威LC20和LC50剂量处理幼虫后,3个品系的呼吸速率均明显提高,汰选抗性品系呼吸速率提高幅度明显大于其他2个品系。在LC20剂量下3个品系呼吸速率峰值均出现在2h前后,10h后恢复到正常水平;在LC50剂量下敏感品系没有明显差异;而田间品系和汰选抗性品系分别在药剂处理后4h和6h达到呼吸高峰,汰选抗性品系保持高水平呼吸速率时间长达9h,分别于药后15h和24h恢复到正常水平。这表明用药后抗性品系呼吸速率的提高幅度与小菜蛾的解毒代谢能力有关,这也揭示了昆虫幼虫中毒后能量消耗会随着抗性水平的提高而增加。     

湖南不同地区小菜蛾对药剂敏感性比较

采用浸叶法在室内测定了湖南长沙和怀化地区小菜蛾Plutella xylostella(L.)田间种群对10种药剂的敏感性。结果表明:湖南长沙和怀化地区田间小菜蛾除对丁醚脲和BT制剂仍处于敏感(抗性倍数<3)状态外,对其它8种药剂产生了不同程度的抗药性,其中以长沙地区小菜蛾田间种群对高效氯氰菊酯抗性倍数最高(抗性倍数为33.58)。长沙和怀化种群对药剂的相对毒力倍数以巴丹最低(1.20)而以多杀菌素最高(2.59)。     

Application of synthetic herbivore‐induced plant volatiles causes increased parasitism of herbivores in the field

We previously reported that Cotesia vestalis (Hymenoptera, Braconidae), a parasitoid of diamondback moth (DBM) (Plutella xylostella; Lepidoptera, Plutellidae) larvae, was attracted to volatiles from crucifer plants infested by moth larvae kept in a desktop acrylic box, and that a blend of four DBM‐induced plant volatiles was responsible for this attraction. In this study, using a specially designed dispenser to release the four compounds, we demonstrated that the wasp was attracted to intact komatsuna plants (Brassica rapa var. perviridis). The experiments were performed in a climate‐controlled room, which was approximately 1000 times larger than the acrylic box used previously. Similarly, using the dispenser in the field, C. vestalis females were attracted to intact komatsuna plants with the dispenser from a distance of three metres. We also examined the effect of the volatile blend on the incidence of parasitism of DBM larvae in the field. Three small containers containing DBM‐infested komatsuna plants with dispensers, and three control containers containing only infested plants (control) were arranged in two lines running perpendicular to a komatsuna field in which both DBM larvae and C. vestalis populations were maintained, at distances of 12, 30 and 70 m. The results showed that the incidence of DBM parasitism was significantly higher in containers containing dispensers than in the control containers, suggesting that the blend could potentially be applied to DBM control in agroecosystems.     

Characterization of a myrosinase cDNA from Brassica parachinensis and its defense role against Plutella xylostella after suppression

Abstract In Brassicaceae, myrosinase catalyzes the hydrolysis of glucosinolate and plays an important role in anti‐herbivore defense. We have cloned and characterized the full‐length complementary DNA of myrosinase gene from Brassica parachinensis that exhibits high sequence identity with myrosinase genes from other Brassica species. To investigate the role of this myrosinase in defense against the diamondback moth (Plutella xylostella), we constructed an RNA‐interference (RNAi) cassette expressing a double‐stranded RNA that targeted myrosinase and transfected it into B. parachinensis. Myrosinase was suppressed in the resulting transgenic plants. Diamondback moth larvae feeding on transgenic plants had lower larval and pupal weights, longer pupal duration, and lower fecundity than those feeding on non‐transgenic plants, suggesting that the diamondback moth has adapted to the glucosinolate‐myrosinase defensive system. Therefore, the suppression of myrosinase is a potential approach for controlling the diamondback moth.     

Immunosuppression induced by expression of a viral RNase enhances susceptibility of Plutella xylostella to microbial pesticides

Abstract Polydnaviruses are a group of insect DNA viruses and are characterized in their segmented genome that is located in the chromosome(s) of host wasps. A polydnavirus, Cotesia plutellae bracovirus (CpBV), encodes a viral ribonuclease (RNase) T2 in a specific segment #3 (CpBV‐S3). This study tested its effect on gene expression associated with host immune responses in the diamondback moth, Plutella xylostella. Micro‐injection of CpBV‐S3 into nonparasitized larvae induced expression of its two encoded genes, CpBV‐ORF301 (=CpBV‐RNase T2) and CpBV‐ORF302. In response to a bacterial challenge, four antimicrobial peptide genes (hemolin, gloverin, cecropin and lysozyme) and six phenoloxidase (PO)–associated genes (proPO‐activating proteinase, PO, serine proteinase homolog and serpins 1–3) were up‐regulated in their expressions. However, the transient expression of CpBV‐S3 suppressed the expressions of cecropin, PO and serpin 1. Double‐stranded RNA specific to the viral RNase T2 could specifically knockdown the viral gene expression and restored the three gene expressions suppressed in the larvae injected with CpBV‐S3. The inhibitory activity of the viral RNase T2 on the target genes was further proven by the suppression of PO activation in response to bacterial challenge in the larvae injected with CpBV‐S3. This immunosuppression by the expression of the viral RNase T2 resulted in significant increase of pathogen susceptibility of P. xylostella against Bacillus thuringiensis or baculovirus infection.