赤拟谷盗章鱼胺受体3(TcOctβR3)cDNA克隆、表达及功能

【目的】章鱼胺信号系统在调节昆虫行为和生理过程中具有至关重要的作用。赤拟谷盗(Tribolium castaneum)作为一种模式昆虫,被广泛用于解析昆虫生长发育及生理等调控机制的研究工作。本研究以赤拟谷盗为对象,旨在明确章鱼胺受体在调节赤拟谷盗行为和生理方面的功能。【方法】根据Gen Bank登录的相关序列信息(XP_008198078),利用RT-PCR技术克隆赤拟谷盗章鱼胺受体基因Tc OctβR3的c DNA序列。利用在线生物信息学分析软件预测该基因的开放阅读框、编码的氨基酸序列以及跨膜结构域等信息,基于邻接法构建该基因与其他昆虫相关序列的系统发育树,明确系统进化关系。分别提取赤拟谷盗各发育阶段(卵、幼虫、蛹和成虫)、不同组织(中枢神经系统、脂肪体、中肠、后肠、马氏管、精巢和卵巢)以及饥饿胁迫后的RNA,以赤拟谷盗核糖体蛋白S3(Tc RPS3)为内参基因,采用实时定量PCR技术分析该基因在赤拟谷盗不同发育阶段、不同组织以及在饥饿胁迫下的表达模式。运用哺乳动物异源表达系统在人胚胎肾细胞HEK293中瞬时表达Tc OctβR3,进而利用第二信使c AMP含量测定技术分析Tc OctβR3与配体的结合能力。最后,通过体外合成赤拟谷盗Tc OctβR3的双链RNA,利用RNA干扰以及轨迹球行为分析等技术探究该基因的生理功能。【结果】序列分析结果表明,赤拟谷盗Tc OctβR3开放阅读框全长1 305 bp,编码434个氨基酸,序列中含有G蛋白偶联受体典型的7个跨膜结构域。基于邻接法构建的系统发育树表明,该基因编码的蛋白质与小蜂甲(Aethina tumida)的OctβR3亲缘关系最近。实时定量PCR分析结果表明,Tc OctβR3在赤拟谷盗各发育阶段均有表达,尤其在低龄幼虫期转录水平最高,而在其他发育阶段表达量无显著差异;在赤拟谷盗不同组织中,Tc OctβR3在中枢神经系统的表达量显著高于其他组织;赤拟谷盗幼虫在经饥饿处理24 h的过程中,Tc OctβR3的表达量呈先下降后上升的趋势,且在处理6 h的表达量最低,为对照的0.47倍,在16 h表达量最高,为对照的1.80倍,最后恢复到正常水平。通过HEK293细胞异源表达Tc OctβR3后,c AMP测定结果表明章鱼胺(OA)呈浓度依赖性地激活Tc OctβR3,其EC50为8.68×10-7 mol·L-1,萘甲唑啉(NA)的激动活性最高,其有效中浓度EC50为8.56×10-8 mol·L-1。4种供试生物胺激动剂活性强弱为:萘甲唑啉酪胺(TA)章鱼胺多巴胺(DA)。进一步采用RNA干扰技术的分析结果表明,注射ds RNA能有效抑制Tc OctβR3的表达,沉默效率高达61.5%,但干扰该基因表达后不会影响赤拟谷盗成虫的爬行速度和产卵量。【结论】Tc OctβR3在赤拟谷盗中枢神经系统可能发挥重要作用,能调节幼虫对饥饿胁迫的响应。明确了Tc OctβR3的分子生物学特性,可为将来以其为靶标筛选高效激动剂和抑制剂提供理论依据。     

雷公藤总生物碱对玉米象和赤拟谷盗的种群抑制作用

采用室内生测法研究了雷公藤总生物碱对玉米象和赤拟谷盗的种群抑制作用。结果表明,雷公藤总生物碱能显著抑制两种贮粮害虫种群的增长。雷公藤总生物碱浓度大于500mg/kg,33d时玉米象成虫死亡率高于90.83%,67d时混药饲喂的下一代（F1代）,其种群抑制率在96.70%以上;其中500mg/kg处理组存活成虫在未混药饲喂条件下,其F1代种群抑制率达80.10%。雷公藤总生物碱浓度在100-1000mg/kg范围内,对赤拟谷盗成虫无明显的致死效应,但可显著抑制其种群的增殖。当处理浓度为250mg/kg时,对赤拟谷盗的种群抑制率为54.94%;处理浓度升至500mg/kg时,种群抑制率高达98.02%。     

Feeding‐deterrent activity of α‐asarone isomers against some stored Coleoptera

All isomers of α‐asarone [(E)‐4‐prop‐1‐enyl‐1,2,5‐trimethoxybenzene] were tested for their feeding deterrent activity against adults of Sitophilus granarius and Tribolium confusum and larvae of Trogoderma granarium and Tribolium confusum. (E)‐2‐prop‐1‐enyl‐1,3,5‐trimethoxybenzene exhibited the strongest deterrent activity against all the species tested. The total coefficients of deterrency for this compound were 140.6 and 169.7 for Tribolium confusum adults and larvae, respectively, and 144.9 and 104.6 for larvae of Trogoderma granarium and adults of Sitophilus granarius, respectively. © 2000 Society of Chemical Industry     

一种防治贮粮甲虫的新型种子处理剂-ADAGE

Adage是美国Syngenta公司近年开发成功的一种新型杀虫剂。实验表明，用0.5ppm-4ppm的剂量处理玉米种子，对赤拟谷盗、玉米象和锯谷盗的防治效果因虫种、剂量、处理时间和处理温度而有差异。当处理浓度为4ppm、处理时间为1-6d时，玉米象成虫在22℃、27℃和32℃时的死亡率分别为79．3％－89．3％、91．2％－99．2％和97．5％－100％。而对于赤拟谷盗，处理时间比处理温度更重要，当处理浓度为4ppm、温度为27℃、处理时间为1、2、3、6d时，赤拟谷盗成虫死亡率分别为23.4%、40.1%、60.8%和100％。同样，Adage对锯谷盗的防治效果也明显受到处理时间的影响。当处理浓度为4ppm、温度为32℃、处理时间为1、2、3、6d时，锯谷盗成虫死亡率分别为66．0％、88．5％、96．7％和100％。     

几种中药材对抗同贮对赤拟谷盗的影响

对丁香、吴茱萸、花椒单独和分别与千年健混和（1：1）对赤拟谷盗成虫的死亡率及混和物对虫体内可溶性蛋白质的影响进行了研究。结果表明：①丁香、吴茱萸单独防虫的效果优于与千年健的混和物，并且丁香的防虫效果更好，在第5d，成虫死亡率为100％，而花椒与千年健的混合物对赤拟谷盗的防虫效果高于花椒单独使用。②拟谷盗成虫在混和物中死亡率由高到低为：丁香与千年健的混和物＞花椒与千年健的混合物＞吴茱萸与千年健的混合物＞千年健。虫体内的可溶性蛋白质含量由高到低为：吴茱萸与千年健的混和物＞花椒与千年健的混和物＞丁香与千年健的混和物＞千年健，说明赤拟谷盗成虫体内的可溶性蛋白质含量与其死亡率之间不存在的相关关系。在中药材的养护中，可考虑采用花椒与千年健共贮、丁香、吴茱萸来防治赤拟谷盗。     

Laboratory selection of resistance by the red flour beetle,tribolium castaneum (Herbst), to an atmosphere of low oxygen concentration

Adult populations of the red flour beetle,Tribolium castaneum (Herbst), were exposed for 40 generations to an atmosphere containing 99.5% N₂ and 0.5% O₂ at 95% RH, in order to select a strain resistant to the low oxygen concentration (LOC) atmosphere. Selection pressure was maintained at between 50% and 70% mortality. At the 40th generation comparison of sensitivity between the selected strain and the original non-selected strain indicated a resistance factor at the 50% mortality level (LT50) of x 5.2. However, throughout the selections, log-time against probit-mortality curves remained roughly parallel from generation to generation and the slope remained low. These findings indicate a multiplicity of genetic factors that at a high level of selection contribute together towards an adaptation of the insects to survival in the LOC atmosphere. Removal of selection pressure from a sub-population of the selected strain from the 13th to 21st generation revealed that resistance was partially retained with a decrease in resistance factor of 23%. Although the study revealed that the insects were able to develop a strain resistant to hypoxia, exposures were at 95% RH to minimize the desiccation effect. This does not reflect field situations, where ambient relative humidities are generally below 70%.     

利用昆虫病原真菌防治贮粮害虫展望

Only two fungus species,Beauveria bassiana and Metarhizium anisopliae,have been given serious attention as possible microbial controls for stored-product pests.Their host ranges are broad, but their potency for the various target insects varies greatly,and some of the most important pests such as the red flour beetle are very tolerant of fungi.Accordingly, strategies are needed to improve the fungal performance.One such strategy is combination with other environmentally benign treatments such as controlled desiccant dusts and controlled atmospheres. Ironically the relatively dryness of stored-product environments favors fungal efficacy.Desiccation stress renders some insects more vulnerable to fungi.The longevity of fungus spores is also best under dry conditions.The prospects of fungi for control of stored-product pests can be improved by taking advantage of these phenomena and judicious selection of use venues and application strategies.     

两种低氧条件对赤拟谷盗不同虫态的抑制作用

研究了在28℃、75%RH的温湿度条件下,5%和2%低氧储藏条件对赤拟谷盗高龄幼虫、蛹和成虫的抑制作用。在5%和2%氧气条件下暴露15d后,赤拟谷盗高龄幼虫化蛹率分别为95.0%和3.7%,蛹羽化率分别为98.0%和0%,成虫死亡率分别为12.7%和100%。本研究结果为气调储粮防治害虫提供依据。     

Phosphine resistance, respiration rate and fitness consequences in stored-product insects

Resistance to fumigants has been frequently reported in insect pests of stored products and is one of the obstacles in controlling these pests. The authors studied phosphine resistance and its physiological basis in adult insects of 12 populations of Tribolium castaneum (Herbst) (Tenebrionidae), ten populations of Rhyzopertha dominica (F.) (Bostrichidae) and eight populations of Oryzaephilus surinamensis L. (Silvanidae) from Brazil, and the possible existence of fitness costs associated with phosphine resistance in the absence of this fumigant. The bioassays for the detection of phosphine resistance followed the FAO standard method. The production of carbon dioxide and the instantaneous rate of population increase (r(i)) of each population of each species were correlated with their resistance ratios at the LC(50). The resistance ratio at LC(50) in T. castaneum ranged from 1.0- to 186.2-fold, in R. dominica from 2.0- to 71.0-fold and in O. surinamensis from 1.9- to 32.2-fold. Ten populations of T. castaneum, nine populations of R. dominica and seven populations of O. surinamensis were resistant to phosphine. In all three species there was significant association (P < 0.05) between respiration rate and phosphine resistance. The populations with lower carbon dioxide production showed a higher resistance ratio, suggesting that the lower respiration rate is the physiological basis of phosphine resistance by reducing the fumigant uptake in the resistant insects. Conversely, populations with higher r(i) showed lower resistance ratios, which could indicate a lower rate of reproduction of the resistant populations compared with susceptible populations. Thus, management strategies based on the interruption of phosphine fumigation may result in reestablishment of susceptibility, and shows good potential for more effective management of phosphine-resistant populations.     

基于GC-MS和电子鼻的面粉中粮虫快速检测方法

储粮害虫会降低粮食及其产品的重量、品质和营养健康指数,并且我国粮虫检测方式仍然以人工检测为主。为满足储粮害虫快速检测的需求,采用气相色谱-质谱联用仪（GC-MS）获得了赤拟谷盗（Tribolium castaneum（Herbst））的主要特定挥发性有机化合物（VOCs）,根据这些化合物的性质筛选出多个金属氧化物气敏传感器,并以传感器阵列为核心开发了储粮害虫电子鼻检测装置。该装置采集了赤拟谷盗、被赤拟谷盗侵染的面粉、被长头谷盗（Latheticus oryzae Waterhouse）侵染的面粉3种实验对象的气味信息,提取每条响应曲线的相对变化值和相对积分值作为原始特征矩阵（10×2）,使用主成分分析（PCA）和偏最小二乘回归算法（PLSR）对原始特征矩阵进行分析,并通过建立回归预测模型,实现了对面粉中赤拟谷盗和长头谷盗虫口密度的预测。优化后的传感器数量由10个减少至8个,赤拟谷盗样品的两个主成分累计的贡献率为79.4%。基于PLSR的预测模型对面粉中赤拟谷盗的数量有很好的预测效果（校正集：相关系数r=0.88,均方根误差为8.09;验证集：r=0.89,均方根误差为7.75）;该预测模型对面粉中长头谷盗的数量也有很好的预测效果（校正集：r=0.94,均方根误差为5.85;验证集：r=0.94,均方根误差为6.08）。研究结果表明：该装置能够满足判别储粮中不同虫口密度样本的基本需要,并且具有可靠的稳定性。