寄主植物对松墨天牛幼虫体内酶系活性的影响

室内采用在马尾松、黑松、火炬松、湿地松和雪松皮下接入松墨天牛Monochamus alternatus Hope幼虫的方法,探明5种寄主植物对松墨天牛幼虫的存活、钻蛀取食及纤维素酶、酯酶、羧酸酯酶活性的影响。结果表明,5种寄主植物对松墨天牛幼虫存活和钻蛀取食有显著影响,接种于马尾松的天牛幼虫存活率100%;接种于雪松的松墨天牛幼虫存活率仅55.56%。松墨天牛取食不同寄主后,其体内纤维素酶、酯酶和羧酸酯酶活性存在显著差异。其中以取食火炬松的Cx酶活性最高,为(4.5754±0.1975)μmol·g-1(FW)·min-1,C1酶和β-l,4-葡萄糖苷酶活性以取食马尾松的松墨天牛幼虫消化道中最高,分别为(2.8199±0.0510)μmol·g-1(FW)·min-1与(3.0793±0.1733)μmol·g-1(FW)·min-1;而以取食黑松和马尾松的酯酶、羧酸酯酶活性最高,分别为1372.74mmoD/min·μg和744.82mmoD/min·μg。     

松天牛小首螨对极端温度耐受性的驯化效应及低温存储研究

[目的]松天牛小首螨是近年发现的首个能够寄生致死松墨天牛卵的一个螨类新种,研究该螨对极端温度耐受性的驯化效应和低温存储条件,有助于进一步评估该螨的潜在地理分布或应用区域,并为该螨的规模化运输和存储提供基础依据。[方法]通过不同温度条件驯化后,测定松天牛小首螨暴露在极端温度下的存活率,同时比较不同低温条件下存储的松天牛小首螨膨腹体涌出的成螨数量,以及存储的雌成螨的生存情况。[结果]高温驯化可显著提高雌成螨高温暴露存活率,其中30℃ 36 h驯化后的存活率提高了1.26倍;低温驯化可显著提高雌成螨极端低温暴露存活率,其中10℃ 96 h、10℃ 120 h驯化后的存活率分别提高了2.08和2.13倍。以膨腹体为存储对象,低温存储后涌出成螨的数量随着存储时间延长而逐渐显著减少;以雌成螨为存储对象,在10℃条件下存储30 d,存活率为65.10%,在4℃条件下存储30 d,存活率达84.21%。[结论]松天牛小首螨具有很强的温度适应性,短期温度驯化即可大幅提升其对极端温度的耐受性。膨腹体适合作为该螨短期低温存储对象,雌成螨则适合作为中长期低温存储对象。     

松墨天牛和光肩星天牛染色体核型

以松墨天牛Monochamus alternatus Hope和光肩星天牛Anoplophora glabripennis Motschulsky为研究对象,以各种天牛的最适条件和最佳材料进行染色体的制备和观察。试验结果如下:松墨天牛的最佳试验材料为7日龄蛹的精巢或卵巢,染色体数目2n=20,性别决定机制为Xyp,有大型染色体5对,中型染色体4对,性染色体在其大小排列中位于最末位,为中型染色体,染色体组式为5L+4M+Xyp;光肩星天牛的最佳试验材料为初羽化成虫的精巢或卵巢,染色体数目2n=20,性别决定机制为Xyp,有大型染色体6对,中型染色体3对,性染色体在其大小排列中位于最末位,但也为中型染色体,染色体组式:6L+3M+Xyp。     

厦门市松墨天牛生物学特性Ⅰ:生活史

于2002—2004年在厦门同安设立试验点对松墨天牛进行饲养及观察。结果表明,松墨天牛在厦门以2a发生3代为主,少数1a发生2代,世代重叠现象明显,几乎全年可见幼虫,以3—5龄幼虫在蛀道内越冬或以低龄幼虫在韧皮部越冬。2a发生3代的松墨天牛有3个羽化高峰,1a发生2代的有2个高峰期,二者的第1个羽化盛期在4月相叠加,所以4月份的成虫数量最多,占全年羽化成虫总数的31％,此后一直到8月下旬,都有较多的成虫羽化。     

鱼藤酮对松墨天牛产卵和取食行为的影响

为探索松墨天牛防治的新方法,实验了鱼藤酮对松墨天牛产卵和取食行为的影响及鱼藤酮对松墨天牛5龄幼虫的触杀作用。结果表明,鱼藤酮对松墨天牛具有显著的产卵忌避作用和成虫取食的干扰作用。产卵忌避作用和拒食作用随鱼藤酮浓度的增大而增强,处理浓度为1 000 mg/L时,鱼藤酮对成虫的产卵忌避率为75.63 %,处理后12 h松墨天牛的选择性和非选择性拒食率分别为72.91%和69.50%。鱼藤酮对松墨天牛5龄幼虫的触杀作用不明显。因此,利用鱼藤酮防治松墨天牛,一方面可以通过抑制其取食减少松材线虫的传播;另一方面通过忌避产卵可减少下代虫口。     

松墨天牛幼虫体内神经毒性酯酶和乙酰胆碱酯酶对甲胺磷敏感性的比较

松墨天牛Monochamus alternatus Hope是传播松材线虫Bursaphelenchus xylophilus Nickle的主要媒介昆虫,在其化学防治中经常使用有机磷杀虫剂,而这类杀虫剂有些可通过作用于神经毒性酯酶（neurotoxic esterase, NTE）使敏感的脊椎动物产生迟发性神经毒性。为了深入理解有机磷杀虫剂对害虫毒杀作用机理,增强对昆虫体内NTE活性的了解,我们在松墨天牛中开展了相关的研究。通过差异测定法和经典的乙酰胆碱酯酶（acetylcholinesterase, AChE）活性测定方法,分别测定了松墨天牛幼虫体内NTE和AChE的活性。结果发现,在松墨天牛体内存在NTE活性,其头部的NTE活力为2.80±0.30 nmol·min^-1·mg^-1,比脂肪体高出了近10倍,但仍较鸟类等脊椎动物为低。体内实验显示,松墨天牛体内NTE可被甲胺磷所抑制; 但在体外实验条件下,甲胺磷对来自松墨天牛头部的NTE活性并没有产生明显抑制作用,而此浓度下的甲胺磷对AChE表现出较高水平的抑制作用。表明松墨天牛体内NTE和AChE对甲胺磷的敏感性完全不同。     

松墨天牛的生物学特性Ⅱ:生活习性

2002—2004年在厦门同安进行松墨天牛的饲养及观察。结果表明,松墨天牛在厦门以2a3代为主,少数1a2代,2a3代的松墨天牛有3个羽化高峰期,1a2代的有2个高峰期,二者的第1个羽化盛期在4月份相叠加,所以4月份的成虫数量最多,占全年羽化成虫总数的31％,此后一直到8月下旬,都有较多的成虫羽化。羽化后的成虫从蛹室向外咬圆形的羽化孔道飞出,有的当天即取食,有的时隔1—2d取食,最初喜食当年生松枝的嫩皮,个别也取食微量松针,性成熟后食1-3年生松枝、树皮补充营养,而且昼夜均取食。成虫补充营养5—7d后开始交尾,可多次重复交配,成虫的产卵与交尾是交错进行的,卵产于刻槽内靠形成层的韧皮部,一个刻槽一般产1粒卵,个别的有2—3粒,大多数无卵,无卵的刻槽约占60％-70％。雌成虫咬啃刻槽后常在树干上留下泪滴状松脂。幼虫孵化后取食松树内皮和韧皮部,在树皮外可见白色纤维状、褐色粉末状排泄物,3龄以后蛀入木质部,老熟后做一椭圆形蛹室化蛹,蛹期8—15d。世代重叠现象明显,几乎全年可见幼虫,以3—5龄幼虫在蛀道内越冬或以低龄幼虫在韧皮部越冬。     

松墨天牛成虫交配与产卵行为的观察

观察了养虫笼中松墨天牛Monochamus alternatusHope的交配和产卵行为。描述了雌、雄虫在交配过程中的多种行为,雌、雄虫间的交配时间可持续23~540 min,平均约200 min;有重复交配现象,重复交配次数可达2~18次;每次交配持续时间为2~100 s。雌虫背负雄虫,边取食边产卵;也可脱离雄虫单独产卵。雌虫在寄主枝条上咬槽产卵,通常日产卵1~2粒,最多3粒。产卵期的长短和产卵量的多少取决于雌虫个体寿命的长短。     

松墨天牛的全球潜在分布区分析

松墨天牛MonochamusalternatusHope分布在亚洲东部,是松材线虫Bursaphelenchusxylophilus(SteinerandBuhrer)在亚洲最有效的昆虫媒介,同时也是重要的蛀干害虫。利用CLIMEX模型分析松墨天牛分布区的气候限制因子,并在全球范围预测它的潜在分布区。模型分析结果表明,温度和降水是松墨天牛分布区的主要气候限制因子。温度在30°N以北地区和30°S以南地区主要表现为冷胁迫,在非洲中部、南亚和澳大利亚北部表现为热胁迫。有效积温不足可能是限制松墨天牛向北扩散的主要原因。降水在中国西北地区、非洲中北部、澳大利亚中部和西部与美国西部主要表现为干胁迫。降水量对分布区范围影响不大。预测结果表明,松墨天牛的全球潜在分布区远远大于实际分布范围。松墨天牛在东半球的潜在分布区包括亚洲东部和南部地区、地中海沿岸、非洲的中部和南部以及澳大利亚的东部和南部,在亚洲热带的潜在分布区1年3代,地中海地区1年1代,非洲1年2～3代,在澳大利亚主要1年1代。松墨天牛在西半球的潜在分布区主要集中在美国南部和东部沿海地区,中美洲以及南美洲的广大地区,美国主要1年1代,中美洲1年2～3代,南美洲主要1年2代。     

松墨天牛对寄主树木的产卵选择

在室内采用选择行为方法,研究了松墨天牛Monochamus alternatus对几种松属植物的产卵选择行为。结果表明,松墨天牛对黑松Pinus thunbergii的选择性最强,对马尾松P.massoniana的选择性最弱,二者间差异显著,它们与火炬松P. taeda和湿地松P. elliottii间无明显差异。在黑松和马尾松的被松材线虫危害木与健康木之间,松墨天牛明显选择被害木产卵;具产卵痕枝段和虫粪处理过的枝段,对松墨天牛的产卵有显著抑制作用,松墨天牛明显地选择对照枝段产卵。     

Effects of spatio-temporal intervals between newly-hatched larvae on larval survival and development inMonochamus alternatus (Coleoptera: Cerambycidae)

The effects of distance between hatching larvae on survival and development were investigated inMonochamus alternatus. Two newly-hatched larvae were inoculated intoPinus densiflora bolts at a distance of 2.5 cm or 10 cm, simultaneously or at an interval of 2 weeks. Some larvae were inoculated singly as a control. When larvae were inoculated simultaneously, mortality of the closely-inoculated larvae was significantly higher than that of distantly-inoculated larvae. Such high mortality was identified as due to conspecific bites. When the two larvae were inoculated asynchronously, the first-inoculated larvae killed some second-inoculated larvae but were never killed by them. Consequently, mortality was higher in second-inoculated larvae than in first-inoculated larvae. In particular, there was a significant difference in mortality between them when the larvae had been inoculated closely. The mortality of second-inoculated larvae was higher in the closely-inoculated group than in the distantly-inoculated group although there was no significant difference between them. In the case of two simultaneously-inoculated larvae, the initial distance between them had no significant effect on the development and growth in the early larval stage. When the larvae were inoculated asynchronously, the first-inoculated larvae grew more quickly than singly-inoculated control larvae.     

松墨天牛漆酶基因MaLac1的克隆、原核表达及表达谱分析

【目的】本研究旨在克隆并鉴定松墨天牛Monochamus alternatus内源漆酶基因MaLac1,分析其在松墨天牛不同发育阶段的表达水平,为进一步明确MaLac1功能提供依据。【方法】基于松墨天牛肠道转录组测序数据,通过RACE克隆松墨天牛MaLac1基因的全长cDNA序列,并对其进行生物信息学分析;将该基因与pET-32a载体链接构建表达载体pET-MaLac1,导入大肠杆菌Escherichia coli Rosetta (DE3)使其表达;使用qPCR检测MaLac1基因在松墨天牛不同发育阶段(低龄幼虫、老熟幼虫、蛹、雌成虫和雄成虫)肠道中的表达差异。【结果】克隆获得松墨天牛MaLac1的cDNA全长序列(GenBank登录号:KY073340)。MaLac1开放阅读框全长2 067 bp,编码一个含688个氨基酸的蛋白质,预测分子量为78.34 kD,等电点为5.30。SignalP 4.1 Server预测MaLac1在N端包含一个15个氨基酸的信号肽。序列比对分析表明,MaLac1具有典型的昆虫漆酶基因特征,与赤拟谷盗Tribolium castaneum漆酶基因的氨...     

携带与未携带松材线虫的松墨天牛转录组差异表达基因分析

【目的】本研究旨在建立携带与未携带松材线虫Bursaphelenchus xylophilus的松墨天牛Monochamus alternatus成虫各组织的转录组数据库,揭示松墨天牛对松材线虫响应的转录组整体表达特征。【方法】以携带和未携带松材线虫的松墨天牛成虫的表皮、气管和脂肪体为材料,采用Illumina HiSeq^TM 2000测序平台开展转录组测序,利用Trinity软件对RNA-Seq数据进行从头组装,利用NCBI数据库进行基因注释。通过DEG-Seq分析携带和未携带松材线虫的松墨天牛成虫中的差异表达基因,对上调表达基因进行GO和KEGG代谢途径富集分析,并通过实时荧光定量PCR技术对部分差异表达的基因包括ENV, CDK1, HSP70-C, HSP70, HSP75, DUO, PABPN1和IGFP基因的表达水平进行验证。【结果】测序过滤后共获得55 059条单基因簇(unigene),平均长度为1 536 bp。将unigene与数据库中的序列进行BLASTX比对,成功注释24 354条unigenes,其中4 022条unigene在GO数据...     

松褐天牛的交配行为

采用室内试验和野外观察相结合的方法,对松材线虫病的主要媒介昆虫松褐天牛Monochamus alternatus Hope的交配行为进行了研究。结果表明： 松褐天牛一次完整的交配包括相遇抱对、插入输精和配后保护3个阶段,在交配过程中雄虫有多次插入输精现象发生。室内试验中共观察到松褐天牛的交配123次,松褐天牛一次完整的交配过程平均需时63.49 min,其中输精前的抱对时间平均为1.68 min,交配过程中每次输精插入时间平均为57.60 s,配后保护时间为15.18 min。松褐天牛在开始交配的4天内平均交配5.15次,不同雄性个体所获得的交配机会差异很大。松褐天牛的交配行为表现出强烈的雄性竞争现象,雄虫能根据雌虫或自身的交配经历调整交配投入,当雌虫或者雄虫是初次交配时,总输精时间和插入输精的次数显著大于与有交配经历的雌虫或雄虫交配时的输精时间和插入输精次数。田间松褐天牛的交配行为与室内观察结果基本一致。     

孢悬液浓度和宿主体型大小对球孢白僵菌对松墨天牛幼虫的致病力的影响

【目的】球孢白僵菌Beauveria bassiana是一种被广泛用于害虫生物防治的生防菌。本研究探讨了孢悬液浓度和宿主体型大小对球孢白僵菌对松墨天牛Monochamus alternatus 幼虫的致病力的影响,旨在为松墨天牛的生物防治提供科学基础。【方法】分别用0.5% 吐温-80(CK)以及1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸和1×10⁹孢子/mL的球孢白僵菌孢悬液接种松墨天牛4龄幼虫,统计接种后15 d内幼虫死亡率和染菌率。同时用最佳浓度(1×10⁹孢子/mL)的球孢白僵菌孢悬液接种体型大小分别为100~150, 200~220, 300~320, 400~420, 500~520和600~650 mg/头的松墨天牛幼虫,测定接种后20 d内幼虫的死亡率和染菌率。【结果】接种1×10⁵~1×10⁹孢子/mL的球孢白僵菌后,松墨天牛4龄幼虫起初活动自如,后在头部出现烧灼状伤并且体色逐渐变红,最后周身长满菌丝。不同浓度下,随球孢白僵菌孢子浓度升高,松墨天牛4龄幼虫校正死亡率和校正染菌率增加。接种1×10⁶~1×10⁹孢子/mL球孢白僵菌孢悬液15 d的松墨天牛4龄幼虫累计校正死亡率均可达到100%,1×10⁷, 1×10⁸和1×10⁹孢子/mL浓度下,松墨天牛达到100%校正死亡率所需时间最少。接种0 (CK), 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸和1×10⁹孢子/mL球孢白僵菌孢悬液的松墨天牛4龄幼虫校正染菌率在第15天分别为0, 20.00%, 86.67%, 90.00%, 96.67%和100.00%,表现为白僵菌孢子浓度越高,染菌率越高。1×10⁹孢子/mL的接种浓度下,松墨天牛幼虫个体越大,天牛幼虫死亡率和染菌率越高。表现在第20天时,体型大小为100~150, 200~220, 300~320, 400~420, 500~520和600~650 mg/头的幼虫的死亡率分别为76.67%, 76.67%, 66.67%, 93.33%, 100.00%和100.00%,染菌率分别为60.00%, 63.33%, 60.00%, 86.67%, 96.67%和100.00%。【结论】球孢白僵菌悬浮液浓度对松墨天牛幼虫的死亡和侵染有显著影响,表现为随孢子浓度的增加而增加;同时,松墨天牛幼虫个体越大,死亡率和染菌率越高。研究结果对开展利用球孢白僵菌防治松墨天牛具重要借鉴和指导意义。     

Effects of temperature on Anoplophora glabripennis (Coleoptera: Cerambycidae) larvae and pupae

Developmental thresholds, degree-days for development, larval weights, and head capsule widths for each larval instar and the pupal stage of Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) were studied at eight constant temperatures (5, 10, 15, 20, 25, 30, 35, and 40°C) for two source populations (Ravenswood, Chicago, IL [IL], and Bayside, Queens, NY [NY]). The estimated lower threshold temperature for development of instars 1-5 and the pupal stage was near 10°C and was near 12°C for the higher instars. Developmental rate was less temperature sensitive for instars 5-9 compared with instars 1-4. Development for all but the first instar was inhibited at constant temperatures >30°C, and all instars failed to develop at 40°C. Although the two source populations had similar responses to temperature, IL larvae were heavier than those from NY. Temperature and its influence on larval weight had profound impacts on whether a larva proceeded to pupation. Based on the temperature effects detailed here, larval development and pupation should be possible in most of the continental United States where suitable hosts are available. These data can be used to develop a degree-day model to estimate beetle phenology; however, at least 2°C should be added to air temperatures to adjust for the mediation of temperature by the wood. These data provide a basis for predicting the potential geographical range of this species and for developing phenological models to predict the timing of immature stages, both of which are important for management programs.     

Cold tolerance and cold hardening strategy of the Japanese pine sawyer Monochamus alternatus (Coleoptera: Cerambycidae)

The Japanese pine sawyer, Monochamus alternatus , is an important pine forest pest and vector transmitting the pine wilt nematode that causes pine wilt disease. Low temperatures in autumn, winter and spring often differentially affect mortality of M. alternatus larvae. In this paper, we mainly compared the differences of mortality and cold hardening of larvae from different seasons, based on supercooling point (SCP) and cumulative probability of individuals freezing (CPIF). The cold hardening of the larvae from autumn, winter and spring seasons were largely different. Correlations between mortality and CPIF of autumn and spring larvae were highest on day 1/4, and gradually decreased with prolonged exposure duration. This beetle's death mainly resulted from freezing in short exposure duration. However, the correlation between mortality and CPIF of winter larvae increased gradually with the prolonged exposure duration. Death did not mainly result from freezing in long exposure duration. Autumn larvae are more susceptible and adaptable than winter and spring larvae. Winter larvae have a slight freeze-tolerance trend. Our research showed that M. alternatus came into complex cold-hardening strategies under natural selection. Freeze avoidance is the primary strategy; with prolonged exposure duration to above SCP or < 0 °C, chill tolerance is more important; this is followed by freeze tolerance during harsh winters.     

温度对松褐天牛传递松材线虫数量的影响

松材线虫病是一种重要的检疫性病害,松褐天牛是传播松材线虫的主要媒介昆虫.本实验研究了温度对松褐天牛传递松材线虫数量的影响.设置22℃、28℃和34℃ 3个温度梯度,结果表明松褐天牛成虫的寿命随温度升高而缩短,其中雌虫寿命短于雄虫,且羽化进度不太一致,多数松褐天牛携带的松材线虫量在1000～9999条之间.通过对松褐天牛取食传入松枝的松材线虫量的追踪记录发现,随着温度升高,传播线虫的高峰时段随之提前.34℃高温下松褐天牛的传递率最低,表明高温对松材线虫病的发生具有抑制作用.     

Temperature Effects on the Transmission of Bursaphelenchus xylophilus (Nemata: Aphelenchoididae) by Monochamus alternatus (Coleoptera: Cerambycidae)

The object of this research was to investigate the effects of ambient temperature on the transmission of the pinewood nematode, Bursaphelenchus xylophilus, by its vector, Monochamus alternatus. Ninety M. alternatus were reared individually at one of three constant temperatures (16, 20, and 25 °C). As the ambient temperature decreased from 25 to 16 °C, longevity of vectors decreased, nematode transmission efficiency decreased, and the peak period of nematode transmission was delayed and its peak height decreased. Low temperature may inhibit the transmission process, and this inhibition could be partially responsible for preventing pine wilt disease from devastating pine forests in cool regions.