CDNA cloning and characterization of S6 kinase and its effect on yolk protein gene expression in the oriental fruit fly Bactrocera dorsalis (Hendel)

p70 S6 kinase (S6K), a serine/threonine protein kinase, is a downstream target of target of rapamycin (TOR) gene and an important regulator of protein synthesis responsible for cell growth and reproduction. In this study, a S6K gene, named BdS6K (GenBank Accession No. GQ203802), was isolated from the oriental fruit fly Bactrocera dorsalis (Hendel). Quantitative RT-PCR showed that BdS6K mRNA is expressed at a higher level in egg than in other developmental stages, as well as in ovary than in fat body. Downregulation of BdS6K activity by rapamycin treatment in larval stage resulted in the developmental defects of larvae, pupae, and adults, with a reduced yolk protein (YP) expression in the fat body throughout the first reproductive cycle with a substantial reduction in ovary size, and also repressed the egg development in female fruit fly. Knockdown of BdS6K gene by RNA interference in the adult significantly decreased the YP expression. These observations support the involvement of BdS6K signaling in the regulation of the YP synthesis and egg development in B. dorsalis.     

橘小实蝇交配行为观察

在实验室内对橘小实蝇Bactrocera dorsalis（Hendel）的交配行为进行了观察。描述了橘小实蝇交配中雄虫的“求偶场”,雄雄相遇、雌雌相遇、雌雄相遇时的相互反应,雌雄虫的交配行为与交配后行为。     

植物果实、颜色和形状对橘小实蝇产卵选择的影响

橘小实蝇Bactrocera dorsalis(Hendel)是近几年入侵江苏南部地区,严重危害当地水果、蔬菜的重要害虫。为深入了解其在该地区果树、蔬菜上的产卵特性,进行植物果实、颜色和形状对其产卵选择影响的研究。实验室中"纸杯法"的产卵选择试验结果表明,橘小实蝇对不同水果的产卵偏好性由高到低依次为:柑橘、香蕉、芒果、枣、石榴、苹果、桃子、梨、李、葡萄、柿;对不同蔬菜的产卵偏好性由高到低依次为:丝瓜、豇豆、苦瓜、黄瓜、茄子、南瓜、青椒、番茄。对不同寄主颜色和形状的产卵选择特点为,雌虫常选择桔黄色、绿色,以及圆球形的寄主产卵,而较少选择黑色、方形的寄主产卵。从试验结果可以看出,寄主果实散发出的气味,及其颜色和形状对橘小实蝇的产卵选择有影响。     

土壤含水量对桔小实蝇蛹期存活的影响

浸水时间、土壤含水量对桔小实蝇Bactroceradorsalis(Hendel)蛹的存活及成虫羽化的影响的研究结果表明:(1)不同浸水时间对桔小实蝇蛹存活率的影响不同。浸水时间越长,蛹存活率越低;浸水对初期蛹的影响较大,蛹龄越大,浸水作用效果越低。浸水时间(TW)、蛹期(AP)和蛹存活率(SP)之间关系符合以下模型:SP=0.4165-0.00307TW+0.2290AP-0.00004496T2W-0.0002528A2P+0.00008258TWAP。(2)土壤含水量对桔小实蝇成虫羽化影响明显。当土壤含水量较低或较高时,羽化率都明显受到抑制。相对含水量在30%～80%之间,蛹的羽化率较高。相对含水量(Ws)和成虫羽化率(E)之间存在以下关系:E=0.396+0.01985W-0.000181W2。     

桔小实蝇气味结合蛋白BdorOBP2的cDNA克隆、组织表达及配基结合特性

为了研究桔小实蝇Bactrocera dorsalis气味结合蛋白(odorant-binding proteins, OBPs)参与其嗅觉识别过程中的功能及其与植物气味的结合特性, 本研究克隆了桔小实蝇的一个气味结合蛋白基因, 命名为BdorOBP2（GenBank登录号为KC773766）, 并对该基因进行了原核表达。BdorOBP2开放阅读框长447 bp, 编码148 个氨基酸, 具有典型的6个半胱氨酸位点。定量PCR结果显示, 桔小实蝇BdorOBP2在不同组织中均有表达, 其中头部中的表达量最高, 翅中表达量最低（为头部表达量的63%±6%）。构建了BdorOBP2原核表达载体, 诱导并获得了重组BdorOBP2并进行了亲和层析纯化。最后以N-苯基-1-萘胺（N-phenyl-1-naphthylamine, 1-NPN）为荧光探针, 利用荧光竞争结合实验测定了重组BdorOBP2与7种主要寄主水果气味物质的结合能力, 发现其对多数酯类和醛类化合物亲合力较强, 亲合力最强的气味物质为反-2-己烯醛和β-紫罗兰酮, 结合常数KD分别为9.96和15.37 μmol/L。本研究结果可为高效地开发和设计桔小实蝇的嗅觉引诱剂配方提供一定的理论依据和参考。     

病原线虫对桔小实蝇种群的控制作用

通过室内和田间实验研究了昆虫病原线虫对桔小实蝇Bactrocera (Bactrocera) dorsalis (Hendel)的控制作用。室内实验结果表明,供试的3种线虫的4个品系（小卷蛾斯氏线虫Steinernema carpocapsae All品系与A24品系,夜蛾斯氏线虫Steinernema feltiae SN品系和嗜菌异小杆线虫Heterorhabditis bacteriophora H06品系）,以小卷蛾斯氏线虫All品系对桔小实蝇的侵染力最强,其3天的LD₅₀和LD₉₅分别为35.0和257.1条/cm²土壤。按300条/cm²土壤的量施用,小卷蛾斯氏线虫All品系对当代桔小实蝇的控制效果为86.3%。用以虫期作用因子组建的生命表方法评价了小卷蛾斯氏线虫All品系对田间桔小实蝇下代种群的控制作用,结果表明,按300条/cm²土壤的量施用线虫,对照杨桃园的桔小实蝇种群趋势指数为105.9,而处理杨桃园的桔小实蝇种群趋势指数下降为15.5;小卷蛾斯氏线虫All品系对桔小实蝇的干扰控制指数为0.146,即线虫处理果园的下代种群密度仅为对照果园的14.6%。     

橘小实蝇的发生特点与综合防治技术

橘小实蝇(Bactrocera dorsalis)在福建省寄主范围很广,并严重为害寄主。它的主要为害期是每年的4-11月。生产实践中采用综合治理与农业防治技术有效地控制其为害。     

拒避-诱杀组合技术对橘小实蝇产卵的影响

本文研究了以矿物油(mineral spray oil)乳剂为保护剂、"猎蝇"蛋白质毒饵为诱杀剂组成的拒避-诱杀组合技术对橘小实蝇Bactrocera dorsalis (Hendel)产卵的控制作用。结果显示:橘小实蝇在采用拒避-诱杀组合技术保护的供试香蕉上平均产卵孔数为0.000±0.000个,果实完好率达到100%,而单独使用"猎蝇"蛋白质毒饵的平均产卵孔数为27.000±1.732个,果实完好率仅为5.55%,表明该组合技术对橘小实蝇产卵的控制效果显著。在100倍～300倍浓度范围内,矿物油浓度越高,拒避-诱杀组合对橘小实蝇产卵的控制效果越好;随着虫口密度的增加,毒饵使用量和施用面积也须相应增加,才能取得更好的防治效果。     

桔小实蝇对敌百虫抗性稳定性及再增长趋势

通过室内试验,研究了用LC₅₀及LC₉₀两种浓度敌百虫汰选14代建立的不同程度高抗性水平的桔小实蝇Bactrocera dorsalis抗性品系TrR₅₀和TrR₉₀在停止使用药剂后的抗性衰退规律,以及抗性衰退到原有水平的1/3时再用药汰选的抗性再增长规律。结果表明：两抗性品系对敌百虫抗性均不稳定,但抗性衰退速率不同,TrR₉₀完全隔离药剂4代后,抗性已衰退至原有水平的1/3,而TrR₅₀完全隔离药剂7代后,抗性才衰退至原有水平的1/3,但随后两品系抗性衰退均减缓,到19代后抗性均还处于低水平抗性阶段,无法衰退至敏感水平。通过对其抗性衰退趋势进行方程拟合,结果表明两品系抗性衰退均符合S型曲线模型。抗性再增长试验结果表明：桔小实蝇两抗性品系对敌百虫抗性再增长趋势不同,TrR₅₀继续使用药剂汰选,经过6代选育,抗性迅速上升,接近原有的抗性水平,随后保持平稳增长; 而TrR₉₀继续使用药剂汰选,经过9代选育,抗性才迅速上升,经过12代选育,抗性才接近原有的抗性水平。通过对两品系抗性再增长趋势进行方程拟合,结果表明两品系抗性再增长趋势不同,TrR₅₀品系抗性再增长趋势符合逻辑斯蒂模型,而TrR₉₀品系则符合二次曲线模型。     

Inferences on the population structure and colonization process of the invasive oriental fruit fly, Bactrocera dorsalis (Hendel)

The phytophagous insects of the Tephritidae family offer different case histories of successful invasions. An example is Bactrocera dorsalis sensu stricto, the oriental fruit fly which has been recognized as a key pest of Asia and the Pacific. It is known to have the potential to establish adventive populations in various tropical and subtropical areas. Despite the economic risk associated with a putative stable presence of this fly, the genetic aspects of its invasion process have remained relatively unexplored. Using microsatellite markers we have investigated the population structure and genetic variability in 14 geographical populations across the four areas of the actual species range: Far East Asia, South Asia, Southeast Asia and the Pacific Area. Results of clustering and admixture, associated with phylogenetic and migration analyses, were used to evaluate the changes in population genetic structure that this species underwent during its invasion process and establishment in the different areas. The colonization process of this fly is associated with a relatively stable population demographic structure, especially in an unfragmented habitat, rich in intensive cultivation such as in Southeast Asia. In this area, the results suggest a lively demographic history, characterized by evolutionary recent demographic expansions and no recent bottlenecks. Cases of genetic isolation attributable to geographical factors, fragmented habitats and/or fruit trade restrictions were observed in Bangladesh, Myanmar and Hawaii. Regarding the pattern of invasion, the overall genetic profile of the considered populations suggests a western orientated migration route from China to the West.     

利用微卫星标记初步分析橘小实蝇4个地理种群的遗传多态性

橘小实蝇Bactrocera dorsalis(Bactrocera)(Hendel)是我国重要的检疫性害虫,对果蔬生产及其国际贸易造成很大影响。作者运用微卫星分子标记技术,用6对微卫星引物对采自我国福建、广东、云南3个地区及邻近国越南橘小实蝇种群间的遗传关系进行了初步分析,结果显示上述4个橘小实蝇地理种群间存在一定的遗传差异,地理隔离是造成这一差异的主要原因。     

矿物油乳剂拒避橘小实蝇的有效组分分析

高品质的矿物油乳剂可用于有机食品生产,在害虫综合治理中有良好的应用前景。矿物油是包含多种化学成分的混合物,其组成成分和比例决定其理化性质并关系到其防治病虫害的效果。已有研究报道表明,矿物油乳剂对许多害虫有拒避作用,但其机理及相关性组成成分尚未研究清楚。本实验测定了4种矿物油乳剂即SK绿颖农用喷洒油、加德士-路易夏用油、安波尔喷洒油、法夏乐石蜡油（分别简称为SK,Caltex,Ampol和Citrole油）和Caltex油＜120℃,＞120℃,＜105℃,105~120℃及SK油＜110℃、＞110℃等6种分子蒸馏组分的正构烷烃碳数当量nCy值、碳数分布值及其对橘小实蝇Bactrocera dorsalis的产卵拒避效果。室内生物测定结果显示,橘小实蝇在SK,Caltex,Ampol和Citrole油0.5%水乳液及清水浸蘸处理后的香蕉上平均产卵孔数分别为17.850±1.282,3.400±0.630,15.850±1.439,20.650±1.212和19.050±1.155,在同样方法处理的阳桃上平均产卵孔数分别为15.500±3.969,3.000±0.707,13.750±4.131,17.250±4.385和62.000±4.708。橘小实蝇在6种分子蒸馏组分0.5%水乳液处理后的香蕉上的平均产卵孔数分别为4.400±1.166,8.200±0.583,5.400±1.720,2.400±1.077,4.800±1.594和12.200±2.63,显示不同矿物油的拒避效果差异显著（P<0.05）,拒避效果最好的矿物油的正构烷烃碳数当量nCy值在nC23左右.矿物油对橘小实蝇的产卵拒避效果与其相应的碳数分布值的Spearman等级相关性分析显示,其拒避效果与矿物油中的C23组分的含量呈显著正相关关系（P<0.05）。这可为进一步分析矿物油乳剂拒避害虫的作用成分和探索其机理提供参考。     

Inducing and isolation of antibacterial peptides from oriental fruit fly, Bactrocera dorsalis Hendel

One antibacterial activity fraction from an immunized dipteran insect, Bactrocera dorsalis, was isolated and purified by prepurification, ion‐exchange chromatography, gel filtration chromatography and reverse‐phase high performance liquid chromatography (HPLC). The final purified fraction was checked on the Smart system HPLC and was judged as a pure fraction. The results of physical and biological analysis revealed that this fraction is heat stable and showed strong activities against Gram‐positive bacterial growth. It possesses antibicrobial peptide properties and is worth further investigation.     

Population dynamics of the oriental fruit fly, Bactrocera dorsalis （Diptera： Tephritidae） in the Kunming area,southwestern China

Population dynamics of Bactrocera dorsalis Hendel （Diptera： Tephritidae） were studied through pheromone trapping over 4 years （1997, 1999, 2000, 2003） in the Kunming region, a high plateau area in southwestern China. B. dorsalis immigrates from southern Yunnan to Kunming each year, and occurs during early May through November. Annual trap captures recorded an increase in the B. dorsalis populations from May to July, when they peaked in abundance, and a decline until November. No flies were detected from November to April. The fruit flies had two generations. There was considerable overlapping due to the continuous arrival of immigrating flies during the summer months. Annual capture rates were significantly related to numbers of flies caught in July when peak captures were recorded; whereas the peak captures, in turn, positively depended on numbers of flies recorded in May, the first month of fly appearance in the current year. It suggested that the annual population abundance was mainly dependent on the size of the initial emigrating population. A daily average temperature of 18℃ was probably the threshold temperature required for the flies to undertake long-range dispersal, which partially explained the start of the fly in May each year on this high plateau. Under field conditions, the fruit flies can withstand 13℃ as a daily average temperature. No flies were recorded in any of the study years at a daily temperature colder than 10 ℃.     

球孢白僵菌两种施用方式对侵染桔小实蝇的影响及田间的防治效果

室内研究了球孢白僵菌Beauveria bassiana不同施用方式对侵染桔小实蝇Bactrocera dorsalis的影响,以及田间喷施球孢白僵菌防治桔小实蝇的效果。结果表明,两种诱集感染方式桔小实蝇成虫存活率分别为40%、41.7%,差异不显著;添加性引诱剂在白僵菌侵染桔小实蝇的过程中没有起到明显地促进感病的作用,只是缩短了病程,IIPC为0.41-0.44,说明两种诱集感染方式对桔实蝇成虫有一定的控制作用。三种施菌方式的桔小实蝇羽化率、成虫死亡率与对照差异均显著;以IIPC为评价指标,施菌方式对桔小实蝇各因子的影响大小顺序为：成虫存活率 >羽化率>化蛹率,其中以对成虫存活率的影响最大。应用化学防治和球孢白僵菌田间防治桔小实蝇的效果表明,施用2500倍40%毒死蜱效果最好,控制作用达到71.9%,其次是喷施2.0×108孢子/mL球孢白僵菌孢子悬浮液,防效达46.9%,施用2500倍4.5%高效氯氰菊酯防治效果达最差, 防效仅37.5%。     

桔小实蝇不同发育阶段过冷却点的测定

对桔小实蝇Bactrocera dorsalis (Hendel)不同发育阶段的过冷却点进行了测定。结果表明：同一虫期个体间的过冷却点出现不同程度的变异,但均服从正态分布。不同虫期的过冷却点差异显著,其中蛹的过冷却点最低（-12.2℃～-15.0℃）。老熟幼虫的过冷却点为-8.1℃。成虫的过冷却点最低值为7日龄雄虫（-10.5℃）和雌虫（-10.1℃）,最高值为60日龄雄虫（-5.9℃）和雌虫（-6.4℃）,但同一发育时期的雌、雄成虫之间的过冷却点没有差异。测定结果提示蛹期最有可能是该虫在温带地区越冬的虫态。     

桔小实蝇抗敌百虫品系的实验种群生物学比较研究

【目的】研究明确桔小实蝇 Bactrocare dorsalis (Hendel) 抗药性品系与敏感品系的基本生物学特征以及相对适合度、内禀增长率等种群生物学参数差异。【方法】实验室条件下筛选出对敌百虫具中抗和高抗的桔小实蝇品系,观察记录其与敏感品系的生物学特性,比较三者之间的主要生物学参数。【结果】桔小实蝇敏感品系与2个抗性品系的卵、幼虫和蛹的发育历期、化蛹率、产卵前期、雌雄虫寿命、存活时间及雌虫比率均无显著差异。中抗品系卵孵化率和成虫羽化率分别为68.33%和93.73%,均显著低于敏感品系（分别为88.33%和97.93%）和高抗品系（分别为86.67%和98.21%）。与敏感品系的产卵量864.61粒/雌和高抗品系的产卵量750.70粒/雌相比,中抗品系的产卵量降低至630.87粒/雌。3个品系雌虫日产卵量动态规律比较一致,均呈开始产卵后短时间内进入产卵盛期,继而产量达到高峰,之后产卵量波动下降。但是与敏感品系相比,2个抗性品系雌虫较早进入产卵高峰。对成虫存活率动态拟合方程参数c值均大于1,符合I型存活曲线基本模型,表明在实验室理想条件下3个品系成虫均能达到其平均寿命。敏感品系种群趋势指数（I）最高,为339.41,其次为高抗品系（I =307.82）,最小为中抗品系（ I =175.79）,表明对敌百虫产生抗性抑制了桔小实蝇种群的增长,中抗品系抑制程度更大。敏感和高抗品系实验种群净增殖率较高,分别为327.89和284.29;中抗品系显著较低,为217.49。2个抗性品系内禀增长率（r_m）和周限增长率（λ）间无显著差异,且均显著大于敏感品系,世代历期（T）则显著缩短。基于种群净增殖率获得的敌百虫高抗和中抗品系相对适合度分别为0.8670和0.6633。【结论】在敌百虫的选择压力下,桔小实蝇抗药性品系的世代周期、中抗品系卵的孵化率和蛹的羽化率显著降低,其他多项生物学特征无显著变化。抗性品系的繁殖力和种群世代增长量受到抑制,以中抗品系更为明显;但与敏感品系相比,抗性品系种群增长潜力更大。     

云南桔小实蝇五个地理种群的遗传分化研究

对云南省瑞丽、景洪、化念、元江、河口的桔小实蝇5个地理种群共27个个体的线粒体细胞色素氧化酶Ⅰ(COⅠ) 基因进行了部分序列测定。序列的碱基变化中转换明显多于颠换、无碱基的插入和缺失。5个地理种群中共有27个多态位点和23种单倍型,其中2种为共享单倍型。桔小实蝇5个地理种群的Fst值在0.0364～0.1364之间(P>0.05), Nm值在3.88～13.25之间。对其所有单倍型聚类分析发现,单倍型在系统树中的分布散乱、混杂,没有显示出明显的地理分布族群。分析认为5个地理种群间已存有一定程度的遗传分化,但分化的程度还比较低。造成5个种群这种遗传分化的因素主要与地理隔离有关,而种群间低程度的遗传 分化估计与桔小实蝇在云南的发展历史及生态适应有关。     

Use of oligonucleotide arrays for molecular taxonomic studies of closely related species in the oriental fruit fly (Bactrocera dorsalis) complex

Molecular markers derived from intron sequence polymorphisms have been identified to augment and improve existing tools available for the analysis of closely related species found in the Bactrocera dorsalis species complex. Arrays of allele‐specific oligonucleotides incorporating these markers have been developed to allow both for rapid taxonomic identification and direct genotyping of individual specimens in single hybridization experiments.     

多杀菌素和荧光桃红B对橘小实蝇取食的影响

40mg/kg多杀菌素(spinosad)或1000、3000mg/kg荧光桃红B(Phloxine-B)均不影响3日龄橘小实蝇雌Bactrocera dorsalis(Hendel)、雄虫对蔗糖的取食量(P>0.05);与蒸馏水对照和马拉硫磷对照相比,40mg/kg多杀菌素或1000mg/kg荧光桃红B对雌、雄虫吐食率也无显著影响(P>0.05),但3000mg/kg荧光桃红B对雌、雄虫吐食率有极显著影响(P<0.01)。40mg/kg多杀菌素或1000mg/kg荧光桃红B对6日龄橘小实蝇雌、雄虫对蔗糖的取食量与蒸馏水对照无差异(P>0.05);3000mg/kg荧光桃红B对雌虫的取食量与蒸馏水对照有显著差异(P<0.05),与马拉硫磷对照无差异(P>0.05),对雄虫的取食量与蒸馏水对照无显著差异(P>0.05),与马拉硫磷对照有显著差异(P<0.05)。与蒸馏水、马拉硫磷相对照,40mg/kg多杀菌素或1000mg/kg荧光桃红B对雌、雄虫的吐食率均无显著影响(P>0.05),而3000mg/kg的荧光桃红B对雌、雄虫的吐食率均有极显著影响(P<0.01)。