褐飞虱若虫中肠凋亡细胞的检测

为揭示褐飞虱Niloparvata lugens Stl若虫在发育过程中中肠的凋亡细胞,使用末端脱氧核苷酸转移酶介导的dUTP-生物素断端标记法(TUNEL)进行中肠组织切片检测,结果表明,1～5龄若虫中肠分别存在2%～5%的凋亡细胞。利用4′,6-二脒基-2-苯基吲哚二盐酸(DAPI)染色法检测表明,存在Ⅰ,Ⅱa和Ⅱb期凋亡的细胞核,其特征包括染色体浓缩、边缘化及细胞核碎裂。透射电子显微镜检测结果表明,早期凋亡的细胞呈现染色质浓缩、边缘化特征,晚期凋亡的细胞出现细胞核碎裂、形成凋亡小体及细胞质空泡化等。本研究揭示了在正常发育过程中褐飞虱若虫中肠有少量的细胞发生了凋亡。通过人工干预的方式调控中肠细胞的凋亡进程有可能使之成为防治该水稻害虫的新靶标。     

褐飞虱抗药性研究现状

褐飞虱Nilaparvata lugens(Stal)对杀虫剂产生抗药性是其近年来暴发频繁的重要原因。文章综述国内外关于褐飞虱抗药性的研究成果,包括褐飞虱抗性测定方法、抗药性的发展、交互抗性、抗性遗传、抗性机理及抗性治理等。田间褐飞虱种群对新烟碱类药剂产生不同程度的抗药性,其中对吡虫啉产生高水平到极高水平抗性,对氯噻啉和噻虫嗪分别产生中等水平和低水平的抗药性,对呋虫胺和烯啶虫胺仍然处于敏感性阶段。此外,褐飞虱种群对噻嗪酮(昆虫生长调节剂)产生低水平到中等水平抗性。长期大面积使用化学药剂是褐飞虱产生抗药性的重要原因。因此,必须加强褐飞虱的抗性治理,以延缓其抗药性进一步发展。     

褐飞虱抗吡虫啉品系生物适合度研究

毒力测定结果显示 ,虽然褐飞虱NilaparvatalugensSt l田间品系对吡虫啉还没有表现出明显的抗性 ,但室内筛选品系已经出现了一定水平的抗性 ,抗性品系 1 (R1)和抗性品系 2 (R2 )的抗性倍数分别为 2 5 3 8和 68 92。通过建立抗性品系、田间种群和敏感品系的生命表 ,发现抗性品系适合度显著下降 ,而且R2 品系下降的幅度明显大于R1品系。存在显著变化的因素有低龄若虫存活率、成虫羽化率、交配率、产卵量和孵化率下降 ,雌虫寿命缩短 ;R2 品系还表现为卵历期延长 ,3龄若虫至 5龄若虫存活率下降。R2 品系产卵高峰期迟 ,高峰期日虫量显著低于敏感品系和田间种群。用种群数量趋势指数 (I)来确定抗性品系的相对生物适合度 ,发现与敏感性品系相比 ,R1品系和R2 品系的相对适合度分别为0 60 9和 0 2 45。     

The population characteristics of the brown plant hopper, Nilaparvata lugens, in the Philippines

Abstract. 1. Population characteristics of the brown planthopper, Nilaparvata lugens (Stål), were investigated over eight growing seasons on rainfed rice in the Philippines.
2. Three nymphal generation peaks were observed in most seasons. The third peak was not always the largest and peaks varied considerably in size from season to season.
3. Generation peaks were less distinct at a second sampling site within an irrigation system, possibly associated with increased asynchrony of planting and the consequent increased immigration potential.
4. Mortality was highest for eggs and first instar nymphs. Egg to adult survivorship was estimated as 1–12%.     

Characterization of neutral lipases revealed the tissue-specific triacylglycerol hydrolytic activity in Nilaparvata lugens

The lipid metabolism plays an essential role in the development and reproduction of insects, and lipases are important enzymes in lipid metabolism. In Nilaparvata lugens, an important insect pest on rice, triacylglycerol hydrolytic activities were different among tissues, with high activity in integument, ovary, and fat body, but low activity in intestine. To figure out the tissue-specific triacylglycerol hydrolytic activity, we identified 43 lipases in N. lugens. Of these 43 lipases, 23 belonged to neutral lipases, so this group was selected to perform further experiments on triacylglycerol hydrolysis. The complete motifs of catalytic triads, β9 loop, and lid motif, are required for the triacylglycerol hydrolytic activity in neutral lipases, which were found in some neutral lipases with high gene expression levels in integument and ovary, but not in intestine. The recombinant proteins of 3 neutral lipases with or without 3 complete motifs were obtained, and the activity determination confirmed the importance of 3 motifs. Silencing XM_022331066.1, which is highly expressed in ovary and with 3 complete motifs, significantly decreased the egg production and hatchability of N. lugens, partially through decline of the lipid metabolism. In summary, at least one-third of important motifs were incomplete in all neutral lipases with high gene expression in intestine, which could partially explain why the lipase activity in intestine was much lower than that in other tissues. The low activity to hydrolyze triacylglycerol in N. lugens intestine might be associated with its food resource and nutrient components, and the ovary-specific neutral lipases were important for N. lugens reproduction.     

Characterization of a Nilaparvata lugens (Stål) brummer gene and analysis of its role in lipid metabolism

The brummer (bmm) genes encode the lipid storage droplet‐associated triacylglycerols (TAG) lipases, which belong to the Brummer/Nutrin subfamily. These enzymes hydrolyze the ester bonds in TAG in lipid metabolism and act in insect energy homeostasis. Exposure to some agricultural chemicals leads to increased fecundity, which necessarily involves lipid metabolism, in some planthopper species. However, the biological roles of bmm in planthopper lipid storage and mobilization have not been investigated. Here, the open reading frame (ORF) of bmm (Nlbmm) was cloned and sequenced from the brown planthopper (BPH; Nilaparvata lugens). The ORF is 1014 bp encoding 338 amino acid residues. Nlbmm contained patatin domains and shared considerable evolutionary conservation with other insect bmms. Nlbmm is highly expressed in the fat body, consistent with its roles in lipid metabolism. Injection with Nlbmm double‐stranded RNA (dsNlbmm) led to reduced Nlbmm mRNA accumulation, but did not influence expression of several genes related to lipid synthesis including acyl‐CoA‐binding protein (ACBP), acetyl‐CoA carboxylase (ACC), and a lipophorin receptor (LpR). Nlbmm knockdown led to increased TAG contents in whole bodies, accumulation of total fat body lipid, and decreased hemolymph lipid content. Nlbmm knockdown did not influence the synthesis and distribution of glycerol. We infer that Nlbmm acts in TAG breakdown and fat metabolism in N. lugens.     

褐飞虱ABCB基因的克隆与表达分析

三磷酸腺苷结合盒转运蛋白(ATP-binding cassette,ABC)广泛分布于各种生物体中,是最大的膜蛋白家族之一。在昆虫中,ABC转运子不仅在分子转运过程中有重要的功能,同时在杀虫剂抗性、代谢和发育中都起到重要的作用。本研究克隆和鉴定了褐飞虱Nilaparvata lugens St l ABCB基因(NlABCB),结果表明NlABCB基因全长1468 bp,含有一个长度为459 bp的开放阅读框,编码153个氨基酸。利用定量PCR检测NlABCB基因在褐飞虱体内的表达,发现NlABCB转录本在褐飞虱的所有发育阶段都有表达,在1龄若虫中表达量最低,随着生长发育表达量逐渐升高。NlABCB在褐飞虱5龄若虫的中肠中表达量最高。研究发现该基因与褐飞虱的生理活动有关。构建NlABCB-dsRNA载体,转化大肠杆菌Escherichia coli HT115后,经IPTG诱导,形成NlABCB-dsRNA,喂养褐飞虱取食含NlABCB-dsRNA人工饲料,发现褐飞虱的存活率明显降低。因此该基因的克隆和功能鉴定,对于作物的抗虫育种,尤其对水稻害虫的生物防治将起到重要的促进作用。     

Carboxylesterase genes in nitenpyram-resistant brown planthoppers,Nilaparvata lugens

Carboxylesterases (CarEs) represent one of the major detoxification enzyme families involved in insecticide resistance. However, the function of specific CarE genes in insecticide resistance is still unclear in the insect Nilaparvata lugens (Stål), a notorious rice crop pest in Asia. In this study, a total of 29 putative CarE genes in N. lugens were identified, and they were divided into seven clades; further, the β-esterase clade was significantly expanded. Tissue-specific expression analysis found that 17 CarE genes were abundantly distributed in the midgut and fat body, while 12 CarE genes were highly expressed in the head. The expression of most CarE genes was significantly induced in response to the challenge of nitenpyram, triflumezopyrim, chlorpyrifos, isoprocarb and etofenprox. Among these, the expression levels of NlCarE2, NlCarE4, NlCarE9, NlCarE17 and NlCarE24 were increased by each insecticide. Real-time quantitative polymerase chain reaction and RNA interference assays revealed the NlCarE1 gene to be a candidate gene mainly involved in nitenpyram resistance, while simultaneously silencing NlCarE1 and NlCarE19 produced a stronger effect than silencing either one individually, suggesting a cooperative relationship in resistance formation. These findings lay the foundation for further clarification of insecticide resistance mediated by CarE in N. lugens.     

Induction of P450 genes in Nilaparvata lugens and Sogatella furcifera by two neonicotinoid insecticides

Nilaparvata lugens and Sogatella furcifera are two primary planthoppers on rice throughout Asian countries and areas. Neonicotinoid insecticides, such as imidacloprid (IMI), have been extensively used to control rice planthoppers and IMI resistance consequently occurred with an important mechanism from the over‐expression of P450 genes. The induction of P450 genes by IMI may increase the ability to metabolize this insecticide in planthoppers and increase the resistance risk. In this study, the induction of P450 genes was compared in S. furcifera treated with IMI and nitromethyleneimidazole (NMI), in two planthopper species by IMI lethal dose that kills 85% of the population (LD₈₅), and in N. lugens among three IMI doses (LD₁₅, LD₅₀ and LD₈₅). When IMI and NMI at the LD₈₅ dose were applied to S. furcifera, the expression changes in most P450 genes were similar, including the up‐regulation of nine genes and down‐regulation of three genes. In terms of the expression changes in 12 homologous P450 genes between N. lugens and S. furcifera treated with IMI at the LD₈₅ dose, 10 genes had very similar patterns, such as up‐regulation in seven genes, down‐regulation in one gene and no significant changes in two genes. When three different IMI doses were applied to N. lugens, the changes in P450 gene expression were much different, such as up‐regulation in four genes at all doses and dose‐dependent regulation of the other nine genes. For example, CYP6AY1 could be induced by all IMI doses, while CYP6ER1 was only up‐regulated by the LD₅₀ dose, although both genes were reported important in IMI resistance. In conclusion, P450 genes in two planthopper species showed similar regulation patterns in responding to IMI, and the two neonicotinoid insecticides had similar effects on P450 gene expression, although the regulation was often dose‐dependent.     

单季稻田的蜘蛛群落及其与褐飞虱的相关性研究

通过对南京地区４种不同类型单季稻田蜘蛛群落的结构及其与褐飞虱的相关性研究,结果表明,该地区蜘蛛群落由１０科２１种组成,其中草间小黑蛛、食虫瘤胸蛛、拟水狼蛛为优势种。物种丰富度、多样性化防田低于生防田,而均匀性则施药田大于不施药田。不同移栽时间的稻田,丰富度、多样性、均匀性迟栽田低于早栽田。蜘蛛与褐飞虱各发育阶段的相关性分析表明,蜘蛛与褐飞虱长翅成虫相关性好,而与低龄若虫相关性差;蜘蛛与褐飞虱的相关性主要由褐飞虱田间第１次若虫高峰时的蜘蛛量和蛛虱比决定,模拟的回归方程为：ｒ＝ － ０．１９９７＋ ０．００７５６ｘ１＋ ０．３３７１ｘ２（Ｆ＞ Ｆ０．０２３）。     

褐飞虱对吡虫啉抗性监测方法的研究

从结果的稳定性、准确性和操作方便等角度出发 ,建立了适合于监测褐飞虱 (NilaparvatalugensSt l)对吡虫啉抗性的稻苗浸根法。与其他方法相比 ,该方法除具有简单易行的特点外 ,还表现出很好的监测准确性。通过建立不同虫态和不同若虫龄期敏感基线以及对桂林、安庆、东台 3个地区的抗性监测 ,发现 3地褐飞虱对吡虫啉都处于敏感性下降阶段 ,安庆和东台地区的敏感性弱低于桂林     

大尺度下褐飞虱种群空间结构初步分析

褐飞虱是我国主要的水稻害虫,有着复杂的空间结构.地统计学可用来分析空间相关变量的结构和插值模拟空间相关变量的分布.运用地统计学中的变差函数和克立格插值法对广东省褐飞虱种群3个主要世代在早稻田间的空间结构进行分析和模拟的结果表明,大尺度的褐飞虱田间种群的空间分布为聚集型,不同世代褐飞虱种群的局部空间结构有差异,聚集的范围分别为400、200和205km;空间分布模拟地图能反映褐飞虱种群在田间的具体分布情况,广东西部地区每一世代褐飞虱的发生密度均高于东部地区.     

褐飞虱在香稻上的发生与产卵特征

研究褐飞虱NilaparvatalugensStl在香稻上的危害与产卵特征,结果表明香稻田中褐飞虱的自然种群密度基数远高于非香型稻。从7月22日至8月31日香稻田中褐飞虱的自然种群密度一直增长,在武香988和鄂香1号田间的若虫分别从7月22日的7头丛和3头丛上升至8月31日的126头丛和73头丛。香型稻田间的褐飞虱的自然种群密度增长慢于非香型稻田间。香稻与非香稻田间褐飞虱成虫出现的高峰日相差不大。香稻田中褐飞虱产卵株率高于非香型稻田,其中最高的为鄂香1号,达到74.27%。香稻田间株平均产卵块数几乎为非香型稻上的2倍,但是香稻上的卵死亡率(鄂香1号为39.24%)显著高于非香型稻上的(鄂中5号为27.58%,两优培九为27.21%),且产卵部位有一定差异。鄂香1号第Ⅲ叶鞘上卵块最多,占总数的60.98%,武香988的第Ⅱ和第Ⅳ叶鞘上产卵较多,第Ⅰ叶鞘上没有稻飞虱产卵。非香型稻鄂中5号上产卵最多的是第Ⅳ叶鞘,第Ⅰ和第Ⅱ叶鞘上没有褐飞虱产卵。两优培九除第Ⅰ和第Ⅴ叶鞘上没有褐飞虱产卵外,第Ⅱ、第Ⅲ和第Ⅳ叶鞘上均有褐飞虱产卵,而且卵量差异不大。     

大尺度下褐飞属种群空间结构初步分析

褐飞虱是我国主要的水稻害虫,有着复杂的空间结构,地统计学可用来分析空间相关变量的结构和插植模拟空间相关变量的分布,运用地统计学中的变差函数和克立格插值法对广东省褐飞虱种群3个主要世代在早稻田间的空间结构进行分析和模拟的结果表明,大尺度的褐飞虱田间种群的空间分布为聚集型,不同世代群在田间的具体分布情况,广东西部地区第一世代褐飞虱的发生密度均高于东部地区。     

褐飞虱乙酰胆碱酯酶基因全长cDNA的克隆及序列分析

利用反转录聚合酶链式反应(RT_PCR)结合快速扩增cDNA末端(RACE)技术克隆了褐飞虱Nilaparvata lugens 乙酰胆碱酯酶基因cDNA。该cDNA全长2 467 bp,包含一个1 938 bp的开放阅读框(GenBank登录号AJ852420); 在推导出的646个氨基酸残基的前体蛋白中, N端的前30个氨基酸残基为信号肽,随后的616个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为69 418 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser242,Glu371和His485),以及在亚基内形成二硫键的6个半胱氨酸完全保守; 位于催化功能域的14个芳香族氨基酸中有10 个完全保守。该酶的氨基酸序列与黑尾叶蝉的同源性最高,达83%。对来自23种昆虫（包括褐飞虱）的30个乙酰胆碱酯酶的聚类分析显示,褐飞虱的乙酰胆碱酯酶与其中6个Ⅱ型乙酰胆碱酯酶（AChE2）同属一个支系; 此外,只存在于昆虫AChE2中的超变区及特异的氨基酸残基,也存在于褐飞虱的乙酰胆碱酯酶中。以上结果表明,所克隆的褐飞虱的乙酰胆碱酯酶基因是一个与黑腹果蝇的orthologous型基因同源的AChE2基因。     

褐飞虱与白背飞虱共栖时的互作效应

用笼罩试验方法,研究了褐飞虱和白背飞虱共栖时的互作效应．结果表明,在９６ ｈ内,随时间的延长,２种飞虱对不同稻株的栖息率渐趋增加,最终达２１．５～２５．０％,不表 现种间差异;脉冲试验使白背飞虱的雌性率和短翅率提高 ２２． １％和 ３９８． １％;排除试验使 白背飞虱的增殖、雌性率和雌虫短翅率各下降 ８． ８％、２０． ８％和 ４８． ０％;种间作用对增殖。 性比和翅型产生正的或负的效应．文中还讨论了种间作用存在的意义和方式．     

褐飞虱对抗性水稻品种Mudgo个体致害性指标

在28±1℃下,观测了褐飞虱Nilaparvata lugens羽化24?h内短翅型雌成虫在敏感水稻品种TN1及抗性品种Mudgo上的寿命及体重增量。结果表明,取食抗性品种时,试虫的平均增重与寿命之间表现出明显的正相关。寿命在7天内的个体,存活期间平均体重增量为负值,而存活7天以上的个体则平均增重明显。进一步研究了初羽化的褐飞虱长翅型雌成虫在TN1及Mudgo上取食第1～4天的体重增量、寿命及产卵量,结果发现试虫在Mudgo上的这3个生物学参数之间存在明显正相关。其中,寿命及体重增量较好地反映出害虫个体对抗性品种的致害能力,可作为个体致害性指标。根据研究结果,提出将羽化后在Mudgo上存活7天以上或最初4天内体重增量大于0.1mg的雌成虫定为能够致害该抗性品种的个体。     

褐飞虱对马拉硫磷的抗性遗传和交互抗性研究

毒力测定显示,抗马拉硫磷品对杀螟松、二嗪磷、异丙威和仲丁威都有明显的交互抗性产生,但对醚菊酯和吡虫啉没有表现出显的交互抗性。抗性遗传研究显示,正交和反交后代F1和F1'的显性值D分别为0.4014和0.3780,说明抗性主效基因是不完全显性的。通过自交后代F2和回交后代BC的LD-p线观察及与期望曲线比较,证明抗性遗传由两个或两个以上的基因控制。