Antibiotic exposure perturbs the bacterial community in the small brown planthopper Laodelphax striatellus

Bacteria symbionts in herbivores play an important role in host biology and ecology, and are affected by environmental factors such as temperature, diet, habitat, antibiotics and so on. However, the effects of antibiotics on the microbiome of the small brown planthopper Laodelphax striatellus (SBPH) remain unclear. Here, we studied the effects of tetracycline on the diversity and composition of bacterial colonies in different tissues of SBPH using high throughput sequencing of 16S ribosomal RNA amplicons. Our results show that Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria were most abundant in SBPH, and the genera Asaia and Wolbachia were most abundant in all body parts of SBPH. Antibiotic treatment had persistent effects on the composition of the SBPH microbiome. Tetracycline depleted the population of Firmicutes, Bacteroidetes, Tenericutes and Fusobacteria, and nearly 100% eliminated Wolbachia, Bacteroides and Abiotrophia in SBPH. Together, these results suggest that antibiotic exposure affects the bacteria symbionts of different body parts in SBPH and will facilitate future studies of the bacterial symbionts of arthropod hosts.     

High temperature determines the ups and downs of small brown planthopper Laodelphax striatellus population

Small brown planthopper, Laodelphax striatellus (Fallén) numbers usually drop sharply in the summer and revive quickly in the autumn. However, it is unclear whether and how the high temperature plays a role in this process. The effects of durations of heat exposure (33°C) on life‐history traits were examined here. Exposure of adults for 1 day during the oviposition stage led to a very low survival of nymphs. The average longevity of L. striatellus exposed for 1–31 days from oviposition was significantly longer than that of the control (27°C). Short‐term (1–5 days) heat exposure of the third instar nymphs did not significantly influence eclosion, but exposure of the fourth instar nymphs significantly increased eclosion. Lifespan from egg to adult was significantly lengthened when the third instar nymphs were exposed to heat for 2–15 days, or the fourth instar were exposed for 10 days. The preoviposition period was prolonged by heat exposure of the third or fourth instar nymphs. Short‐term heat exposure of less than 3 days of the third or fourth instar nymphs did not restrict fecundity, but when the exposure duration exceeded 5 days the total eggs per female and hatchability decreased. Exposure to high temperature increased the brachypter rate of adults. In summary, low survival and slowing development under heat exposure resulted in population decline in the summer, and the relatively high fecundity and brachypter rate led to quick revival in autumn. Temperature in the summer determines the rise and fall in numbers of L. striatellus.     

灰稻虱的饲养和饲毒

灰稻虱的饲养 ,虫源可从田间直接捕获繁殖 ,饲料用稻、麦、稗均可 ,但以稗草为最佳。饲毒须用低龄若虫在罹病小麦或水稻上吸毒 3天以上 ,无毒虫饲毒后须经过 1 5天以上的循徊期后才能用于感染接种。所用的带毒病株可直接从田间寻获 ,若需自己制备 ,可将通过了循徊期的带毒灰稻虱接种到麦苗或稻苗 (3～ 5叶期为宜 )上传毒 ,待病苗症状表现后用于饲毒。本文详细介绍了 1～ 3代灰稻虱饲养饲毒的方法和步骤。     

高温及水稻类型对灰飞虱种群的影响

近年来,灰飞虱Laodelphax striatellus(Fall n)在江浙等地呈明显上升态势。采用不同温度及水稻类型处理灰飞虱若虫,获得灰飞虱种群在不同处理组合下的存活率、发育进度及抗寒力。结果表明,35℃的高温下,灰飞虱各龄若虫无论在杂交籼稻还是粳稻上,其发育进度均比25~30℃推迟2~7d。35℃高温下若虫死亡率较高,并且不能正常羽化。粳稻上饲养的灰飞虱若虫的存活率明显高于杂交籼稻,但水稻类型和温度对灰飞虱存活率的影响不存在明显的互作作用。在适宜温度下(27~30℃)用杂交籼稻饲养的灰飞虱,其过冷却点及结冰点均要比在高温(35℃)或低温(25℃)下饲养的低。在26~30.5℃变温条件下,杂交籼稻上饲养的灰飞虱的过冷却点和结冰点显著低于粳稻上饲养的,而且不同温度和水稻类型上的灰飞虱的过冷却点均低于零下10℃。可见江浙稻区冬季低温对灰飞虱的越冬已不存在制约作用。温度和水稻类型的变化是近年灰飞虱暴发成灾的重要原因。     

南昌地区灰飞虱的生活史、繁殖和越冬生物学特性

为了探明灰飞虱Laodelphax striatellus (Fallén)在南昌地区的生物学特性, 本研究在实验室和自然条件下系统调查了该虫的年生活史, 温度对其生长发育、 繁殖、 性比及翅型分化的影响, 及其越冬生物学。结果表明, 灰飞虱在南昌一年发生4~7代。在18~32℃, 卵的发育历期随温度升高逐渐缩短。若虫的发育历期在18~28℃随温度升高逐渐缩短, 但若虫的发育历期在30和32℃时显著长于28℃的发育历期（P=0.000﹤0.05）。越冬个体的若虫期为143~187 d。卵和若虫的发育起点温度分别为10.17℃和7.51℃。在室外, 7月中旬高温下孵化的第4代若虫的发育历期也明显延长, 显示了高温诱导的夏季休眠现象。在18~28℃, 产卵前期随温度升高而逐渐缩短, 当温度上升到30℃时, 其产卵前期比26和28℃下有所延长。产卵期在22℃最长, 30℃下最短。成虫在20~24℃下的寿命最长。在28℃下, 平均每雌产若虫量最大。不论在室内还是在室外, 雌雄比均接近1∶1。在18~32℃, 羽化的成虫均以长翅型占绝对优势。在自然条件下, 越冬代和第6代羽化的成虫以短翅型占优势, 其他各代仍以长翅型占优势。在自然条件下, 9月中旬孵化的若虫就有少量个体滞育越冬, 10月中旬后孵化的若虫全部进入越冬。越冬若虫的龄期为1-5龄。本研究为该虫发生的预测及有效防控提供基础资料。     

红眼突变灰飞虱的生物学特性和交配优势

灰飞虱Laodelphax striatellus是我国重要水稻害虫之一, 表型变异可能影响灰飞虱的配偶选择性和种群特性。本实验采用生命表构建法研究了红眼突变灰飞虱的繁殖力、 内禀增长率等种群生命参数, 然后以1红眼雌虫、 1红眼雄虫和1纯合黑眼雄虫（♀rr×♂rr♂BB）或1红眼雄虫、 1红眼雌虫和1纯合黑眼雌虫（♀rr♀BB×♂rr）配对模式以及不同比例红眼成虫群体配对模式研究红眼突变对灰飞虱交配竞争力的影响。结果表明红眼突变灰飞虱除若虫死亡率（17.1%）显著高于正常个体（10.3%） (P<0.05)外, 其他生物学指标如繁殖力、 卵孵化历期、 若虫发育历期、 成虫寿命、 内禀增长率等与正常个体无显著差异。但红眼突变对灰飞虱配偶选择性有一定的影响。♀rr×♂rr♂BB和♀rr♀BB×♂rr组合F1代红眼和黑眼分离比例卡方试验值为18.15和14.99, 大于理论值。当红眼与黑眼个体以2︰8比例配对时, 红眼灰飞虱雄虫存在交配优势, 其后代红眼个体数大于期望值。这些结果为进一步研究灰飞虱红眼突变机理和利用红眼基因标记研究灰飞虱遗传分化奠定了基础     

灰飞虱海藻糖酶基因的克隆及RNA干扰效应

RNA干扰（RNAi）不但可以用于研究基因的功能, 还可以通过沉默靶标基因干扰特定的生命过程。因此, 通过深入研究, 发掘高效专一性靶基因和RNAi技术, 有可能开辟针对性的害虫RNAi防控新途径。本研究通过灰飞虱Laodelphax striatellus转录组数据分析并结合RACE技术, 克隆了灰飞虱两种海藻糖酶的全长基因, 分别命名为LSTre-1和LSTre-2, 其GenBank登录号分别为JQ027050和JQ027051。它们均具有海藻糖酶基因的典型特征, 与已报道的其他昆虫的海藻糖酶基因具有很高的相似性, 并表现出一定的虫种亲缘关系。其中LSTre-1为水溶性海藻糖酶基因, 全长2 042 bp, 开放阅读框编码602个氨基酸, 前端有25个氨基酸的信号肽, 但无跨膜结构域; LSTre-2为膜结合型海藻糖酶基因, 全长2 619 bp, 开放阅读框编码618个氨基酸, 前端有26个氨基酸的信号肽, 有2个疏水性跨膜结构域。利用喂食法研究2种海藻糖酶基因dsRNA对灰飞虱的致死效应, 发现靶向水溶性酶基因的干扰效应略高于靶向膜结合型的, 但两种海藻糖酶基因的dsRNA都可以显著抑制灰飞虱海藻糖酶基因的表达, 降低其活力, 还能显著抑制试虫的生长, 大幅增加试虫死亡率。 结果提示, 通过适宜途径干扰海藻糖酶基因可以开发防治灰飞虱的新途径。     

灰飞虱唾液腺三大解毒酶家族的转录组分析

灰飞虱Laodelphax striatellus （Fallén）是危害多种禾本科经济作物的重要刺吸式害虫。唾液腺对刺吸式口器昆虫取食植物尤其重要, 其分泌的唾液可以帮助刺吸式口器昆虫刺穿植物、 消化食物、 解毒植物的次生物质。细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）、 谷胱甘肽S 转移酶（glutathione S transferase, GST）和羧酸酯酶（carboxylesterase, CarE）是昆虫主要的解毒酶系。为了分析解毒酶基因在灰飞虱唾液腺中的表达谱, 本研究对灰飞虱成虫唾液腺进行转录组测序、 重头组装和注释, 并与豌豆蚜Acyrthosiphon pisum和西方蜜蜂Apis mellifera的同源蛋白进行系统发育分析。发现有9个谷胱甘肽S 转移酶（glutathione S transferase, GST）基因、 22个羧酸酯酶（carboxylesterase, CarE）基因和39个细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）基因在灰飞虱唾液腺中表达。通过对同源蛋白进行系统发育分析, 发现灰飞虱唾液腺大部分的CarE是参与消化/解毒和激素/信息素的加工, 而参与神经/发育的CarE很少; 灰飞虱唾液腺表达的P450基因远远少于豌豆蚜和西方蜜蜂基因组的P450基因数, 且只有CYP6和CYP4家族的成员; GST家族在3种昆虫的保守性最高。研究结果为灰飞虱对寄主植物和杀虫剂的适应性研究奠定了基础。     

灰飞虱体内一种酵母类共生菌的分子鉴定

为明确稻飞虱体内酵母类共生菌（yeast-like symbiont,YLS）的种类,采用超速离心的方法分离纯化灰飞虱Laodelphax striatellus（Fallén）体内YLS,用真菌的通用引物对其18S rDNA、5.8S-ITS rDNA全长序列进行扩增。结果得到一条分子量约为2 340 bp的序列。序列同源性分析表明,该菌与Noda等所报道的类酵母菌的18S rDNA序列差异较大（同源性只有89.6％）,而与季也蒙毕赤酵母Pichia guilliermondii有99.8％的同源性。原位杂交（ISH）和巢氏PCR均证明该菌存在于灰飞虱脂肪体和卵内,但数量较少。因此,灰飞虱体内YLS除了Noda等报道的类酵母菌外,尚存在另外一种季也蒙毕赤酵母菌。     

基于微卫星标记的中国东北地区灰飞虱遗传变异及种群遗传结构分析

【目的】本研究旨在明确我国东北地区灰飞虱Laodelphax striatellus种群遗传变异及种群遗传结构,阐明种群间遗传分化与基因流。【方法】利用9对微卫星引物对采自我国东北地区15个地理种群的375头灰飞虱样品进行测序与分析;利用GeneAlex6.51, GENEPOP4.0.9和STRUCTURE 2.3.4等软件分析灰飞虱地理种群间的遗传多样性、遗传分化、基因流及种群遗传结构。【结果】在所分析的375头灰飞虱个体中,各位点平均有效等位基因数Na=6.898;总体上,灰飞虱不同地理种群遗传多样性较高(平均观测杂合度Ho=0.548;平均期望杂合度He=0.582),各种群间基因流较低(Nm=0.660)。UPGMA聚类树、PCoA及STRCTURE分析结果表明,东北地区灰飞虱种群分为两分支:吉林(JL)和沈阳(SY2012,SY2013和SY2014)种群聚为一支;其余种群聚为一支。AMOVA分析结果表明,灰飞虱遗传变异主要来自种群内(87%),种群间变异水平较低(13%)。【结论】中国东北地区灰飞虱遗传多样性较高,不同地理种群存在一定程度的遗传分化,且基因交流较低,存在一...     

灰飞虱Bursicon基因的克隆、序列分析及在不同发育阶段的表达

Bursicon是通过G蛋白受体调节昆虫表皮硬化及展翅的功能蛋白, 它在昆虫蜕皮后的表皮硬化过程中起着关键作用。为探讨灰飞虱Laodelphax striatellus的 bursicon的功能, 利用RT-PCR和RACE技术克隆获得1 126 bp的bursicon α和761 bp的bursicon β全长序列, 将其分别命名为Lsburs-α和Lsburs-β。生物信息学分析表明： Lsburs-α开放阅读框长483 bp, 编码160个氨基酸, 该蛋白具有2个N-豆蔻酰化位点、 3个酪蛋白激酶Ⅱ磷酸化位点以及2个蛋白激酶C磷酸化位点。Lsburs-β开放阅读框长417 bp, 编码138个氨基酸, 该蛋白具有2个N-豆蔻酰化位点、 3个酪蛋白激酶Ⅱ磷酸化位点以及1个酪氨酸激酶磷酸化位点。qRT-PCR结果表明： Lsburs-α和Lsburs-β在灰飞虱各龄期均有转录表达, 并在若虫期随龄期增加呈上升趋势, 在羽化期达到峰值, 成虫期表达量逐渐降低。结果提示bursicon与灰飞虱蜕皮后的外表皮硬化关系密切。本文结果为深入研究bursicon的功能、受体调节和信号通路等奠定了基础。     

毒死蜱亚致死剂量对灰飞虱致害性和繁殖力的影响

为科学用药防治灰飞虱Laodelphax striatellus (Fallén)提供理论依据, 采用麦秆浸渍法测定毒死蜱对灰飞虱成虫的室内活性, 并用Parafilm小袋法和滤纸漏斗法测定毒死蜱亚致死剂量对灰飞虱蜜露排泄量、 虫体增重、 产卵量等指标的影响。Parafilm小袋法的测定结果表明, 经毒死蜱亚致死剂量（0.21 mg/L有效成分）处理的灰飞虱单雌蜜露分泌量和虫体增重分别比对照增加10.99%和22.22%, 但与对照相比无显著差异（P>0.05）; 单雌产卵量为79.6±26.4个, 比对照增加12.27%, 差异达到极显著水平（P<0.01）。滤纸漏斗法的测定结果表明, 处理过的灰飞虱雌成虫蜜露分泌面积为119.74±5.90 mm², 比对照增加13.06%, 差异达到显著水平（P<0.05）。毒死蜱亚致死剂量处理后对灰飞虱蜜露分泌量、 虫体增重和单雌产卵量均表现不同程度的促进作用, 说明毒死蜱亚致死剂量可能会导致灰飞虱致害性加剧。本研究结果对深入研究杀虫剂对灰飞虱种群的亚致死效应具有重要的参考价值。     

灰飞虱高带毒（RSV）群体酵母双杂交cDNA文库的构建

为了研究灰飞虱Laodelphax striatellus Fallén与水稻条纹病毒（rice stripe virus, RSV）互作机制, 本研究构建了灰飞虱高带毒群体酵母双杂交cDNA文库。以实验室筛选的灰飞虱高带毒群体为材料, 分离纯化mRNA, 反转录合成双链cDNA, 并连接三框型接头, 层析柱分级纯化。采用同源重组反应制备三框型cDNA入门文库, 再通过同源重组将入门文库转移到Gateway兼容载体pGADT7-DEST上, 构建获得酵母双杂交cDNA文库。检测结果表明： 文库库容量为3.68×10⁷ cfu, 扩增文库滴度为2.62×10¹⁰ cfu/mL; 文库重组率大于95%, cDNA插入片段平均长度>1 kb, 达到了标准cDNA文库的要求。灰飞虱高带毒群体酵母双杂交cDNA文库的构建为开展昆虫介体与水稻条纹病毒互作机制的研究奠定了基础。     

Cytoplasmic incompatibility in allopatric field populations of the small brown planthopper, Laodelphax striatellus, in Japan

The small brown planthopper, Laodelphax striatellus (Homoptera, Delphacidae), shows reproductive cytoplasmic incompatibility between laboratory-cultured strains. Wild planthoppers, were crossed for the purpose of examining the incompatibility principle in allopatric field populations. On the basis of the incompatibility recorded in field populations, Japanese planthopper populations were divided into two groups: one from the north-eastern part and one from the western part of Japan. Crosses between the females of the former group and males of the latter gave infertile eggs, showing unidirectional incompatibility. A small number of eggs from incompatible pairs, however, developed normally.

---

Résumé Laodelphax striatellus présente une incompatibilité cytoplasmique reproductive entre souches de laboratoire. Des individus de la nature ont été croisés pour examiner l'incompatibilité entre populations allopatriques récoltées dans les champs. Sur la base de l'incompatibilité observée avec les populations naturelles, les populations japonaises ont été divisées en deux groupes: un pour la partie nordest du Japon, l'autre pour la partie occidentale. Les croisements entre femelles du premier groupe et mâles du second ont donné des oeufs stériles, montrant une incompatibilité unilatérale. Un petit nombre d'oeufs de couples incompatibles se sont cependant développés normalement.

     

Identification of a fatty acid synthase gene (FAS1) from Laodelphax striatellus planthoppers contributing to fecundity

Fatty acid synthase (FAS) is a multifunctional enzyme that plays an important role in the formation of fatty acids. The fatty acids take part in many processes, such as cell signaling and energy metabolism, and in insects they are important in both cuticular hydrocarbon (CHC) formation and reproduction. Here we characterized the sequence structure and function of an FAS from the small brown planthopper (SBPH), Laodelphax striatellus. The full-length open reading frame (ORF) sequence of LsFAS1 was 7122 bp, encoding a predicted protein of 2373 amino acid residues. There were 7 functional domains in the LsFAS1 protein sequence. Gene expression screening by real-time quantitative polymerase chain reaction (RT-qPCR) showed that LsFAS1 was expressed in all developmental stages. Relative expression was highest at the 4th-instar and female adult stages. Among different tissues, the expression level of LsFAS1 in the ovary was the highest. Phylogenetic analysis showed that LsFAS1 clustered in a clade with 2 FASs from Nilaparvata lugens. Furthermore, these 3 FASs are related to cockroach BgFAS and locust LmFAS. After RNA interference-mediated knock-down, most treated insects died at eclosion. In addition, the lifespan of dsFAS1-treated female adults was shorter than that of the dsGFP-injected control, and offspring production decreased. Also, the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) genes decreased. Virgin females dissected at days 2 and 4 post-eclosion showed many matured oocytes in planthoppers treated with dsGFP but not with dsFAS1. These data highlight the importance of LsFAS1 in SBPH, including a role in reproduction.     

灰飞虱发育起点温度及有效积温的探讨

利用培养箱在恒温条件下饲养灰飞虱Laodelphax striatellus Fallén,测定了卵、若虫、成虫繁殖前和全世代发育历期,用直线回归法计算了灰飞虱各虫态和全世代的发育起点温度和有效积温分别为10.06、6.43、10.29、8.08℃和102.3、365.2、87.5、552.1日·度。并根据有效积温法则预测了该虫在济宁市1年完成的代数为4～5代。     

Effect of anaesthesia with carbon dioxide on vectoring ability and survival of the planthopper Laodelphax striatellus

This paper describes the effects of carbon dioxide anaesthesia on the ability of the planthopper Laodelphax striatellus to subsequently acquire and transmit maize rough dwarf reovirus (MRDV) and barley yellow striate mosaic rhabdovirus (BYSMV), both propagative in the vector. Anaesthesia reduced the acquisition of MRDV by about 45%, but did not affect acquisition of BYSMV. In serial transfer transmission tests, anaesthesia caused interruption of transmission of both MRDV and BYSMV in about 11% of infectious insects; the effect may be due to impaired ability to find the phloem. The effect was stronger on third instar nymphs than on adults. Transmission of MRDV was resumed 3 days after anaesthesia, but none of the insects that stopped transmitting BYSMV resumed it. The survival of insects which ceased to transmit BYSMV was significantly lower than that of hoppers that continued to transmit. No other effects on survival were detected. A possible interaction of carbon dioxide with BYSMV in L. striatellus is discussed, in the light of effects of carbon dioxide on Drosophila melanogaster infected by some rhabdoviruses.