基于16S rRNA高通量测序的灰飞虱体内细菌群落结构及多样性分析

【目的】探明灰飞虱Laodelphax striatellus体内细菌的群落结构和种类多样性。【方法】采用Illumina MiSeq测序平台对新羽化24 h的灰飞虱雌雄成虫体内细菌16S rRNA的V3-V4变异区序列进行高通量测序,应用USEARCH和Silva等软件和数据库来统计样品序列数目和操作分类单元(operational taxonomic unit,OTU)数量,分析灰飞虱雌雄成虫体内细菌的种类组成、丰度、Alpha多样性及其差异。【结果】灰飞虱雌、雄成虫样本分别获得29 333和25 919条有效序列,根据序列相似性进行聚类分析分别获得55和57个OTUs。其中,雌、雄成虫两类样本共有OTU数目20个,特有的OTU数目分别为35和37个。基于OTU物种分类分析,雌雄成虫样本中细菌种类一共覆盖7个门、15个纲、23个目、33个科、56个属和73个种。在门、纲、目和科分类阶元上,雌雄成虫两类样本均以变形菌门(Proteobacteria,雌99.96%/雄99.16%)、α-变形菌纲(Alphaproteobacteria,雌97.76%/雄97.84%)、红螺菌目(Rhodospirillales,雌83.92%/雄53.21%)和醋酸杆菌科(Acetobacteraceae,雌83.90%/雄53.17%)中的细菌为优势菌。在属分类阶元上,灰飞虱雌雄成虫体内优势菌为醋酸杆菌科未分类属(unclassified Acetobacteraceae),沃尔巴克氏体属Wolbachia次之,后者在雌、雄成虫体内丰度分别为13.81%和44.52%。在种水平上,灰飞虱雌、雄成虫体内特有细菌分别为21和32个种,但两类样本中性别特有种的丰度普遍极低,均不足0.5%。【结论】灰飞虱雌雄成虫间细菌群落组成和种类多样性存在差异。本研究为进一步挖掘灰飞虱体内关键微生物并解析其生物学功能及其利用等方面奠定了基础。     

三种稻飞虱体内类酵母共生菌18S rDNA和 ITS 5.8S rDNA序列克隆及进化分析

为研究水稻3种主要害虫灰飞虱Laodelphax striatellus、 褐飞虱Nilaparvata lugens和白背飞虱Sogatella furcifera体内类酵母共生菌（yeast-like symbiotes, YLS）的种属地位及与寄主的进化关系, 测定了其体内YLS的18S rDNA及ITS-5.8S rDNA的全长序列。基于3种稻飞虱体内YLS的18S rDNA序列比对表明, 褐飞虱YLS和白背飞虱YLS的一致性比其与灰飞虱YLS的高（褐飞虱YLS和白背飞虱YLS为98.91%, 灰飞虱YLS和褐飞虱YLS为95.74%, 灰飞虱YLS和白背飞虱YLS为96.02%）, 而基于ITS-5.8S rDNA序列比对, 灰飞虱YLS和白背飞虱YLS的一致性比其与褐飞虱YLS的要高（白背飞虱YLS和灰飞虱YLS为99.57%, 灰飞虱YLS和褐飞虱YLS为91.91%, 白背飞虱YLS和褐飞虱YLS为90.46%）。基于真菌18S rDNA和ITS-5.8S rDNA的系统发育树均表明, 3种稻飞虱体内YLS与其他已知真菌进化关系较远。本研究证实了昆虫真菌类共生菌与寄主形成了长期的进化关系, 从而形成了不同于已知真菌的分类地位。     

Phylogenetic position of yeast-like symbiotes from three rice planthoppers (Homoptera: Delphacidae ) based on 18S rDNA and ITS-5.8S rDNA sequences

为研究水稻3种主要害虫灰飞虱Laodelphax striatellus、褐飞虱Nilaparvata lugens和白背飞虱Sogatella furcifera体内类酵母共生菌( yeast-like symbiotes,YLS)的种属地位及与寄主的进化关系,测定了其体内YIS的18SrDNA及ITS-5.8S rDNA的全长序列.基于3种稻飞虱体内YLS的18S rDNA序列比对表明,褐飞虱YLS和白背飞虱YLS的一致性比其与灰飞虱YLs的高(褐飞虱YLS和白背飞虱YLS为98.91％,灰飞虱YLS和褐飞虱YLS为95.74％,灰飞虱YLS和白背飞虱YLS为96.02％),而基于ITS-5.8S rDNA序列比对,灰飞虱YLS和白背飞虱YLS的一致性比其与褐飞虱YLS的要高(白背飞虱YLS和灰飞虱YLS为99.57％,灰飞虱YLS和褐飞虱YIS为91.91％,白背飞虱YLS和褐飞虱YLS为90.46％).基于真菌18S rDNA和ITS-5.8S rDNA的系统发育树均表明,3种稻飞虱体内YLS与其他已知真菌进化关系较远.本研究证实了昆虫真菌类共生菌与寄主形成了长期的进化关系,从而形成了不同于已知真菌的分类地位.     

褐飞虱不同致害性种群体内共生菌18S rDNA部分序列比较

分离纯化了褐飞虱3种不同致害性种群体内类酵母共生菌 (yeast-like symbionts, YLS),并对其18S rDNA基因序列进行了比较。结果表明,褐飞虱3种不同致害性种群体内类酵母共生菌18S rDNA均扩增出600 bp左右的片断。依据获得的18S rDNA特异性序列,结合已知真菌的18S rDNA部分序列,构建了不同宿主的YLS分子系统树。结果显示, 褐飞虱3种不同致害性种群体内的YLS同属子囊菌亚门（Ascomycotina）的核菌纲（Pyrenomycetes）,并与此纲中的Hypomyces chrysospermus亲缘关系相对最近。     

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。     

灰飞虱体内类酵母共生菌异常毕赤酵母的分离培养及其对杀菌剂的敏感性

【目的】随着杀虫剂等化学农药的大规模使用,灰飞虱Laodelphax striatellus的抗药性不断增强,防治效果下降,因此寻找新的防治方法尤为重要。本研究探索使用杀菌剂来减少灰飞虱体内共生菌的数量,以降低灰飞虱的活力,为"抑菌防虫"提供理论依据。【方法】对灰飞虱体内的类酵母共生菌(yeast-like symbiotes,YLS)进行离体培养并鉴定,测定离体培养YLS对不同浓度不同种类杀菌剂的敏感性,将对其抑制作用明显的杀菌剂喷施于麦苗,待灰飞虱取食后统计灰飞虱死亡率、体重,并通过荧光定量PCR检测体内共生菌的数量变化。【结果】离体培养得到1株YLS,经碳源同化实验和18S r DNA分子鉴定,将该菌株归为异常毕赤酵母Pichia anomala,以变性梯度凝胶电泳(denatured gradient gel electrophoresis,DGGE)技术验证了该菌株在灰飞虱体内的存在。对杀菌剂敏感性的测定结果显示,70%丙森锌和50%戊唑醇+25%肟菌酯对P.anomala抑制效果明显,而氟吡菌胺+霜霉威盐酸盐(687.5 g a.i./L)与40%嘧霉胺对P.anomala的抑制效果较差。用对P.anomala抑制效果明显的两种杀菌剂(70%丙森锌和50%戊唑醇+25%肟菌酯)以800 mg/L浓度喷施麦苗,灰飞虱取食喷施麦苗后的死亡率分别达到46.7%与63.3%,显著高于对照,而体重比对照组显著低。荧光定量检测结果表明,灰飞虱体内3种共生菌(Noda菌、季也蒙毕赤酵母和异常毕赤酵母)的数量在使用了各种杀菌剂后均显著降低。【结论】结果说明,体外喷施杀菌剂对灰飞虱体内共生菌有抑制作用,进而对灰飞虱的生长存活产生影响,这为杀菌剂与杀虫剂的协同使用防治灰飞虱奠定基础。     

三种稻飞虱成虫体内酵母类共生菌的形态差异

试验采用冷冻切片技术结合显微摄像系统观察法研究了酵母类共生菌在3种稻飞虱成虫体内的存在状态、形态特性以及种类和组成.光学显微镜观察证实,3种稻飞虱成虫的头部和胸部均未观察到共生菌,在其腹部脂肪体中有大量酵母类共生菌,且以出芽进行无性繁殖,并伴有菌胞的出现.3种稻飞虱成虫体内共生菌的形态和组成明显不同.其中,褐飞虱体内的共生菌个体较大,以梭形、杆状和卵形为主,分别占共生菌总数的30.7%、53.5%和15.1%;灰飞虱和白背飞虱体内的共生菌个体较小,以卵形为主,分别占共生菌总数的93.4%和94.7%.此外,褐飞虱成虫体内的各类共生菌都显著大于灰飞虱和白背飞虱成虫体内的同种类型个体,且灰飞虱和白背飞虱体内的同种共生菌个体大小之间也存在一定差异.     

贵州害竹飞虱二新种记述(同翅目:飞虱科)

记述我国贵州省为害竹子的飞虱２新种,即基褐异脉飞虱Ｓｐｅｃｉｎｅｒｖｕｒｅｓ ｂａｓ９ｉｆｕｓｃａ Ｃｈｅｎ ｅｔ Ｌｉ,ｓｐ．ｎｏｖ和望谟竹飞虱Ｂａｍｂｕｓｉｐｔｈａｇａ ｗａｎｇｍｏｅｎｓｉｓ Ｃｈｅｎ ｅｔ Ｌｉ,ｓｐ．ｎｏｖ．。模式标本保存在贵州大学昆虫研究所。     

取食不同寄主植物的灰飞虱对药剂敏感性和解毒酶活性的影响

为了探索取食不同寄主植物的灰飞虱Laodelphax striatellus对药剂敏感性和体内解毒酶活性的影响,本研究在室内采用稻苗浸渍法分别测定了4种杀虫剂对取食水稻、小麦和稗草后的灰飞虱3龄若虫的毒力,同时比较了灰飞虱体内酯酶(ESTs)、谷胱甘肽-S-转移酶(GSTs)和多功能氧化酶(MFO)的活性.结果表明,用5种寄主植物连续饲养灰飞虱3代后,灰飞虱对三氟苯嘧啶的敏感性无明显变化,对吡虫啉、噻虫嗪和溴氰虫酰胺的敏感性存在一定差异但不显著.取食不同寄主植物的灰飞虱体内3种解毒酶活性存在明显差异,取食稗草的灰飞虱体内ESTs和MFO活性最高,取食水稻紫香糯2315的灰飞虱体内GSTs活性最高.研究结果表明取食不同寄主植物可影响灰飞虱对杀虫剂的敏感性和体内解毒酶的活性.     

木糖代谢基因表达水平对酿酒酵母重组菌株产物形成的影响

以Ｅ．ｃｏｌｉ－Ｓ．ｃｅｒｅｖｉｓｉａｅ穿梭质粒ＹＥｐ２４为骨架,将树干毕赤酵母（Ｐｉｃｈｉａ ｓｔｉｐｉｔｉｓ ＣＢＳ６０５４）的木糖还原酶（ＸＲ）基因ＸＹＬ１及木糖醇脱氢酶（ＸＤＨ）基因ＸＹＬ１分别以不同的相对表达方向置于酿酒酵母的乙醇脱氢酶Ｉ（ＡＤＨ１）启动子和磷酸甘油激酶（ＰＧＫ）启动子下,构建不同ＸＹＬ１及ＸＹＬ２的重组质粒。这些重组质粒分别转化酿酒酵母（Ｈ１５８）受体菌。得到的重组菌株     

Identification of Two Species of Yeast-like Symbiotes in the Brown Planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis>

To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed spacer (ITS)–5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that the two different 18S and ITS–5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and ITS–5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS–5.8S rDNA, showed the high similarity to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes.     

Complex co-evolutionary relationships between cicadas and their symbionts

Previous evidence suggests that cicadas lacking Hodgkinia may harbour the yeast-like fungal symbionts (YLS). Here, we reinforce an earlier conclusion that the pathogenic ancestor of YLS independently infected different cicada lineages instead of the common ancestor of Cicadidae. Five independent replacement events in the loss of Hodgkinia/acquisition of YLS and seven other replacement events of YLS (from an Ophiocordyceps fungus to another Ophiocordyceps fungus) are hypothesised to have occurred within the sampled cicada taxa. The divergence time of YLS lineages was later than that of corresponding cicada lineages. The rapid shift of diversification rates of YLS and related cicada-parasitizing Ophiocordyceps began at approximately 32.94 Ma, and the diversification rate reached the highest value at approximately 24.82 Ma, which corresponds to the cooling climate changes at the Eocene–Oligocene boundary and the Oligocene–Miocene transition respectively. Combined with related acquisition/replacement events of YLS occurred during the cooling-climate periods, we hypothesise that the cooling-climate changes impacted the interactions between cicadas and related Ophiocordyceps, which coupled with the unusual life cycle and the differentiation of cicadas may finally led to the diversification of YLS in Cicadidae. Our results contribute to a better understanding of the evolutionary transition of YLS from entomopathogenic fungi in insects.     

Structural diversity of symbionts and related cellular mechanisms underlying vertical symbiont transmission in cicadas

Many insects depend on symbiont(s) for survival. This is particularly the case for sap-feeding hemipteran insects. In this study, we revealed that symbionts harboured in cicadas are diverse and complex, and the yeast-like fungal symbionts (YLS) are present in most cicada species, but Hodgkinia is absent. During vertical transmission, Sulcia became swollen with the outer membrane drastically changed, while Hodgkinia became shrunken and changed from irregular to roughly spherical. Sulcia and/or Hodgkinia were exocytosed from the bacteriocytes to the intercellular space of bacteriomes, where they gathered together and were extruded to haemolymph. YLS and associated facultative symbiont(s) in the fat bodies were released to the haemolymph based on bacteriocyte disintegration. The obligate symbiont(s) were endocytosed and exocytosed successively by the epithelial plug cells of the terminal oocyte, while associated facultative symbiont(s), and possibly also YLS, may take a ‘free ride’ on the transmission of obligate symbiont(s) to gain entry into the oocyte. Then, the intermixed symbionts formed a characteristic ‘symbiont ball’ in the oocyte. Our results suggest that YLS in cicadas represent a new example of a relatively early stage of symbiogenesis in insects and contribute to a better understanding of the diversity and transmission mechanisms of symbionts in insects.     

食料对烟草甲共生菌数量和生长发育的影响

烟草甲Lasioderma serricorne(Fabricius)消化道内含有一种类酵母共生菌,其在烟草甲的营养需求和对外源物质的解毒过程中有重要作用。以麦粒、烟叶、麦片、甘草和面粉为食料饲养烟草甲,研究了食料对烟草甲共生菌数量和发育历期的影响,发现:烟草甲5龄幼虫体内的共生菌数量最多,蛹次之;食料对共生菌的数量有显著的影响,尤其在5龄幼虫期;麦粒饲养的烟草甲发育历期最短、体内的共生菌数量最多,而甘草饲养的烟草甲发育历期最长、体内的共生菌数量最少;共生菌的数量与烟草甲的生长发育历期之间存在着明显的负相关关系。     

Benefits and potential trade‐offs associated with yeast‐like symbionts during virulence adaptation in a phloem‐feeding planthopper

Insect herbivores form symbioses with a diversity of prokaryotic and eukaryotic microorganisms. A role for endosymbionts during host feeding on nutrient‐poor diets – including phloem – is now supported by a large body of evidence. Furthermore, symbiont‐herbivore associations have been implicated in feeding preferences by host races (mainly aphids) on multiple plant species. However, the role of symbionts in mediating herbivore preferences between varieties of the same plant species has received little research attention despite the implications for virulence adaptation to resistant crops. This study investigates the role of yeast‐like symbionts (YLS) in virulence adaptation and host plant switching among populations of the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), that were selected on various rice [Oryza sativa L. (Poaceae)] lines differing in their resistance against herbivores. Planthopper fitness (nymph weight) declined where YLS densities were depleted through heat treatment. However, compared to normal symbiotic planthoppers, the depletion of symbionts did not generally change the relative fitness of planthoppers (each ‘adapted’ to a single natal host) when switched to feed on a range of rice lines (exposed hosts). In some cases, this occurred despite differences in YLS density responses to the various hosts. Furthermore, we detected no fitness costs associated with YLS in adapted populations. Therefore, the results of this study suggest that, whereas YLS are essential for planthopper nutrition, changes in YLS density play little role during virulence adaptation and host plant switching by the brown planthopper.     

Food quality affects wing-form,demographic traits and number of yeast-like symbionts (YLS) in the planthopper vector,Delphacodes kuscheli (Hemiptera: Delphacidae)

Delphacodes kuscheli, the main vector of maize Mal de Río Cuarto disease in Argentina, was used to analyze the effect of food quality on wing-form, demographic traits and number of yeast-like symbionts (YLS). First instar nymphs were caged on the leaf apex or on the ligular zone of oat plants, characterized by having low and high nutritional value respectively, and followed in their development to adulthood. Food quality affected the proportion of wing morphs, with an increased number of brachypterous individuals on the higher food quality zone. Macropterous and brachypterous females from the ligular zone had higher survivorship, shorter development time and higher number of YLS. For males, development time and number of YLS did not differ significantly in individuals fed from both zones, while body and wing length were superior in individuals fed on the ligular zone. Results suggest that females allocate the resources from feeding on a richer zone to reduce the pre-reproductive period and to increase the number of YLS, and males acquire a larger size while maintaining development time and YLS number. This is the first study quantifying the variation of number of YLS according to food quality in a Neotropical planthopper pest.     

Consequences of stress induced by heat on fitness of Delphacodes kuscheli and its yeast‐like endosymbionts

The planthopper Delphacodes kuscheli is the main vector of Mal de Río Cuarto virus in Argentina, disease that severely affects maize production. In this study, we investigated the effects of heat stress on fitness traits and on the number of its obligate yeast‐like symbionts (YLS). The exposition of newly‐hatched nymphs to 35°C for 3 days, a well‐known procedure used to reduce the number of YLS in planthoppers, was applied. To compare different fitness components between control and heat‐treated insects, we estimated nymphal instars development time, nymphal survival, adult body length, longevity, fecundity and fertility. Also, correlates of fitness, as proportion of sexes and wing forms of the emerging adults, were evaluated. In heat‐treated group, the nymphal developmental time increased due to an increase in the fifth instar duration, and the nymphal survival, body length of adults and fecundity were reduced when compared to control. There was a significant association between treatments (control and heat‐treated insects) and wing morphs. The heat treatment successfully reduced the number of YLS in third instar nymphs of D. kuscheli. Our results revealed the negative effect of heat stress on development, survival and reproduction of D. kuscheli and on the load of its YLS endosymbionts suggesting that YLS could play a crucial role in the development and reproduction of these planthoppers.     

Systemic fungicides reduce the abundance of yeast-like symbiotes and the performance of the planthopper Delphacodes kuscheli (Hemiptera: Delphacidae)

Delphacodes kuscheli is the main vector of Mal de Río Cuarto, the most important viral disease of maize in Argentina. This planthopper harbours obligate fungal mutualists, known as yeast-like symbiotes (YLS), which play a key role in the host life cycle. Here, we evaluated the efficacy of six systemic fungicides, prothioconazole (P), prothioconazole & trifloxystrobin (PT), pyraclostrobin & epoxiconazole (PE), pyraclostrobin, epoxiconazole & fluxapyroxad (PEF), picoxystrobin & ciproconazole (PC), and tebuconazole (T) on the reduction of the abundance of YLS in nymphs and newly emerged adults, and the impact of such reduction on the vector performance. All fungicides, except PE, reduced the number of YLS in nymphs treated from 3rd instar five days after starting treatments. When 3rd instar nymphs were allowed to reach adulthood, the fungicides P, PT and PC caused the highest nymphal mortality, significantly reduced the number of YLS in newly emerged adults, lengthened development time and negatively affected morphometric variables of females. There was also a trend towards a higher occurrence of brachypters. In females, the fungicides P, PT and PC caused a greater reduction of YLS when nymphs were treated to these fungicides from 3rd instar compared with nymphs treated from 4th instar. In males, the YLS number was significantly less when nymphs were fed on plants treated with P, PT, PC, and T in 3rd instar. This showed that fungicide treatments caused a greater reduction of YLS when they were applied in early stages of development. Our results provide an effective method to reduce the abundance of YLS in D. kuscheli, contributing to understand the ecological role that these symbionts could be playing in the success of this maize planthopper pest.