Infection density of Wolbachia and incompatibility level in two planthopper species, Laodelphax striatellus and Sogatella furcifera

Wolbachia, a bacterial endosymbiote of arthropods, causes cytoplasmic incompatibility (CI) in many insect species. CI traits were studied in two planthopper species, Laodelphax striatellus and Sogatella furcifera, and Wolbachia densities in these planthopper species were calculated by quantitative PCR methods. The CI level of L. striatellus was quite high and even aged males strongly caused CI. In contrast, S. furcifera showed partial CI, and males lost their ability to cause CI with age. Wolbachia infecting these two planthopper species were the same with respect to the nucleotide sequences of Wolbachia genes, 16S rDNA, ftsZ gene, groE genes, and wsp gene. Two methods for quantitative PCR, one using a DNA sequencer and the other a real-time sequence detection system, were established to calculate the amount of Wolbachia in the planthoppers. The density of Wolbachia in S. furcifera males was quite low. The difference in CI levels between the two planthopper species seems to be due to different amounts of Wolbachia infecting males.     

Development and preliminary application of a triplex real‐time polymerase chain reaction assay for evaluating predation on three planthoppers in a rice ecosystem

A multiplex real‐time quantitative polymerase chain reaction (PCR) assay was developed to simultaneously detect the DNA of three rice planthoppers, that is, Sogatella furcifera (Horváth) (white‐backed planthopper), Nilaparvata lugens (Stål) (brown planthopper) and Laodelphax striatellus (Fallén) (small brown planthopper), in the gut of their predators. The sets of primers and ALLGlo probes were targeted to the regions of internal transcribed spacer 2 (ITS2) genes in nuclear ribosomal DNA (rDNA). The sensitivity, specificity and interference test for the multiplex real‐time quantitative PCR assay were analysed. The assay's detection limits were 100, 1000 and 100 copies for the white‐backed planthopper, the brown planthopper and the small brown planthopper, respectively. The specificity tests showed no cross‐reactivity with genomic DNA from 30 other dominant herbivores, saprophagous insects and predators from rice ecosystem for each planthopper species. The assay was used in a preliminary study of predation events on the three planthoppers by three major spiders viz., Pardosa pseudoannulata (Bösenberg et Strand), Ummeliata insecticeps (Bösenberg et Strand) and Tetragnatha maxillosa Thorell which each differ in their preferred microhabitat as well as their predatory habits in rice field, and the results showed their predation on each planthopper species could be well evaluated using this method. Therefore, the multiplex real‐time quantitative PCR assay provides a new tool to study the mechanisms of prey shifting and natural regulation of the three rice planthoppers by generalist predators in rice ecosystem.     

半滑舌鳎雌性特异AFLP标记CseF783的克隆及其在遗传性别鉴定中的应用

半滑舌鳎性别控制和全雌育种等研究领域中迫切需要一种能够快速鉴定鱼类个体遗传性别的有效方法。文章采用AFLP技术, 利用选择性引物组合(E-ACT/M-CAA)从半滑舌鳎中筛选到一条雌性特异的AFLP标记。对该标记进行二次PCR扩增、琼脂糖凝胶回收、克隆、测序。分析表明, 序列全长为791 bp, 与GenBank中的序列无同源性。以该雌性特异AFLP标记DNA序列为模板, 设计了一对特异的PCR引物, 成功地将其转化为SCAR(Sequence characterized amplified regions)标记, 并在100尾已知性别的半滑舌鳎个体(雌雄各50尾)中进行验证, 结果表明, 该SCAR标记在所有雌性个体中均扩增得到一条长度为324 bp的DNA条带, 而在49尾雄性个体中均扩增不到该DNA条带(有1尾雄性个体例外), 证明该SCAR标记是雌性特异的, 并可用于半滑舌鳎个体遗传性别鉴定。随后, 利用该SCAR标记检测了3日龄半滑舌鳎幼苗, 结果表明, 雌性个体比例为41.7%。     

基于rDNA ITSl和ITS2序列的褐飞虱、白背飞虱和灰飞虱的分子鉴定

本研究测定了褐飞虱 Nilaparvata lugens、白背飞虱 Sogatella furcifera 和灰飞虱Laodelphax striatellus 的rDNA ITSl和ITS2的序列,以探讨这3种稻飞虱的分子鉴定方法.3种飞虱的ITSI和ITS2侧翼区(18S,5.8S和28S)序列相对稳定,但ITS1和ITS2序列在3种飞虱中变异较大.ITS1在所分析的438个位点中可变位点达294个,ITS2在分析的403个位点中可变位点为177个.根据3种飞虱rDNA的ITS1和ITS2序列设计了特异性引物,应用特异性引物对样品进行了PCR扩增,分析发现3种飞虱ITS1区的特异性引物扩增效果不理想.而ITS2区的特异性引物可以稳定地扩增出明显的目的DNA条带.因此,采用ITS2区的特异性引物可以对3种飞虱进行快速的分子鉴定.     

稻飞虱天敌螯蜂研究进展

稻飞虱（褐飞虱Nilaparvata lugens、白背飞虱Sogatella furcifera、灰飞虱Laodelphax striatellus）是世界性重要的水稻Oryza sativa L.害虫之一,给水稻生产造成了重大经济损失。化学防治一直是控制稻飞虱的主要途径,但长期使用化学药剂使稻飞虱产生抗药性,并引起害虫增殖等诸多弊端,迫切需要有效的生物防控手段进行控害。螯蜂是稻飞虱若虫和成虫期重要天敌,兼具捕食与寄生的双重习性,在控制稻飞虱种群数量方面发挥着重要的作用,然而,国内外有关螯蜂的研究报道仍然偏少。本文综述了我国稻飞虱天敌螯蜂常见种类、生物学特性、控害效果及其影响因子,分析了存在的问题,旨在为进一步开发利用螯蜂资源提供参考。     

Analysis of Yeast-Like Symbiote Diversity in the Brown Planthopper (BPH), Nilaparvata lugens Stål, Using a Novel Nested PCR-DGGE Protocol

Yeast-like symbiotes (YLS) are endosymbionts that are intimately associated with the growth, development, reproduction of their host, the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). However, it is unclear how many species of YLS are found within N. lugens, and how they are related to each other. Traditional methods or simple amplification based on 18S rDNA sequence does not reliably identify new species quickly and efficiently. Therefore, a novel nested PCR-denaturing gradient gel electrophoresis (DGGE) strategy was developed in this article to analyze the YLS of brown planthopper using a nested PCR protocol that involved the 18S rDNA gene and the 5.8S–ITS gene using fungal universal primers. The nested PCR protocol was developed as follows: firstly, the 18S rDNA gene, and 5.8S–ITS gene were amplified using fungal universal primers. Subsequently, these products were used as a template in a second PCR with primers ITS1GC–ITS2, ITS1FGC–ITS2, and NFGC-NR, which was suitable for DGGE. Using this highly specific molecular approach, we found several previously detected fungi: Noda, Pichia guilliermondii, Candida sp., and some previously undetected fungi, such as Saccharomycetales sp., Debaryomyces hansenii, and some uncultured fungi. In conclusion, the nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a new tool for uncovering fungal endosymbiont diversity within planthoppers.     

Sry-negative XX sex reversal in the American cocker spaniel dog

The Sry gene product serves an important function in male sex determination through testis induction. However, testicular development has been reported in SRY-negative XX sex reversed humans. XX sex reversal of the American cocker spaniel, inherited as an autosomal recessive trait, may be a homolog of this disorder. The purpose of this study was to determine whether the Sry high mobility group (HMG) box is present in genomic DNA of affected dogs. Conserved Sry HMG box and hypoxanthine phosphoribosyltransferase (HPRT) sequences were used as primers in polymerase chain reactions. A 167 bp Y-specific canine Sry HMG box sequence was cloned from genomic DNA of normal male dogs. Internal primers generated a 104 bp Sry HMG box product from normal males, but not from females or XX sex reversed dogs. Parallel reactions generated an HPRT product from all dogs. Results indicate that the Sry HMG box is absent in genomic DNA of XX sex reversed dogs. We speculate that activation of the testis differentiation cascade in the absence of Sry in this model is due to a mutant autosomal gene. © 1995 Wiley-Liss, Inc.     

Identification of sex in hop (Humulus lupulus) using molecular markers.

The rapid identification of sex in the dioecious hop (Humulus lupulus) is important for the breeding of this cultivated plant because only unfertilized flowers of the female plants are used as an ingredient in the production of beer. It is thought that a sex-chromosome mechanism controls the development of male or female plants. We have compared pools of male and female plants derived from a hop cross to identify molecular markers associated with the Y or male-specific chromosome. Of 900 functional RAPD primers, 32 revealed fragments specific for male plants that were absent in female plants of this cross. Subsequently, the 32 positive primers were tested on unrelated male and female plants. Three of these 32 primers were specific for the Y chromosome in all lines. The Y-specific product derived from one of these primers (OPJ9) was of low copy in hybridization experiments and predominantly present in male plants. Primers developed from the DNA sequence of this product provide a marker for rapid sex identification in crosses of hop by means of PCR.     

基于rDNA ITS1和ITS2序列的褐飞虱、白背飞虱和灰飞虱的分子鉴定

本研究测定了褐飞虱Nilaparvata lugens、白背飞虱Sogatella furcifera和灰飞虱Laodelphax striatellus的rDNA ITS1和ITS2的序列, 以探讨这3种稻飞虱的分子鉴定方法。3种飞虱的ITS1和ITS2侧翼区（18S, 5.8S和28S）序列相对稳定, 但ITS1和ITS2序列在3种飞虱中变异较大。 ITS1在所分析的438个位点中可变位点达294个, ITS2在分析的403个位点中可变位点为177个。根据3种飞虱rDNA的ITS1和ITS2序列设计了特异性引物, 应用特异性引物对样品进行了PCR扩增, 分析发现3种飞虱ITS1区的特异性引物扩增效果不理想, 而ITS2区的特异性引物可以稳定地扩增出明显的目的DNA条带. 因此, 采用ITS2区的特异性引物可以对3种飞虱进行快速的分子鉴定。     

Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.     

Improved template representation in cpn60 polymerase chain reaction (PCR) product libraries generated from complex templates by application of a specific mixture of PCR primers

Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples. The development and optimization of this specific primer mixture represents an improvement of established methods and a significant advance in the ability to generate cpn60 PCR product libraries that more closely represent the sequence diversity in complex templates.     

Sexing of in vitro produced ovine embryos by duplex PCR

The aim of this article was to develop a fast and easy duplex polymerase chain reaction (PCR) method, for sex determination of ovine in vitro produced embryos prior to implantation. We tested the approach with 107 samples of autosomal cells (oviductal sheep cells and male lamb fibroblasts), divided into three groups for each sex according to the number of cells employed (30, 5, 2, respectively). We then used the test on 21 embryos at blastocyst stage. On the same day the embryos were transferred in pairs into 11 recipient synchronized ewes. The PCR utilized two different sets of primers: the first pair recognized a bovine Y-chromosome-specific sequence (SRY), that showed 100% homology with the corresponding sequence of the ovine Y-chromosome and is amplified in males only. The second pair recognized the bovine 1.715 satellite DNA (SAT) which was amplified in all ovine samples but, when submitted to the GenBank database did not show homology with any of the reported ovine sequences. However, after sequencing, ovine amplification product showed 98% homology with the bovine specific satellite sequence. The autosomal samples were amplified with 85.0% efficiency and 91.2% accuracy, while amplification was successful with all 21 embryos (100% efficiency). Eight lambs were born and the sex as determined by PCR corresponded to the anatomical sex in seven (87.5% accuracy). These results confirm that this method can be applied in ovine breeding programs to manipulate sex ratio of offspring.     

Areliable sex determination assay for bovine preimplantation embryos using the polymerase chain reaction

We have developed a polymerase chain reaction (PCR)-based method for accurate sex determination of preimplantation bovine embryos. The method utilizes three different sets of primers in the PCR. The first pair of primers recognizes the bovine-specific satellite sequence that is amplified in both females and males. In addition, two pairs of primers recognize bovine Y chromosome-specific sequences that are amplified in males only. Duplicate embryo extracts were used in the PCR; the first sample was run in the presence of bovine-specific as well as one set of the Y chromosome-specific primers; the second sample was run in the presence of the other male-specific primers. The method has been specifically designed for screening bovine embryos. Based upon examining blood cell DNA from adult males and females, the assay is extremely accurate, as no single incorrect result has occurred yet. Missing samples were easily detected by the absence of the bovine-specific signal. The method has been used for the transfer of bovine embryos on which sex determinations have been performed.     

Two ancient bacterial endosymbionts have coevolved with the planthoppers (Insecta: Hemiptera: Fulgoroidea)

ABSTRACT: BACKGROUND: Members of the hemipteran suborder Auchenorrhyncha (commonly known as planthoppers, tree- and leafhoppers, spittlebugs, and cicadas) are unusual among insects known to harbor endosymbiotic bacteria in that they are associated with diverse assemblages of bacterial endosymbionts. Early light microscopic surveys of species representing the two major lineages of Auchenorrhyncha (the planthopper superfamily Fulgoroidea; and Cicadomorpha, comprising Membracoidea [tree- and leafhoppers], Cercopoidea [spittlebugs], and Cicadoidea [cicadas]), found that most examined species harbored at least two morphologically distinct bacterial endosymbionts, and some harbored as many as six. Recent investigations using molecular techniques have identified multiple obligate bacterial endosymbionts in Cicadomorpha; however, much less is known about endosymbionts of Fulgoroidea. In this study, we present the initial findings of an ongoing PCR-based survey (sequencing 16S rDNA) of planthopper-associated bacteria to document endosymbionts with a long-term history of codiversification with their fulgoroid hosts. RESULTS: Results of PCR surveys and phylogenetic analyses of 16S rDNA recovered a monophyletic clade of Betaproteobacteria associated with planthoppers; this clade included Vidania fulgoroideae, a recently described bacterium identified in exemplars of the planthopper family Cixiidae. We surveyed 77 planthopper species representing 18 fulgoroid families, and detected Vidania in 40 species (representing 13 families). Further, we detected the Sulcia endosymbiont (identified as an obligate endosymbiont of Auchenorrhyncha in previous studies) in 30 of the 40 species harboring Vidania. Concordance of the Vidania phylogeny with the phylogeny of the planthopper hosts (reconstructed based on sequence data from five genes generated from the same insect specimens from which the bacterial sequences were obtained) was supported by statistical tests of codiversification. Codiversification tests also supported concordance of the Sulcia phylogeny with the phylogeny of the planthopper hosts, as well as concordance of planthopper-associated Vidania and Sulcia phylogenies. CONCLUSIONS: Our results indicate that the Betaproteobacterium Vidania is an ancient endosymbiont that infected the common ancestor of Fulgoroidea at least 130 million years ago. Comparison of our findings with the early light-microscopic surveys conducted by Muller suggests that Vidania is Muller's x-symbiont, which he hypothesized to have codiversified with most lineages of planthoppers and with the Sulcia endosymbiont.     

Sexing the Sciuridae: a simple and accurate set of molecular methods to determine sex in tree squirrels, ground squirrels and marmots

We determined the sequence of the male-specific minor histocompatibility complex antigen (Smcy) from the Y chromosome of seven squirrel species (Sciuridae, Rodentia). Based on conserved regions inside the Smcy intron sequence, we designed PCR primers for sex determination in these species that can be co-amplified with nuclear loci as controls. PCR co-amplification yields two products for males and one for females that are easily visualized as bands by agarose gel electrophoresis. Our method provides simple and reliable sex determination across a wide range of squirrel species.     

实验犬布氏杆菌的多重PCR检测与分型鉴定

目的建立实验犬及相关生物制品布氏杆菌的多重PCR检测与分型鉴定方法。方法选择布氏杆菌Omp2基因同源性较高的区域设计引物对布氏杆菌进行多重PCR扩增,扩增结果一致的样本进行酶切以区分不同型,同时进行序列测定,以确定该方法的准确性;然后验证该方法的特异性和敏感性。结果成功扩增得到目的条带,并通过酶切区分五种布氏杆菌;PCR产物与布氏杆菌DNA序列同源性达到99%,并验证了该方法的检测结果。实验结果证明该方法特异性较好,灵敏性为1.8×10^-7μg/mL。结论成功建立布氏杆菌多重PCR检测与分型鉴定方法,所建立的方法特异性好,灵敏度高。本研究对保证实验犬群的质量,保护饲养人员、实验人员的身体健康具有重要意义。     

Genetic basis of enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprint pattern in Sinorhizobium meliloti and identification of S. meliloti employing PCR primers derived from an ERIC-PCR fragment

The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was employed to generate genomic amplification products of Sinorhizobium meliloti strain 2011. Eleven distinctive PCR fragments obtained in PCR reactions by using the ERIC2 primer were cloned and their partial or complete nucleotide sequences established. DNA sequences that extended past the ERIC2 primer region were not conserved among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Thus, repetitive ERIC or ERIC-like sequences seem not to be an integral part of the S. meliloti genome. An amplification product of S. meliloti 2011 was identified which was present in S. meliloti strains but absent in other rhizobial species. Based on the nucleotide sequence information, a pair of PCR primers was designed and used for PCR amplification of sequences of S. meliloti laboratory strains 2011, L5–30, AK631 and 102F34. Nucleotide sequence analysis of the amplification products revealed a 100% DNA sequence conservation. Database searches showed that the DNA fragment putatively encodes the C-terminal part of a protein displaying similarity to 2-hydroxyacid dehydrogenases of various organisms. The newly designed PCR primers should be useful for the rapid identification of S. meliloti isolates. Received: 17 February 1999 / Accepted: 9 April 1999     

A rapid sex-identification test for the forest musk deer (Moschus berezovskii) based on the ZFX/ZFY gene

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.     

Cloning of Taiwan water buffalo male-specific DNA sequence for sexing

Random amplified polymorphic DNA (RAPD) fingerprinting was carried out to investigate the sex-specific DNA sequence for sexing in Taiwan water buffalos. One hundred and forty random primers were used for RAPD-PCR (polymerase chain reaction). One of these primers, OPC-16, produced a 321 bp fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing, a novel male-specific sequence was obtained. Two primers (BuSexOPC16-F and -R) were designed according to the cloned male-specific sequence to amplify the male-specific fragment using PCR for sexing. Sex-specific bands in the gel were represented in the males but none were found in the females when the Taiwan water buffalo genomic DNA samples were amplified with these two primers using PCR. The same results were also obtained from Taiwan yellow, Holstein, Angus, and Hereford cattle samples. This showed that the sex of these five breeds could be easily and effectively determined using the PCR technique.     

稻飞虱翅型纯系后代个体的翅型分化对光周期变化不敏感

【目的】为了明确光周期和遗传因子在稻飞虱翅型分化中的作用, 研究了3种稻飞虱（褐飞虱Nilaparvata lugens、白背飞虱Sogatella furcifera和灰飞虱Laodelphax striatellus）翅型纯系或近纯系在不同光照时数下的翅型分化比率。【方法】以经过5~45代连续翅型筛选后的褐飞虱、白背飞虱和灰飞虱的长翅型和短翅型纯系或近纯系为材料, 在室内分别测定了其在长光照（16和20 h）、短光照(4~12 h)和正常光照(14 h) 3类光周期条件下饲养后, 雌、雄成虫中长翅和短翅个体出现的比率及存活率。【结果】白背飞虱和灰飞虱的长翅型纯系M♂×M♀或短翅型纯系B♂×B♀在不同光周期下的翅型比率均无显著差异（P>0.05）。褐飞虱短翅型近纯系B♂×B♀的雌虫短翅率和成虫总短翅率在不同光周期下也无显著差异（P>0.05）, 但雄虫短翅率在正常光照14 h和短光照4 h下显著高于长光照20 h下的（P<0.05）。当褐飞虱短翅型达到纯系后, 其后代翅型在6~16 h光照条件下无显著差异。褐飞虱长翅型近纯系M♂×M♀的后代虽有短翅个体出现, 但是雌虫和雄虫的各自短翅率在不同光周期下无显著差异（P>0.05), 仅总体短翅率在12 h光照条件下的显著高于16 h下的（P<0.05）。褐飞虱长、短翅型杂交筛选品系M♂×B♀的雌虫短翅率随光照时数的延长而升高; 灰飞虱杂交筛选品系M♂×B♀的短翅雄虫随光照时数的缩短而增多（P<0.05）, 但当筛选代次达到45代时, 这种趋势不再显著。3种稻飞虱长翅型和短翅型纯系或近纯系若虫的存活率会稍低于长、短翅型杂交后代的存活率, 但长、短翅型品系的存活率在6~16 h光照条件下差异不显著(P>0.05)。【结论】稻飞虱翅型分化对光周期的反应受飞虱本身遗传背景的影响, 翅型纯系后代个体的翅型分化对光周期变化不敏感。