黑胸散白蚁（Reticulitermes chinensis Snyder）肠道共生古菌的系统发育分析

摘要：【目的】利用非培养法对黑胸散白蚁（Reticulitermes chinensis Snyder）肠道共生古菌进行系统发育分析。【方法】采用古菌16S rDNA通用引物以黑胸散白蚁全肠DNA为模板扩增共生菌的16S rDNA并建立基因文库,对得到的基因序列进行系统发育分析。【结果】从黑胸散白蚁肠道得到5个不同的16S rDNA序列,它们之间的相似性为93.2%～99.2%,系统发育分析表明这5个16S rDNA序列代表的克隆分别与来源于黑胸散白蚁近缘种,栖北散白蚁和北美散白蚁肠道中的甲烷短杆菌克隆或     

黑胸散白蚁新群体的建立及发展规律

经十年的室内配对饲养观察,黑胸散白蚁 Reticulitermes chinensisSnyder初期群体配对后5—10天开始产卵,胚胎发育期36—46天,幼蚁经历两个龄期,各龄8—13天,工蚁是幼蚁经两次蜕皮后分化出来,具有上颚自如活动,头宽0.71mm以上;前兵蚁蜕一次皮发育为兵蚁需经历10—13天,触角14—15节,头宽0.81—0.82mm,大于2龄工蚁,初期巢群最早3个半月左右产生兵蚁.当年配对的产卵期3至4个半月,产卵量16—45粒.饲养7—8年后的群体开始出现若蚁(长翅成虫的幼期).9、10年群体发育成熟即产生长翅成虫.解剖三个成熟群体中一个巢原始蚁王、蚁后均存在,另两巢发现原始蚁王、蚁后均死亡,其中一巢由群体内自行补充上了翅鳞型母蚁1头,无翅补充型生殖蚁1头(较小,性别不清),翅芽型3头.另一巢补充上了无翅型大腹母蚁1头,翅芽型1头.通过室内长期连续饲养观察,对该种白蚁新建群体发育成熟年龄及其内在因素已有一定的了解.     

黑胸散白蚁腹腺的形态学与细微构造

对黑胸散白蚁(Reticulitermes chinensis Snyder)腹腺整体装片和切片进行了描述.腹腺分前、后两部分.“腹腺前部”有两类分泌细胞和一个中央腔.两类分泌细胞中,一类是圆形,个体较大;另一类细胞突起很长,具扁平的核.复盖于腹腺前部的体壁上有许多小管和一些感器,表明腹腺前部的分泌细胞产物可能是经体壁上的小管或者先贮存于中央腔中,再经体壁小管逸出体外.“腹腺后部”由大的椭圆形分泌细胞组成.根据腹腺前部紧贴于第Ⅴ腹节表皮层,而腹腺后部是可动的,并且复盖于腹腺后部的体壁上无排出小管,作者认为这些细胞的分泌物可能是释放入血淋巴中.腹腺前部及腹腺后部分泌细胞的分泌物可能有不同的机能,有待于进一步研究.     

黑胸散白蚁腹腺的形态学与细微构造

对黑胸散白蚁(Reticulitermes chinensis Snyder)腹腺整体装片和切片进行了描述.腹腺分前、后两部分.“腹腺前部”有两类分泌细胞和一个中央腔.两类分泌细胞中,一类是圆形,个体较大;另一类细胞突起很长,具扁平的核.复盖于腹腺前部的体壁上有许多小管和一些感器,表明腹腺前部的分泌细胞产物可能是经体壁上的小管或者先贮存于中央腔中,再经体壁小管逸出体外.“腹腺后部”由大的椭圆形分泌细胞组成.根据腹腺前部紧贴于第Ⅴ腹节表皮层,而腹腺后部是可动的,并且复盖于腹腺后部的体壁上无排出小管,作者认为这些细胞的分泌物可能是释放入血淋巴中.腹腺前部及腹腺后部分泌细胞的分泌物可能有不同的机能,有待于进一步研究.     

黑胸散白蚁末龄若虫蜕皮羽化行为观察

【目的】了解黑胸散白蚁Reticulitermes chinensis Snyder末龄若虫的蜕皮羽化行为。【方法】室内恒温(26±0.5)℃恒湿(75%±5%)饲养黑胸散白蚁末龄若虫,间隔一定时间进行观察记录。【结果】黑胸散白蚁末龄若虫蜕皮羽化时间持续2~8 h。蜕皮一般从末龄若虫胸腹部交界处背部开始破裂,从腹末、翅末、足端、口器末端或触角端脱落,50%以上的正常羽化蜕皮是从腹末端脱落。初始羽化成虫为白色,随后身体和翅的颜色逐渐加深和变黑,羽化8~12 h后,虫体整体颜色变为黑色。室内观察结果还表明,34.7%的末龄若虫无法正常羽化。【结论】黑胸散白蚁末龄若虫蜕皮羽化持续时间较短,蜕皮一般是从胸腹部交界处背部开始破裂,但可从不同部位脱落,且蜕皮羽化行为容易受到外界环境的影响。     

黑胸散白蚁肠道具内切葡聚糖酶活性共生菌的筛选、鉴定及产酶条件的优化

目的 从黑胸散白蚁肠道内筛选获得具有降解纤维素性能的菌株,并对菌株最佳产酶条件进行优化.方法 采用筛选性培养基进行筛选,通过培养性状、显微观察及16S rDNA部分片段同源性分析进行菌种鉴定,利用正交试验优化该菌株的最佳产酶培养基配方以及单因子试验优化产酶培养条件.结果 通过鉴定,获得的菌株属柠檬酸杆菌属(Citrobacter sp.B03),最适产酶的碳氮源为CMC-Na和蛋白胨.该菌株最佳产酶培养基的配方为CMC-Na5.0 g/L、蛋白胨5.0 g/L、NH4Cl 0.6 g/L、KH2P04 0.9 g/L、MgSO4 0.9 g/L;最佳产酶培养条件为起始pH 5.0,温度35℃,装液量20～ 30 mL/150 mL.结论 经过优化,可将该菌株产生的纤维素酶酶活力从0.184 U/mL提高到0.311 U/mL,该研究结果对纤维素酶的工业开发具有一定的指导意义.     

低等白蚁肠道共生微生物的多样性及其功能

低等白蚁肠道里存在着复杂的微生物区系,包括真核微生物鞭毛虫和原核生物,细菌及古细菌。低等白蚁的后肠以特别膨大的囊形胃及其氢氧浓度的明显梯度分布和丰富的微生物区系为特征,是白蚁进行木质纤维素消化的主要器官。后肠内的鞭毛虫能将纤维素水解并发酵为乙酸,二氧化碳和氢,为白蚁提供营养和能源。系统发育研究表明,低等白蚁肠道共生细菌的主要类群为白蚁菌群1、螺旋体、拟杆菌,低G C mol%含量的革兰氏阳性菌和紫细菌等。而古细菌主要为甲烷短杆菌属的产甲烷菌。共生原核生物与二氧化碳的还原和氮的循环等代谢有关。但肠道共生微生物的具体功能和作用机制还有待进一步的揭示。     

黑胸散白蚁新群体的建立和发展与环境条件的关系

在实验室进行了温度、水湿、食物、土壤、活动空间与新群体建立和发展关系的实验,野外又观察收集了相关资料。结果表明：在20℃-28℃和含水量为50.5%-69.6%的基质中,配对饲养的新群体几乎全部正常发展;30℃以上或基质含水量低于38.2%,新群体难以建立;25℃时工蚁取食的净增量最大;工蚁含水量为79%、构成蚁巢的白蚁排泄物和木材含水量分别为54.8%和52.3%;7种木材饲养新群体,以刺槐饲养的发展最快,香樟和马尾松次之;群体的发展还与土壤和活动空间有密切关系。在此基础上,探讨了白蚁建巢的限制条件,认为迁巢活动和群体分裂是长期适应环境条件的结果,提出了相应的防治对策。     

黑胸散白蚁幼期不同品级的发育和分化

在黑胸散白蚁Reticulitermes chinensis发育和分化过程中,发现有假工蚁、假若蚁两种虫态。假工蚁由6龄和7龄工蚁转化发育而来,假若蚁由4龄和5龄若蚁转化发育而来。显微测量结果表明,黑胸散白蚁的胚后发育主要包括2个龄期的幼蚁期、6个以上龄期的工蚁期、4个龄期的若蚁期和有翅成虫。在此基础上分析了其他虫态的分化来源,发现兵蚁由3～7龄工蚁分化发育而来,翅鳞型和长翅芽型补充生殖蚁由6龄若蚁转化发育而来,短翅芽型补充生殖蚁由4龄和5龄若蚁转化发育而来,微翅芽型补充生殖蚁既可由4～6龄工蚁转化发育而来,又可由假工蚁和假若蚁转化发育而来,无翅型补充生殖蚁由3～7龄工蚁转化发育而来。提出了黑胸散白蚁群体中不同品级个体的可能分化途径。     

黑胸散白蚁幼期不同品级的发育和分化

在黑胸散白蚁Reticulitermes chinensis发育和分化过程中,发现有假工蚁、假若蚁两种虫态。假工蚁由6龄和7龄工蚁转化发育而来,假若蚁由4龄和5龄若蚁转化发育而来。显微测量结果表明,黑胸散白蚁的胚后发育主要包括2个龄期的幼蚁期、6个以上龄期的工蚁期、4个龄期的若蚁期和有翅成虫。在此基础上分析了其他虫态的分化来源,发现兵蚁由3～7龄工蚁分化发育而来,翅鳞型和长翅芽型补充生殖蚁由6龄若蚁转化发育而来,短翅芽型补充生殖蚁由4龄和5龄若蚁转化发育而来,微翅芽型补充生殖蚁既可由4～6龄工蚁转化发育而来,又可由假工蚁和假若蚁转化发育而来,无翅型补充生殖蚁由3～7龄工蚁转化发育而来。提出了黑胸散白蚁群体中不同品级个体的可能分化途径。     

Fluorescence in situ hybridization for intracellular localization of nifH mRNA

Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells.     

Crystal ball: Fluorescence in situ hybridization in the age of super-resolution microscopy

Super-resolution microscopy encompasses a suite of cutting edge microscopy methods able to surpass the resolution limits of light microscopy. The recent commercial availability of super-resolution microscopy is advancing many fields of biology. In this crystal ball forward look, we briefly examine the perspectives of combining super-resolution microscopy and fluorescence in situ hybridization (FISH). We strongly believe, based on first evidence presented here, that using super-resolution microscopy in environmental microbiology has the potential to reshape the way we analyze the results obtained with FISH, by improving both the localization and quantification of target molecules.     

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo.     

Phylogenetic characterization and in situ detection of bacterial communities associated with seahorses (Hippocampus guttulatus) in captivity

Although there are several studies describing bacteria associated with marine fish, the bacterial composition associated with seahorses has not been extensively investigated since these studies have been restricted to the identification of bacterial pathogens. In this study, the phylogenetic affiliation of seahorse-associated bacteria was assessed by 16S rRNA gene sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rRNA analysis. Both methods revealed that Vibrionaceae was the dominant population in Artemia sp. (live prey) and intestinal content of the seahorses, while Rhodobacteraceae was dominant in water samples from the aquaculture system and cutaneous mucus of the seahorses. To our knowledge, this is the first time that bacterial communities associated with healthy seahorses in captivity have been described.     

Karyotype Analysis of Gossypium arboreum × G. bickii by Genome in situ Hybridization

By using genome in situ hybridization (GISH) on root somatic chromosomes of allotetraploid derived from the cross Gossypium arboreum × G. bickii with genomic DNA (gDNA) of G. bickii as a probe, two sets of chromosomes, consisting of 26 chromosomes each, were easily distinguished from each other by their distinctive hybridization signals. GISH analysis directly proved that the hybrid GarboreumxG. bickii is an allotetraploid amphiploid. The karyotype formula of the species was 2n = 4x = 52 = 46m (4sat) + 6sm (4sat). We identified four pairs of satellites with two pairs in each sub-genome. FISH analysis using 45S rDNA as a probe showed that the cross G. arboreumxG. bickii contained 14 NORs. At least five pairs of chromosomes in the G sub-genome showed double hybridization (red and blue) in their long arms, which indicates that chromatin introgression from the A sub-genome had occurred.     

Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes

SSU rRNA genes of oxymonad protists from the hindgut of the wood-feeding termite Reticulitermes flavipes were PCR-amplified using a newly designed oxymonad-specific forward primer and a newly designed reverse primer specific for termite gut flagellates. After cloning, the clone library was sorted into four groups by RFLP analysis and nearly full-length SSU rRNA gene sequences were obtained for representative clones from each group. Phylogenetic analysis revealed that sequences of all four groups formed a monophyletic cluster with the only other existing SSU rRNA gene sequence of oxymonads. Using whole-cell hybridization with clone-specific fluorescently labeled probes, each of the four clone groups could be assigned to a specific morphotype, which were identified as Dinenympha gracilis, Dinenympha fimbriata, and so-far undescribed species of Pyrsonympha and Dinenympha. Our results demonstrate that the morphological variety of oxymonads is not caused by the presence of different developmental stages of the same organism, but that the various morphotypes represent different species.     

基因组原位杂交鉴定牛筋草AA基因组在穇子染色体上的分布及探针长度优化方法

采用基因组原位杂交(Genomic in situ hybridization,GISH)方法研究了牛筋草(Eleusine indica)AA基因组在穇子(E.coracana)染色体上的分布,并探讨了AA、BB基因组的同源关系。用超声波破碎法进行预剪切,以缺口平移法标记的牛筋草总DNA为探针,BB基因组的E.floccifolia(Forssk.)Spreng.总DNA为封阻,与AABB基因组穇子的中期染色体进行杂交。结果表明,牛筋草AA基因组分布在穇子的18条染色体上。不加封阻或加过量封阻均不能鉴别AA基因组,说明AA和BB基因组间的分化程度不大,双方共享的重复序列较多。牛筋草与E.floccifolia总DNA分别用超声波破碎2 min和3 min后,可得到峰值为300-750 bp的DNA片段,这说明不同物种的超声波破碎时间需要调整,以获得合适长度的探针。     

Localization of strawberry (Fragaria x ananassa) and Methylobacterium extorquens genes of strawberry flavor biosynthesis in strawberry tissue by in situ hybridization

Strawberry flavor is one of the most popular fruit flavors worldwide, with numerous applications in the food industry. In addition, the biosynthetic origin of the most important strawberry flavor components, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF), is a challenging research area. DMHF's precursor, 2-hydroxy-propanal (or lactaldehyde), is biosynthesized by the endophytic bacterium Methylobacterium extorquens (M. extorquens). In particular, the alcohol dehydrogenase (ADH) enzymes of M. extorquens are involved in the biogenesis of DMHF precursors since they have the capacity to oxidize the strawberry-derived 1,2-propanediol to lactaldehyde. In this study, the expression of the endophytic ADH and the plant DMHF biosynthesis genes was examined in the tissues of raw and ripe strawberry receptacles by in situ hybridization. The presence of endophytic bacteria was studied in the same tissues by probes targeting bacterial 16S ribosomal ribonucleic acid. Hybridization signals of probes specific for endophytic ADH and plant DMHF biosynthesis genes, as well as bacteria-specific probes, were detected in the same locations. The probes were localized near the plasma membranes or intercellular spaces of cortical and vascular tissues of the receptacle, and intracellularly in the tissues of achenes. By localizing the expression of the endophytic methanol ADH and plant DMHF biosynthesis genes to the same tissues, we have reinforced our original hypothesis that an intimate symbiotic relationship between strawberry and endophytic cells exists and leads to the biosynthesis of DMHF.     

Visualization of initial bacterial colonization on dentine and enamel in situ

Bacterial colonization of dentine is of high relevance in cariology, endodontology and periodontology. The aim of the present in situ study was to establish recent methods for visualization and quantification of initial bacterial adherence to dentine in comparison to enamel. For this purpose, bovine enamel and dentine slabs were fixed on buccal sites of individual upper jaw splints worn by 6 subjects for 30 min, 120 min and 360 min, respectively. Adherent bacteria on the slabs were visualized and quantified with DAPI-staining (4′,6-diamidino-2-phenylindole) and fluorescence in situ hybridization (FISH) of streptococci and eubacteria using the CLSM (confocal laser scanning microscopy) as well as an epifluorescence microscope. In addition, the number of colony forming units was quantified after desorption. Representative samples were processed for SEM (scanning electron microscopy) and TEM (transmission electron microscopy). All methods clearly indicated that a significantly higher number of bacteria adhered to dentine than to enamel. Furthermore, the amount of bacteria on the dentine increased with increasing oral exposure time, but remained rather constant on the enamel. The CLSM allowed visualization of bacteria in the dentinal tubules. Bacteria were found preferentially at the openings of the dentine tubules, but were distributed randomly on the enamel.In conclusion, the adopted methods are suitable for visualization and quantification of bacterial adhesion to dentine. Even the initial bacterial colonization of dentine is much more pronounced than bacterial adherence to the enamel.