Peat moss (Sphagnum) is a non-vascular higher plant with unique xylem-like hyaline (H) cells that are accompanied by photosynthetic chlorophyllous cells. These cellular structures play crucial roles in water storage and carbon sequestration. However, it is largely unknown how peat moss develops the H cells. This study systematically explored the Sphagnum Developmental Cell Atlas and Lineage and classified leaf cell development into two lineages with six stages (S0-S5) based on changes in key cellular traits, including the formation of spiral secondary cell walls (S4) and the presence of water pores (S5). Cell lineage-specific subcellular remodeling was transcriptionally regulated during leaf development, and vacuole-mediated clearance of organelles and cell death led to mature dead H cells. Interestingly, expression of land plant conserved Vascular-related NAC Domain (VND) genes correlated with H cell formation. Overall, these results suggest that the origination of xylem-like H cells is related to VND, likely through the neofunctionalization of vacuole-mediated cell death to attempt xylem formation in peat moss, suggesting potential uncoupling of xylem and phloem cell origins. This study positions peat moss as a potential model organism for studying integrative evolutionary cell biology.

The single-celled eukaryote Euplotes aediculatus was chosen to test and compare the toxic effects of Cu and CuO nanoparticles (NPs). The antioxidant enzymatic activity, morphological changes, and functional groups on the membrane were determined using spectrophotometry, microscopy, and Fourier transform infrared spectroscopy after NPs treatment. The toxicity of the NPs to cells was dose-dependent, and the 24 h-LC50 values of the CuNPs and CuONPs were 0.46 µg/L and 1.24 × 103 µg/L, respectively. These NPs increased the activities of superoxide dismutase, glutathione peroxidase, and catalase and destroyed the cell structure; moreover, the CuNPs were more toxic than the CuONPs. In addition to the higher enzymatic activity, CuNPs also caused nucleoli disappearance, chromatin condensation, and mitochondrial and pellicle damage. The oxidization of the functional groups of the membrane (PO2 − , C–O–C, and δ(COH) of carbohydrates) also confirmed the severe damage caused by CuNPs. Our study showed that oxidative stress and organelle destruction played important roles in the toxic effects of these NPs on this protozoan. Compared with other aquatic organisms, E. aediculatus can be considered a potential indicator at the preliminary stage of environmental pollution.

高压冷冻及冷冻替代技术是一种利用高压和低温对含水组织样品进行瞬间冷冻固定和低温脱水的电镜样品制备技术,它因可以保存更加真实的细胞超微结构而常应用于透射电镜观察和分析.本文利用模式动物小鼠为材料,将这种技术应用于体扫描电镜的样品制备,并与常规化学固定的样品制备进行比较.结果显示在小鼠心肌组织中,高压冷冻及冷冻替代技术可以有效避免常规制备样品中的细胞器膜结构皱缩、细胞质分布不均匀、细胞核膜收缩形成锯齿状、甚至局部断裂等现象,使细胞超微结构更接近其生活时的状态.

The Class Nassophorea is not monophyletic with unsolved relationship of four orders, which calls for discussion to combine morphological features and molecular phylogeny. In the present study, the ultrastructure of Apocolpodidium etoschense in the order Colpodidiida is first studied. Comparisons between orders of Nassophorea were conducted and a discussion of systematics was performed based on a SSU rRNA gene-based phylogeny. The order Colpodidiida and Nassulida shared the following features: Two pairs of alveolocysts in the cortex, the presence of a ''B-cartwheel'' in the distal region of the kinetosome, the presence of cytostomal lamellae and subcytostomal lamellae in the cytopharyngeal basket, and spindle trichocysts with a simple tip. These similarities shape a core group of Nassophorea, which are morphologically and genetically different from the order Microthoracida. Consequently, Microthoracida should be regarded as an independent taxon rather than a member of Nassophorea. Within the core group of Nassophorea, Colpodidiida as an independent order is further validated by its delicate cytopharyngeal basket which lacks nematodesmal lamellae; while the non-monophyly of the order Nassulida might be explained by differentiation of the cartwheels in kinetosomes and the arrangement of kinetosomes with postciliary microtubules in the nassulid organelle 3 within its members.

Although many reports have demonstrated that nanoparticles can have a negative effect on aquatic organisms, the toxic effects on symbiotic organisms remain poorly understood. The present study conducts ultrastructure, enzyme activity, and transcriptomics to assess the toxic effects to the Paramecium bursaria-Chlorella symbiotic system from exposure to copper nanoparticles (CuNPs) for 24 h. We found that in both the host and symbiotic algae, CuNP exposure induced high reactive oxygen species level, which leads to oxidative damage and energy metabolism disorder. Moreover, transmission electron micrographs (TEMs) showed that the symbiotic algae in the cytoplasm of P. bursaria were enveloped in the digestive vacuole and digested, and the level of acid phosphatase activity increased significantly within 24 h, which indicated that the stability of the symbiotic system was affected after CuNP exposure. We speculated that the increased energy demand in the host and symbiotic algae resulted from oxidative stress, precipitating the decrease of the photosynthetic products provided to the host, the digestion of the symbiont, and the destruction of the stable symbiotic relationship. The study provides the first insight into the mechanisms of nanoparticles' toxicity to the symbiotic relationship in the ecosystem, which may help to understand the environmental effects and toxicological mechanisms of nanoparticles.

扫描电子显微(配备能谱仪附件)技术作为表面微观分析的重要手段之一,可充分展现催化剂的表面形貌和微区成分,对催化剂的研制和开发具有重要意义.本文将背散射电子成像与X射线能谱仪结合,研究了 Au-Ni复合催化剂的表面形貌及活性组分的分散状态,并详细探讨了图像清晰度与扫描速度、工作电压、工作距离等的关系.研究发现,在扫描速度10 s/frame、工作电压5 kV、工作距离4 mm条件下,采集背散射电子图像并结合能谱分析,可以获得充分展现催化剂性质的图像,为Au-Ni复合催化剂的催化活性机理研究提供重要的实验数据支撑.该研究提供了一种行之有效且快速方便的、纳米量级的分析准确度及分辨率的表征技术,有助于电镜相关人员更好地理解背散射现象的物理机制.

为探究纳米材料进入水环境后对水生生物的影响,本文利用扫描及透射电子显微镜观察了纳米银和纳米铜对纤毛虫绿草履虫(Paramecium bursaria)细胞超微结构的损伤情况.结果表明:(1)两种纳米材料对细胞结构的损伤方式具有一定的相似性,损伤主要表现在绿草履虫细胞的表膜、线粒体以及细胞核等结构,且损伤程度随时间增加逐渐加重.(2)纳米铜对细胞超微结构造成的损伤程度比纳米银严重.实验结果表明,两种纳米材料均对纤毛虫具有生物毒性,推测其通过氧化应激导致细胞表面及内部结构损伤.

纳米氧化锌(ZnONPs)被广泛应用于医药材料等领域,但不适当的排放会给水生生态环境带来安全隐患.本研究用ZnO NPs对单细胞真核生物绿草履虫(Paramecium bursaria)的急性和慢性毒理进行测试,结合电镜技术、荧光技术以及酶活力测定、DHE荧光探针技术对ZnO NPs的致毒机制进行探索.结果显示ZnO NPs对绿草履虫的24h-LC50和24h-EC50分别为0.39 mg/L和0.04 mg/L,与其他受试动物相比较为敏感.超微结构显示ZnO NPs可对绿草履虫细胞表膜、刺丝泡、线粒体和细胞核等结构造成损伤.酶活力和荧光探针等结果推测ZnO NPs可能是通过诱导活性氧的积累使绿草履虫遭受氧化胁迫继而死亡.另外,ZnO NPs的解离度与锌离子的毒性测定结果表明锌离子是ZnO NPs导致绿草履虫死亡的主要因素,因此通过改性以抑制ZnO NPs的溶解度可以提高纳米材料使用的安全性.

Toxicology tests were carried out by choosing Cu nanoparticles (CuNPs) and CuO nanoparticles (CuONPs) as experimental materials and Euplotes aediculatus as the experimental organism. To investigate the toxicity effect and mechanism of two NPs on E. aediculatus , we determined antioxidant enzyme activity and observed morphologic changes using optical and electron microscopes, combined with the Fourier infrared spectrum technique. The results showed that the 24 h-LC 50 of CuNPs and CuONPs was 0.46 µg/L and 1.24×10 3 µg/L. The movement ability of cells was decreased and surface cilia gradually shed with CuNPs and CuONPs at 24 h-LC 50 . In addition, the cell body swelled and finally ruptured. There were varying extents of damage to the nucleus and mitochondria. With CuNPs, disappearance of nucleoli and condensation of chromatin were observed, while the mitochondria were wrinkled in an irregular shape and cristae were partially fractured. With CuONPs, changes of nucleus were not obvious, and the mitochondria were merely irregular. While neither CuNPs nor CuONPs had major effect on the ultrastructure of the membrane, some functional groups were oxidized with CuNPs, e.g. PO 2 -, C-O-C, δ(COH) of carbohydrates. The 24 h-EC 50 , 48 h-EC 50 and 72 h-EC 50 of CuNPs on E. aediculatus were 2.10×10 -3 µg/L, 7.92×10 -4 µg/L and 2.77×10 -4 µg/L. The EC 50 of CuONPs in same period was 7.20 µg/L, 0.86 µg/L and 0.19 µg/L. The above concentration of CuNPs and CuONPs could increase the activities of SOD, CAT and GPx, which were dose-dependent. The above results indicate that CuNPs and CuONPs inhibited reproduction and caused death. CuNPs were more toxic to E. aediculatus and more destructive to cell structure. Oxidative stress and destruction to cell structures may be toxic mechanisms. The E. aediculatus was more sensitive to CuNPs or CuONPs, and the value of 24 h-LC 50 was much lower than other organisms, so it can be recommended as an indicator for early monitoring in freshwater environment.

本文利用透射电镜观察纳米银对冠突伪尾柱虫(Pseudourostyla cristata)、浮萍棘尾虫(Stylonychia lemnae)、小腔游仆虫(Euplotes aediculatus)三种纤毛虫超微结构的影响,结果表明:(1)浮萍棘尾虫的表膜结构受到破坏,另外两种细胞表膜未受到损伤.(2)冠突伪尾柱虫的线粒体结构破坏最严重,外膜裂解,嵴断裂瓦解;小腔游仆虫的线粒体破坏程度次之,外膜完整,嵴断裂;浮萍棘尾虫线粒体形态较完整.(3)冠突伪尾柱虫细胞核受损最严重,核膜、染色质均有瓦解现象.初步推测,纳米银对纤毛虫具有较大的生物毒性,且毒性作用与纤毛虫种类相关.

An integrated approach considering both morphologic and molecular data is now required to improve biodiversity estimations and provide more robust systematics interpretations in hypotrichs, a highly differentiated group of ciliates. In present study, we document a new hypotrich species, Lamtostyla gui n. sp., collected from Chongming wetland, Shanghai, China, based on investigations using living observation, protargol staining, scanning and transmission electron microscopy, and gene sequencing. The new species is mainly recognized by having a short amphisiellid median cirral row composed of four cirri, three frontoventral cirri, three dorsal kinetids, four to eight macronuclear nodules, and small colorless cortical granules distributed as rosettes around dorsal bristles. Transmission electron microscope observation finds the associated microtubules of cirri and pharyngeal discs of L. gui are distinct from those in other hypotrichs. Morphogenesis of this species indicates that parental adoral membranelles retained intact or partial renewed is a potential feature to separate Lamtostyla granulifera-group and Lamtostyla lamottei-group. Phylogenetic analysis based on small subunit ribosomal RNA (rRNA) gene shows that this molecular marker is not useful to resolve phylogenetic relationships of the genus Lamtostyla, as well as many other hypotrichous taxa. We additionally characterize the internal transcribed spacers (ITS) region and the almost complete large subunit rRNA, which will be essential for future studies aimed at solving phylogenetic problems of Lamtostyla, or even the family Amphisiellidae. As a final remark, the critical screening of GenBank using ITS genes of our organism allows us to recognize a large amount of hypotrichous sequences have been misclassified as fungi. This observation suggests that hypotrichs could be frequently found in fungi-rich environment and overlooked by fungal specialists.

本文利用光学显微镜、电子显微镜技术研究了30 nm粒径圆球形纳米银颗粒及30 nm粒径圆球形纳米氧化锌颗粒对双眉虫(Diophrys sp.)细胞超微结构的影响.结果显示,这两种纳米材料均对细胞结构造成了一些相似的影响,主要表现在:(1)细胞内形成大量空泡;(2)线粒体出现肿胀、内部嵴断裂以及局部结构模糊和变空的现象;(3)细胞内出现大量自噬体,以及自噬后形成的髓鞘样小体.此外,纳米银颗粒处理组还出现细胞核核仁消失,核膜界限模糊的现象;纳米氧化锌处理组还发生了纤毛杆处质膜融合,细胞中产生大量溶酶体的现象.初步推测,纳米银颗粒主要通过影响细胞核功能,进而造成其他生命活动紊乱,而纳米氧化锌颗粒则会诱导细胞凋亡和破坏纤毛器结构从而限制细胞运动.

Ciliated protists can form cysts to resist unfavorable environmental conditions and then excyst when environmental conditions become favorable. This study used electron and light microscopy to investigate the structure of vegetative cells and resting cysts, as well as the encysting and excysting processes of Diophrys oligothrix. For the first time, ampules were revealed beneath the pellicle in the genus Diophrys, and their extrusome types differed between Diophrys species. Membrane-packed discs of diverse shapes were found in the cytoplasm just beneath the pellicle around the cytopharynx and were separated by rows of microtubule units. Beneath the discs, some double-layer microtubule structures were detected as well. During encystment, the ventral ciliature was folded in a ventral cavity of the cell, and the caudal cirri were retracted directly into the cyst in a separate cavity on the dorsal side. In the resting cysts, high autophagic activity occurred, possibly including digestion of membrane-packed discs and ampules. Two macronuclear nodules kept their basic shape, although the chromatin aggregation and fusion region were observed in ultrathin sections. The cyst wall contained two layers, namely, the ectocyst, and endocyst. In mature cysts, basal bodies and ciliary shafts were observed, demonstrating that D. oligothrix forms non-kinetosome-resorbing cysts. The process of excystment occurred in two modes, either with or without participation of a contractile vacuole.