小腔游仆虫形态学、个体发育与分子系统学研究

doi:10.17520/biods.2017045

摘要/Abstract

摘要：

游仆类是纤毛虫中进化最为复杂和高等的一大类群, 为了进一步探索和完善游仆类的多样性, 本研究利用活体观察、蛋白银和银浸法染色技术对采自青岛小西湖的小腔游仆虫(Euplotes aediculatus)的形态学及细胞发生学进行了详尽的研究, 并在完整的形态学及发生学研究基础上, 测定了小腔游仆虫的核糖体小亚基基因(SSU rDNA)序列, 通过序列比较和分子系统树构建等方法, 对小腔游仆虫的系统地位进行了分析。结果表明: 本种鉴别特征为9根额腹棘毛, 5根横棘毛, 2根缘棘毛, 2根尾棘毛, 8列背触毛, double-eurystomus型银线系。发生学特征包括: (1)后仔虫口原基在表皮下独立发生, 前仔虫完全继承老口围带; (2)额-腹-横棘毛原基从左向右按照3:3:3:2:2的模式形成额腹棘毛和横棘毛; (3)前后仔虫最左侧额腹棘毛分别由独立产生的原基形成; (4)缘棘毛原基独立发生; (5)初级背触毛原基来自虫体中部老结构的反分化; (6)前后仔虫尾棘毛分别来自最右侧2列背触毛原基和老背触毛列末端; 这些特征显示出游仆虫属个体发生模式的高度保守性。分子系统分析与形态学数据一致, 即游仆虫属为单元发生, 且小腔游仆虫与艾美游仆虫(Euplotes amieti)、阔口游仆虫(E. eurystomus)和伍氏游仆虫(E. woodruffi)聚在一起。

关键词: 纤毛虫, 小腔游仆虫, 形态学, 细胞发生, 系统学, 核糖体小亚基基因(SSU rDNA)

Abstract

Euplotids are the most complex and highly differentiated group of ciliates. In order to further explore the species diversity of euplotids, the morphology and morphogenesis of Euplotes aediculatus, collected from Small West Lake of Qingdao, were investigated using live observations, protargol, and silver nitrate impregnation. Based on the detailed morphological and morphogenetic data, the small subunit ribosomal rDNA (SSU rDNA) was sequenced for E. aediculatus. The species is characterized by the following features: nine frontoventral, five transverse, two left marginal, and two caudal cirri, eight dorsal kineties, and a silverline system of the double-eurystomus type. The main morphogenetic features during division are as follows: (1) the oral primordium of the opisthe develops de novo within a pouch beneath the cortex, the parental adoral zone of membranelles is entirely kept by the proter; (2) fronto-ventral-transverse cirral anlagen (FVTA) II-VI form cirri in the mode of 3:3:3:2:2; (3) frontal cirrus I/1 of the proter and opisthe is generated from the anlage formed de novo; (4) marginal cirri anlagen are formed de novo; (5) the primary dorsal kinety anlagen are from the dedifferentiation of several bristles in the middle of each parental dorsal kinety; (6) the right-most two dorsal kinety anlagen and parent dorsal kineties contribute to two caudal cirri for the proter and the opisthe, respectively. All of these features showed the high conservation of the ontogenetic process in the genus Euplotes. Sequence comparison and phylogenetic analyses based on SSU rDNA revealed a close relationship among Euplotes aediculatus, E. eurystomus, E. amieti, and E. woodruffi, which is consistent with their similar morphology.

Key words: ciliates, Euplotes aediculatus, morphology, morphogenesis, phylogeny, small subunit ribosomal rDNA (SSU rDNA)

张雪, 王玉蕊, 樊阳波, 罗晓甜, 胡晓钟, 高凤 (2017) 小腔游仆虫形态学、个体发育与分子系统学研究. 生物多样性, 25, 549-560. DOI: 10.17520/biods.2017045.

Xue Zhang, Yurui Wang, Yangbo Fan, Xiaotian Luo, Xiaozhong Hu, Feng Gao (2017) Morphology, ontogeny and molecular phylogeny of Euplotes aediculatus Pierson, 1943 (Ciliophora, Euplotida). Biodiversity Science, 25, 549-560. DOI: 10.17520/biods.2017045.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2017045

https://www.biodiversity-science.net/CN/Y2017/V25/I5/549

图/表 7

图1 小腔游仆虫的活体、蛋白银染色及银浸法染色的形态。A: 典型个体腹面观; B、C: 纤毛图式腹面(B)和背面(C)观; D、E: 腹面(D)和背面(E)银线系。AZM: 口围带; CC: 尾棘毛; CV: 伸缩泡; FVC: 额腹棘毛; Ma: 大核; MC: 缘棘毛; PM: 口侧膜; TC: 横棘毛。比例尺: 100 µm。

Fig. 1 Morphology of Euplotes aediculatus in vivo, after protargol and silver nitrate impregnation. A, Ventral view of a representative individual; B and C, Ventral (B) and dorsal (C) view of the infraciliature and nuclear apparatus; D and E, Silverline system on ventral (D) and dorsal side (E); AZM, Adoral zone of membranelles; CC, Caudal cirri; CV, Contractile vacuole; FVC, Frontoventral cirri; Ma, Macronucleus; MC, Marginal cirri; PM, Paroral membrane; TC, Transverse cirri. Scale bar = 100 µm.

图2 小腔游仆虫的形态结构。A-E: 小腔游仆虫的活体照片; F, G: 蛋白银染色照片; H, I: 银浸法染色照片。A: 典型个体腹面观, 箭头示领状结构; B, C: 不同个体腹面观示不同体型, B图箭头指示伸缩泡, 无尾箭头指示背面肋突; D: 示虫体背面围绕背触毛的皮层颗粒; E: 虫体后部腹面观, 示缘棘毛和分叉的尾棘毛; F, G: 纤毛图式腹面(F)和背面(G)观; H, I: 腹面(H)和背面(I)银线系, H图箭头指示伸缩泡。比例尺: 100 µm。

Fig. 2 Morphology of Euplotes aediculatus. A-E, Photomicrographs in vivo; F and G, Photomicrographs after protargol; H and I, Photomicrographs after silver nitrate impregnation. A, Ventral view of a representative individual, arrow points to collar; B and C, Ventral views, showing different body shapes, arrow in picture B points to contractile vacuole, arrowheads in picture B point to ribs in the back; D, Dorsal view, showing the cortical granules arranged around the dorsal cilia; E, Posterior portion of an individual, showing the caudal cirri (arrows) and the left marginal cirri; F and G, Ventral (F) and dorsal (G) view of the infraciliature and nuclear apparatus; H and I, Silverline system on ventral (H) and dorsal side (I), arrow in picture H points to contractile vacuole. Scale bar = 100 µm.

表1 小腔游仆虫青岛小西湖种群的形态学数据。所有数据均来自蛋白银染色制片。

Table 1 Morphometric characterizations of Euplotes aediculatus from Small West Lake of Qingdao based on protargol-stained specimens

特征 Character	最小值 Minimum	最大值 Maximum	平均值 Mean	中值 Median	标准差 Standard deviation	变异系数 Coefficient of variation	个体数 Number of cells
体长 Length of body (μm)	115	142	128.3	129	7.25	5.7	20
体宽 Width of body (μm)	76	101	92.2	93.5	6.28	6.8	20
口区长 Length of adoral zone (μm)	79	98	86.3	86.5	4.74	5.5	20
口围带小膜数目 Number of adoral membranelles	47	55	51.1	51.5	1.90	3.7	20
额腹棘毛数目 Number of frontoventral cirri	9	9	9	9.0	0	0	19
横棘毛数目 Number of transverse cirri	5	5	5.0	5.0	0	0	20
缘棘毛数目 Number of marginal cirri	2	2	2.0	2.0	0	0	20
尾棘毛数目 Number of caudal cirri	2	2	2.0	2.0	0	0	20
背触毛列数 Number of dorsal kineties	8	8	8.0	8.0	0	0	20
中央背触毛列毛基体数目 Number of dikinetids in mid-dorsal kinety	21	26	21.9	21.0	1.36	6.2	20
最左侧背触毛列毛基体数目 Number of dikinetids in the leftmost dorsal kinety	15	23	18.5	18.0	1.73	9.4	20

图3 小腔游仆虫细胞发生个体银染制片的显微照片。A: 发生早期个体, 示后仔虫口原基出现, 短箭头示前后仔虫的额-腹-横棘毛原基; B: 发生早中期个体腹面观, 短箭头示额-腹-横棘毛原基增粗并开始发生断裂, 长箭头示前后仔虫的缘棘毛原基; C: 发生中期个体腹面观, 示额-腹-横棘毛原基断裂形成棘毛, 短箭头示前后仔虫的最左侧一根额腹棘毛, 长箭头示缘棘毛原基开始断裂形成缘棘毛; D: 发生早中期个体背面观, 短箭头示新形成的背触毛原基; E: 发生中期个体腹面观, 短箭头示额-腹-横棘毛片段化基本完成并开始迁移, 长箭头示前后仔虫新的缘棘毛; F: 发生后期个体腹面观, 短箭头示前后仔虫的横棘毛, 长箭头示最左侧一根额腹棘毛, 此时大核呈棒状; G: 发生后期个体背面观, 短箭头示前仔虫新形成的尾棘毛; H: 发生末期个体腹面观, 示新形成棘毛基本迁移至其最终位置, 大核开始发生分裂。DKA: 背触毛原基; LMC: 左缘棘毛; Ma: 大核; OP: 后仔虫口原基; UMA: 波动膜原基。比例尺: 70 µm。

Fig. 3 Photomicrographs of Euplotes aediculatus during morphogenesis after protargol impregnation. A, Ventral view of an early divider, showing the oral primordium in opisthe, arrowheads mark the frontal-ventral-transverse cirral streaks of both dividers; B, Ventral view of an early-middle divider, arrowheads show the differentiation of frontal-ventral-transverse cirral anlagen, arrows marks the marginal anlagen; C, Ventral view of a mid-stage divider, arrowheads demonstrate the leftmost frontal cirrus (I/1) in both proter and opisthe, arrows marks the differentiation of marginal anlagen; D, Dorsal view of an early-middle divider, arrowheads show the formation of dorsal kineties anlagen; E, Ventral view of another mid-stage divider, arrowheads show the differentiation of frontal-ventral-transverse cirral anlagen almost complete, arrows point to the new marginal cirri of both daughter cells; F, Ventral view of a late divider, noting the macronucleus become a short strand, arrowheads show the transverse cirri, arrows mark the leftmost frontal cirri (I/1); G, Dorsal view of another late divider, arrowheads show the newly formed caudal cirri in the proter; H, Ventral view of a last stage divider, showing cirri almost in their final position and the division of the macronucleus. DKA, Dorsal kineties anlagen; LMC, Left marginal cirri; Ma, Macronucleus; OP, Opisthe’s oral primordium; UMA, Undulating membrane anlagen. Scale bar = 70 µm.

图4 基于核糖体小亚基基因序列构建的最大似然树。本文新测序列加粗显示。节点置信值以最大似然树/贝叶斯树顺序给出。黑点表示满分置信值(100/1.00), “*”表示贝叶斯树与最大似然树拓扑结构不同。比例尺代表每100个碱基位点中有5个碱基替代。

Fig. 4 Maximum likelihood (ML) tree based on small subunit ribosomal DNA sequences. Newly characterized sequence in this study is shown in bold. The numbers at the nodes represent the support values of ML/Bayesian inference (BI). Fully supported (100/1.00) branches are marked with solid circles. Asterisks reflect the disagreement between ML and BI. All branches are drawn to scale. The scale bar corresponds to five substitutions per 100 nucleotide positions.

图5 7个Euplotes aediculatus种群及1个E. eurystomus种群的SSU rDNA碱基差异比较。数字代表比对后的碱基所在位置, 缺失位点用(-)表示, 比对一致的碱基用(.)表示。

Fig. 5 Unmatched sites from SSU rDNA sequence alignment of seven Euplotes aediculatus populations and one E. eurystomus population. Numbers indicate the positions of nucleotides in the alignment, missing sites are represented by dashes (-), and matching sites are marked with dots (.).

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