中国绿水螅分子系统发生地位的初步探讨

目的:初步探讨中国绿水螅(Hydra sinensis)分子系统发生地位以及水螅属内部各类群系统发生关系。方法:采用酚-氯仿法提取中国绿水螅总DNA,扩增线粒体COI和16S r RNA基因片段并进行DNA序列测定,再利用Clustal及MEGA等生物信息学分析软件进行系统发生分析。结果:在本研究重建的所有系统发生树中,中国绿水螅始终与绿水螅Hydra viridissima的不同种群一起构成绿水螅单系群。同时,棕色水螅群的单系性被基于COI基因的NJ树以及基于16S r RNA基因的NJ树和ML树支持,唯独基于COI基因的ML树不支持棕色水螅群的单系发生。在基于COI基因的ML树中纤弱水螅族在系统树的基部独立为一支系,而绿水螅群和其他棕色水螅群水螅一起组成另一支系,提示纤弱水螅族水螅的系统发生地位值得进一步探讨。值得注意的是,根据本文的结果,棕色水螅群内3族的划分仍然有一定疑问。基于COI基因的NJ树和ML树支持普通水螅族、寡水螅族和纤弱水螅族各自族内的单系发生,但16S r RNA基因的NJ树和ML树中仅普通水螅族水螅聚为单系群,而寡水螅族和纤弱水螅族水螅各自并非单系发生。结论:把水螅属划分为绿水螅群及棕色水螅群有一定的合理性,但棕色水螅群内寡水螅族、普通水螅族和纤弱水螅族3族的划分还有待商榷。     

强壮水螅的特征及其与寡水螅的种间差别问题

一种广泛分布于黑龙江省的大型具柄水螅被暂定为强壮水螅Hydra robusta(IT(?) 1947)。这一中国品系与IT(?)(1947)在日本报告的新种主要特征相同,包括具有精巢乳头。只是精巢乳头不稳定。在实验室条件下第一次有性生殖时每个雄体都发生精巢乳头,但第二次有性生殖时,同一群的后代可全部失去精巢乳头。这是介于强壮水螅与寡水螅Hydra oligactis Pallas(1766)之间的中间性状。从而导致作者做出结论,认为过去其他学者所主张的,以精巢乳头的有无做为鉴别特征来区分强壮水螅和寡水螅是不可靠的。因此,本文又检验了此种水螅的体细胞染色体,证明2n=30,其中第二对染色体上有明显近中位置的次缢痕,最小染色体的长度为最大染色体长度的二分之一以上。这些可做为此种水蝗的鉴别特征。对Niiyama(1944)和Datta(1970)在寡水螅上所做的染色体研究也做了比较及讨论。     

Taxonomy of the European Hydra (Gnidaria: Hydrozoa): a re-examination of its history with emphasis on the species H. vulgaris Pallas, H. attenuata Pallas and H. circumcincta Schulze

The name Hydra attenuata Pallas is currently applied to the wrong animal. The common brown polyp, which is widely called H. attenuata, was described by Pallas (1766) as Hydra vulgaris. The name H. attenuata Pallas originally referred to an uncommon pale polyp, currently known as H. circumcincta Schulze. The history of this confusion is analysed here. The taxonomy of hydra was in disarray during the 18th and 19th centuries, and was clarified in 1917 with the monograph of Schulze. But Schulze misapplied the name for the common hydra, H. vulgaris, to an unusual form and thus was led to assign the name of a rare hydra, H. attenuata, to the common type. Schulze redescribed the rare, pale hydra that Pallas had named H. attenuata as H. circumcincta. The correct name of the common European brown, stalkless hydra is thus H. vulgaris Pallas, 1766. The name H. attenuata has priority for the uncommon pale hydra, but because of disuse of this application of the name, the pale hydra should be recognized by the current, generally accepted binominal H. circumcincta Schulze, 1914.     

Genome sizes and chromosomes in the basal metazoan Hydra

Hydras belong to one of the earliest eumetazoan animal groups, but to date very little is known about their genome sizes, gene numbers, and chromosomes. Here we provide genome size estimates and corresponding karyotypes for five Hydra species. Nuclear DNA contents were assessed by slide-based Feulgen microphotometry. Hydra oligactis possesses the largest genome of 1450 Mbp, followed by similar 1 C capacities in H. carnea (1350 Mbp), H. vulgaris (1250 Mpb) and H. circumcincta (1150 Mbp). The smallest genome of 380 Mbp was determined in H. viridissima. While the number of chromosomes is identical in all five Hydra species (2n = 30), the size of the chromosomes is strictly correlated to the size of the genome, with H. viridissima having conspicuously small chromosomes. The taxonomic and evolutionary significance of the C-value and chromosomal size variation in this ancient group of metazoans as well as its impact on genomic organization and forthcoming genome projects are discussed.     

Three new genera and five new species of misophrioid copepods (Crustacea) from anchialine caves on Indo-West Pacific and North Atlantic Islands

Hydra lirosoma sp. nov. is a small, hermaphroditic species possessing broad holotrichous isorhiza nematocysts. The spherical embryo and cobbled embryotheca set it apart from similar species. Hydras with broad holotrichous isorhizas form a distinct cluster of species, but one which was not explicitly represented in the three genera ( Pelmatohydra, Hydra and Chlorohydra ) recognized by Schulze (1917, Archiv für Biontologie, 4(2 ): 29). Schulze failed to recognize this group because of the small number of species known at that time. The hydras that are known now fit naturally into four clusters of species termed, in this paper, the oligactis, vulgaris, viridissima, and braueri groups.     

Taxonomy of the European Hydra (Cnidaria: Hydrozoa): a re-examination of its history with emphasis on the species H. vulgaris Pallas,H. attenuata Pallas and H. circumcincta Schulze

The name Hydra attenuata Pallas is currently applied to the wrong animal. The common brown polyp, which is widely called H. attenuata, was described by Pallas (1766) as Hydra vulgaris. The name H. attenuata Pallas originally referred to an uncommon pale polyp, currently known as H. circumcincta Schulze. The history of this confusion is analysed here. The taxonomy of hydra was in disarray during the 18th and 19th centuries, and was clarified in 1917 with the monograph of Schulze. But Schulze misapplied the name for the common hydra, H. vulgaris, to an unusual form and thus was led to assign the name of a rare hydra, H. attenuata, to the common type. Schulze redescribed the rare, pale hydra that Pallas had named H. attenuata as H. circumcincta. The correct name of the common European brown, stalkless hydra is thus H. vulgaris Pallas, 1766. The name H. attenuata has priority for the uncommon pale hydra, but because of disuse of this application of the name, the pale hydra should be recognized by the current, generally accepted binominal H. circumcincta Schulze, 1914.     

Variation in green hydra. A description of three cloned strains of Hydra viridissima Pallas 1766 (Gnidaria: Hydrozoa) isolated from a single site

Three different strains of green hydra were isolated in successive years from a single site in the River Frome, Bristol, England. The strains resembled each other in important taxonomic characters, and were identified as Hydra viridissima by their possession of smooth-walled single-chambered embryothecas, but were smaller, possessed smaller nematocysts and produced fewer eggs per individual than the Western European holotype. It is possible that English green hydra represent a geographical subspecies.
The three strains could be distinguished from each other by differences in rates of bud production and in characters associated with their symbiotic algae. These differences are considered varietal, and it is suggested that H. viridissima is a polytypic species. The possible origins of these variations and their significance is discussed.     

Identification of a new member of the GLWamide peptide family: physiological activity and cellular localization in cnidarian polyps

KPNAYKGKLPIGLWamide, a novel member of the GLWamide peptide family, was isolated from Hydra magnipapillata. The purification was monitored with a bioassay: contraction of the retractor muscle of a sea anemone, Anthopleura fuscoviridis. The new peptide, termed Hym-370, is longer than the other GLWamides previously isolated from H. magnipapillata and another sea anemone, A. elegantissima. The amino acid sequence of Hym-370 is six residues longer at its N-terminal than a putative sequence previously deduced from the cDNA encoding the precursor protein. The new longer isoform, like the shorter GLWamides, evoked concentration-dependent muscle contractions in both H. magnipapillata and A. fuscoviridis. In contrast, Hym-248, one of the shorter GLWamide peptides, specifically induced contraction of the endodermal muscles in H. magnipapillata. This is the first case in which a member of the hydra GLWamide family (Hym-GLWamides) has exhibited an activity not shared by the others. Polyclonal antibodies were raised to the common C-terminal tripeptide GLWamide and were used in immunohistochemistry to localize the GLWamides in the tissue of two species of hydra, H. magnipapillata and H. oligactis, and one species of sea anemone, A. fuscoviridis. In each case, nerve cells were specifically labeled. These results suggest that the GLWamides are ubiquitous among cnidarians and are involved in regulating the excitability of specific muscles.     

普通水螅(无鞘螅目,水螅科)中国一新纪录种

对采自中国广东省珠江流域(114°E,23°N)的一种淡水水螅进行连续两年的培养与观察,并扩增了其线粒体细胞色素氧化酶亚基Ⅰ(COⅠ)的基因序列.通过形态学分析及CO Ⅰ基因序列的鉴定确定其为普通水螅Hydra vulgaris Pallas,1766,为中国一新纪录种.     

光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶活力的影响

单细胞真核绿藻在中国水螅(Hydra sinensis)内胚层皮肌细胞中共生是有较高科研价值的特殊生物学现象。水螅宿主细胞为共生藻提供CO2、氮源及矿物质,而共生藻通过光合作用可能为宿主提供碳水化合物等有机物营养,因此水螅与共生藻间代谢流是以共生藻光合作用为中心,但基于代谢流二者间的互作机制目前尚未阐明。水螅通过营养积累进行出芽生殖,从母体脱落的芽体数量间接反映水螅营养积累的相对量。而光暴露时长能影响共生绿藻光合作用,如果共生藻的确能向水螅细胞转移光合作用产物,那光暴露时长应该能间接影响水螅的营养积累、从而进一步影响水螅无性出芽生殖。为证实该假说,本研究应用种群累积培养法,观察了光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶(SOD和CAT)活力的影响。结果显示,光周期对中国水螅种群增长具有明显的影响。培养15 d后,所有实验组水螅的种群密度均为正增长,其中8L∶16D(在一个24h周期内光暴露8 h、黑暗16 h)实验组种群密度最大、而0L∶24D(持续黑暗)实验组种群密度最小。另外,随着光暴露时长的增加,中国水螅SOD及CAT活力整体均呈下降趋势。结果表明,从光周期对中国水螅无性出芽生殖及两种抗氧化酶活力的影响来看,中国水螅对光周期的生理学响应较为敏感,这个现象可能源于共生绿藻能通过向宿主细胞转移光合作用产物的方式为水螅提供营养补充。     

Germline stem cells and sex determination in Hydra

The sex of germline stem cells (GSCs) in Hydra is determined in a cell-autonomous manner. In gonochoristic species like Hydra magnipapillata or H. oligactis, where the sexes are separate, male polyps have sperm-restricted stem cells (SpSCs), while females have egg-restricted stem cells (EgSCs). These GSCs self-renew in a polyp, and are usually transmitted to a new bud from a parental polyp during asexual reproduction. But if these GSCs are lost during subsequent budding or regeneration events, new ones are generated from multipotent stem cells (MPSCs). MPSCs are the somatic stem cells in Hydra that ordinarily differentiate into nerve cells, nematocytes (stinging cells in cnidarians), and gland cells. By means of such a backup system, sexual reproduction is guaranteed for every polyp. Interestingly, Hydra polyps occasionally undergo sex-reversal. This implies that each polyp can produce either type of GSCs, i.e. Hydra are genetically hermaphroditic. Nevertheless a polyp possesses only one type of GSCs at a time. We propose a plausible model for sex-reversal in Hydra. We also discuss so-called germline specific genes, which are expressed in both GSCs and MPSCs, and some future plans to investigate Hydra GSCs.     

Quantification of cell-cycle distribution and mitotic index in Hydra by flow cytometry

The applicability of flow cytometry (FCM) to analyse cell-cycle distribution and mitotic cells in Hydra oligactis and Hydra vulgaris is demonstrated. The freshwater polyps H. vulgaris and H. oligactis are well-accepted animal models for studying cell proliferation, regeneration and differentiation. Disintegrated animals were labelled for FCM analysis according to the method of Nuesse et al. [(1990) Flow cytometric analysis of G(1) and G(2)/M-phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements. Cytometry 11, 813]. Proliferation and regeneration experiments, in the absence or presence of the oligopeptide head activator, were quantified. Cell-cycle analysis of different parts of the animals shows low proliferation in the head region and high proliferation in the gastric and foot regions. Cell-cycle analysis of different parts of Hydra, comparison of H. oligactis and H. vulgaris, as well as pharmacological treatment, yielded results that are in agreement with prior microscopic analysis. Our results demonstrate that FCM is an appropriate technique for quantifying proliferation in this animal model. It can be used for basic research on development, regeneration and differentiation as well as for innovative drug investigation and toxicology studies.     

Germ cells in Hydra oligactis males. I. Isolation of a subpopulation of interstitial cells that is developmentally restricted to sperm production

Single clones of interstitial cells were generated and analyzed to determine if one interstitial cell has the capacity to differentiate both somatic and germ cells. Such clones were produced by using hydroxyurea to selectively eliminate interstitial cells from normal Hydra oligactis males. The number of animals devoid of interstitial cells within the population was determined by staining whole animals with toluidine blue which renders the interstitial cells visible. The number of animals containing single clones of interstitial cells was then estimated using single hit Poisson statistics. In treatments which rendered 60-80% of the population devoid of interstitial cells, the majority of the animals containing interstitial cells lost the ability to produce somatic cells, including nerves and nematocytes, but retained the capacity to produce sperm. This result strongly suggests the presence of a separate germ line in hydra.     

Light and electron microscopic localization of a monoclonal antibody in neurons in situ in the head region of Hydra

A mouse monoclonal antibody to Hydra attenuata was used to demonstrate immunoreactive product in neurons in situ, in both whole mount and sectioned hypostomes and tentacles of H. oligactis and H. littoralis. Immunoreactive cells were concentrated around the mouth and scattered along the length of the tentacles. In the hypostome, nerve cells sent one or more processes orally and the others aborally but the processes were more distinctly stained in H. oligactis. A thin strand of five to six perihypostomal neurons was present close to the hypostome-tentacle junction. In the tentacles, neurons with long processes contacted up to five different batteries of nematocysts. Neural processes were associated with nematocyst batteries in three ways: 1) forming a perikaryal loop to encircle a centrally located stenotele, 2) branching at a distance from the perikaryon to contact a variety of nematocysts, and 3) terminal branching by one or more neurons with contacts on one to several nematocysts within a battery. Immunocytochemical localization of neurons in Hydra by light microscopy was correlated for the first time with electron microscopy. Peroxidase-antiperoxidase (PAP)-positive sensory cells were concentrated around the mouth opening. PAP-positive ganglion cells were predominant in the tentacles. Sensory cells were elongate or spindle-shaped (unipolar), triangular with two oppositely directed processes (bipolar), and multipolar (tripolar or tetrapolar) with one of the processes extending to the epidermal surface. Ganglion cells were either unipolar or bipolar or multipolar, with neurites paralleling the mesoglea and occasionally having processes abut on it.     

Nucleotide sequence of an actin-encoding gene from Hydra attenuata: structural characteristics and evolutionary implications

We have determined the complete nucleotide sequence of an actin-encoding gene from Hydra attenuata as well as partial sequences of cDNA clones from two additional actin-encoding genes. The gene from the genomic clone contains a single intron, and has promoter and polyadenylation signals similar to those found in other species. The hydra genome has a very A + T-rich base composition (71%). This is reflected in the codon usage of the actin-encoding genes, which is strongly biased towards codons having A or T in the third position. The hydra actin-encoding gene family consists of three or more transcribed genes, two of which are very closely related to each other and probably arose by a recent gene duplication. Hydra actin, like other invertebrate actins, is more similar to the non-muscle isotypes of vertebrates than to the vertebrate muscle actins. Hydra actin is more similar to animal actins than to those of plants or fungi, which is consistent with the view that all metazoans arose from a single protist ancestor.     

Identification and characterization of the epithelial polarity receptor ”Frizzled” in Hydra vulgaris

The Wnt signaling pathway plays an important role in the specification of cell patterning during development in many species. Here we report the isolation and characterization of a putative Wnt receptor, Frizzled, in Hydra vulgaris. Analysis of the amino acid sequence of Frizzled in hydra reveals that this receptor contains many strong sequence similarities to other known Frizzled receptors. Hydra divergence is estimated to have occurred about one billion years ago; thus comparison of the Frizzled sequence of hydra with that of other species is likely to provide important information on the structure and function of those highly conserved regions. Northern and Southern blotting reveal that the Frizzled receptor in hydra has a 2.34-kb message size, and that it is encoded by a single gene. In situ hybridization using hydra frizzled as a probe reveals that the receptor message is restricted to the endoderm in adult hydra. This distribution supports the hypothesis that the Frizzled receptor is functioning in a pathway that controls cell differentiation in hydra. Received: 24 September 1999 / Accepted: 7 December 1999     

Analysis of a foot regeneration deficient strain of Hydra oligactis.

A foot regeneration deficient strain of Hydra oligactis with altered size regulation was investigated. Analysis of the concentration of four morphogenetically active substances in Hydra oligactis showed that the foot regeneration deficiency was mainly due to a drastically reduced foot activator concentration. Foot activator was distributed as a very steep gradient in Hydra oligactis leading to a more severe impairment of foot regeneration the closer to the head cutting was done. The concentration of foot inhibitor was comparable, that of head activator slightly increased and that of head inhibitor reduced compared to Hydra vulgaris. Studies at the cellular level implied that the enlarged gastric region was due to an elevated level of inhibition hinting at a shift in the ratio between bound and free head inhibitor in this animal.     

Nerve cells in hydra: monoclonal antibodies identify two lineages with distinct mechanisms for their incorporation into head tissue

The relationship between populations of nerve cells defined by two monoclonal antibodies was investigated in Hydra oligactis. A population of sensory nerve cells localized in the head (hypostome and tentacles) is identified by the binding of antibody JD1. A second antibody, RC9, binds ganglion cells throughout the animal. When the nerve cell precursors, the interstitial cells, are depleted by treatment with hydroxyurea or nitrogen mustard, the JD1+ nerve cells are lost as epithelial tissue is sloughed at the extremities. In contrast, RC9+ nerve cells remain present in all regions of the animal following treatment with either drug. When such hydra are decapitated to initiate head regeneration, the new head tissue formed is again free of JD1+ sensory cells but does contain RC9+ ganglion cells. Our studies indicate that (1) nerve cells are passively displaced with the epithelial tissue in hydra, (2) JD1+ sensory cells do not arise by the conversion of body column nerve cells that are displaced into the head, whereas RC9+ head nerve cells can originate in the body column, (3) formation of new JD1+ sensory cells requires interstitial cell differentiation. We conclude from these results that the two populations defined by these antibodies are incorporated into the h ad via different developmental pathways and, therefore, constitute distinct nerve cell lineages.