Regulation of Asexual Reproduction of the Planarians Dugesia tigrina

We studied asexual reproduction of planarians under the natural and artificial photoperiodic conditions. It was shown that light inhibits the fission of planarians, while darkness stimulates it. The diurnal dynamics of the fission of planarians demonstrated a circadian rhythm. This rhythm is stable, which is expressed when the conditions are experimentally changed: constant darkness, unnatural rhythm of light-darkness succession). However, this stability is affected at the time zone change. The planarians are adapted to new conditions and begin to fission at once in correspondence with the new diurnal regime.     

鲁中山区东亚三角头涡虫有性生殖过程研究初报

于2005年3-5月、9～11月对生活于鲁中山区的东亚三角头涡虫（Dugesia japonica）的有性生殖过程进行了研究,发现鲁中山区东亚三角头涡虫1年只有1次有性生殖过程。卵囊产出的高峰在4月,卵囊孵化与种群密度最高峰在5月,其有性生殖过程与温度、食物、虫体大小密切相关。经实验观察1个卵囊最多能孵出涡虫幼体10条,最少3条。     

Sexual and Asexual Reproduction in Natural Stands of Populus nigra

The balance between sexual and asexual propagation in the establishment of natural stands of Populus nigra was assessed from the genotypes of trees sampled in different natural stands. One site was completely sampled and genotyping proceeded using STS and RAPD markers. Among 118 trees, only four were vegetative copies of other trees of the site. Isozymes were used in four stands, and a method to detect the presence of vegetative copies in a sample is proposed for these markers. No vegetative copies were found in two cases, and a low number of copies was detected in the other two. Observations are reported for seedlings as well as for different modes of vegetative propagation in natural stands. We conclude that sexual and asexual propagation play complementary roles in the dynamics of this species, and that in most cases the adults originate from seedlings.     

Seasonal changes in the sexualization of the planarian Dugesia ryukyuensis

Asexual individuals in a fissiparous clone of the planarian Dugesia ryukyuensis develop hermaphroditic sexual organs and eventually undergo sexual reproduction instead of asexual reproduction if they are fed with the adults of Bdellocephala brunnea, an oviparous planaria. The experimental sexualization means that the adults of B. brunnea contain a putative sexualizing substance(s), which is the first candidate for the chemical(s) responsible for switching from asexual to sexual reproduction in metazoans. In the present study, the feeding experiment over two consecutive years revealed that the experimental sexualization has seasonal changes. In summer, the asexual individuals were not fully sexualized, though they developed a pair of ovaries. The developing ovaries degenerate if the feeding is stopped. On the contrary, in winter, they developed all the sexual organs. The sexual organs keep developing even if the feeding is stopped after a certain critical point named the point-of-no-return. It was demonstrated that the extreme difference of the sexualization was attributed to the seasonal change of the quality and/or quantity of the sexualizing substance contained in B. brunnea, as well as the minor change of the susceptibility to the sexualizing substance in the asexual individuals. On the other hand, the histological research of B. brunnea revealed that the degree of the maturation of the sexual organs varied extremely through a year. Taking these results into account, we suggest that the production of the sexualizing substance has no direct relation to any particular mature sexual organs.     

Different Responses of Sexual and Asexual Reproduction of Arundinella hirta to Flooding

The sexual and asexual reproductive features of the graminoid species Arundinella hirta growing at riversides of the Jialing River were analyzed.It was found that the total seed mass,seed number per plant,and sexual reproductive allocation of A.hirta decreased with decreasing bank elevation,and plants growing at the lowest elevations of banks subjected to intense flooding did not show sexual reproduction.The total plant biomass and the number of ramifications per plant increased with flooding intensity,which implies that,contrary to sexual reproduction,asexual reproduction of A.hirta was enhanced by flooding.     

日本三角涡虫成体干细胞的异质性研究进展

Neoblasts是日本三角涡虫Dugesia japonica成体体内唯一具有增殖和分化能力的多能干细胞,是其损伤修复和再生的细胞基础。Neoblasts的组成是均质还是异质,长期以来一直存在争议。近年来研究表明,neoblasts具有异质性,由不同的细胞亚群组成。本文从形态、射线抗性、细胞周期及分子标志等4个方面综述了neoblasts异质性研究的最新进展,为将来探讨neoblasts不同亚群功能、相互关系及各亚群在涡虫再生过程中的作用提供借鉴。     

Different Responses of Sexual and Asexual Reproduction of Arundinella hirta to Flooding

The sexual and asexual reproductive features of the graminoid species Arundinella hirta growing at riversides of the Jialing River were analyzed. It was found that the total seed mass, seed number per plant, and sexual reproductive allocation of A. hirta decreased with decreasing bank elevation, and plants growing at the lowest elevations of banks subjected to intense flooding did not show sexual reproduction. The total plant biomass and the number of ramifications per plant increased with flooding intensity, which implies that, contrary to sexual reproduction, asexual reproduction of A. hirtawas enhanced by flooding. Translated from the Journal of Southwest China Normal University (Natural Science), 2005, 30(2) (in Chinese)     

Neoblast-enriched fraction rescues eye formation in eye-defective planarian 'menashi' Dugesia ryukyuensis

Planarians are well known for their remarkable regenerative capacity. This capacity to regenerate is thought to be due to the presence of totipotent somatic stem cells known as ‘neoblasts’, which have particular morphological characteristics. The totipotency of neoblasts was supported by Baguñà's experiment, which involved the introduction of donor cells into irradiated hosts. However, since Baguñà's experiment did not include the use of a phenotypic marker, the donor cells could not be traced. In the current study, a genetic mutant planarian, menashi, an eye‐defective mutant that lacks the pigmented area in the eyes, was established. This planarian is excellent for tracing the fate of cells after their introduction into irradiated hosts. To investigate the differentiation potency more directly, a neoblast‐rich fraction obtained from normal worms was transplanted into an X‐ray‐irradiated menashi strain. Planarians that survive X‐ray irradiation were developed, and we observed the pigment of the area in the eyes of the regenerating planarians. This result suggests that the neoblast‐rich fraction contains cells that can proliferate and differentiate. These cells can replace the cells and structures lost by X‐ray irradiation and ablation, and they can also differentiate into eye pigment cells.     

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities¹. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a ''trunk'' fragment is cut from an intact worm, a new ''head'' will regenerate anteriorly and a ''tail'' will regenerate posteriorly restoring the original ''head-to-tail'' polarity of body structures prior to amputation (Figure 1A).Regeneration is driven by planarian stem cells, known as ''neoblasts'' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity^2-9.The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters¹⁰, represent an important system for studying evolution of the central nervous system^{11, 12} and have behavioral screening potential^{13, 14.} Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs^{8, 9, 15}. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals^16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.     

Switching from asexual to sexual reproduction in the planarian Dugesia ryukyuensis: change of the fissiparous capacity along with the sexualizing process

Asexual worms of fissiparous strain of the planarian Dugesia ryukyuensis switch from asexual to sexual reproduction, if they are fed with sexually mature worms of Bdellocephala brunnea. This suggests that the sexually mature worms have a sexualizing substance(s) that induces the sexuality in the asexual worms. Here, we found by analysis of the sexualization that the cessation of the fission, namely their asexual reproduction, occurs immediately after the acquisition of sexuality. This result suggests that the downstream mechanisms induced by the putative sexualizing substance in B. brunnea become responsible for the cessation of fission. We also found that the decapitation triggers fission in the worms even after the acquisition of sexuality if they are not sexually mature, while the fully sexualized worms never fission even though they are decapitated. This result suggests that the cessation of fission takes place via at least two steps: (1) the mechanisms associated with the cephalic system; (2) other mechanisms independent of cephalic control.     

The Dr-nanos gene is essential for germ cell specification in the planarian Dugesia ryukyuensis

Homologs of nanos are required for the formation and maintenance of germline stem cell (GSC) systems and for gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, alternating between asexual and sexual reproduction; they develop and maintain their somatic stem cells (SSCs) and GCSs from pluripotent stem cells known as neoblasts. We isolated a nanos homolog, Dr-nanos, from the expressed sequence tags (ESTs) of the sexualized form of Dugesia ryukyuensis. We examined the expression of Dr-nanos in asexual and sexualized planarians by in situ hybridization and analyzed its function using RNA interference (RNAi) together with a planarian sexualization assay. A nanos homolog, Dr-nanos, was identified in the planarian D. ryukyuensis. Dr-nanos expression was observed in the ovarian primordial cells of the asexual worms. This expression increased in proportion to sexualization and was localized in the early germline cells of the ovaries and testes. In X-ray-irradiated worms, the expression of Dr-nanos decreased to a large extent, indicating that Dr-nanos is expressed in some subpopulations of stem cells, especially in GSCs. During the sexualization process, worms in which Dr-nanos was knocked down by RNAi exhibited decreased numbers of oogonia in the ovaries and failed to develop testes, whereas the somatic sexual organs were not affected. We conclude that Dr-nanos is essential for the development of germ cells in the ovaries and testes and may have a function in the early stages of germ cell specification, but not in the development of somatic sexual organs.     

Characterization of novel genes expressed specifically in the sexual organs of the planarian Dugesia ryukyuensis

The planarian Dugesia ryukyuensis reproduces both asexually (fissiparous) and sexually (oviparous) and can switch from the asexual mode to the sexual mode. By feeding with mature Bdellocephala brunnea oviparous worms, the fissiparous worms, which do not possess sexual organs, can be converted to fully sexualized worms in a process termed sexualization. As sexualization proceeds, the sexual organs are formed uniformly and five stages (stages 15) of the process have been identified histologically. In order to clarify the sexualization process, we attempted to isolate the genes expressed specifically at stage 5 by the differential display method. We isolated five genes expressed in the testis and two genes expressed in the yolk gland, which is an organ specific to sexualized worms. By BLAST search, one of the testis-specific genes was coded as testis-specific alpha-tubulin and two yolk gland-specific genes are similar to ribose-phosphate pyrophosphokinase I and F-box/SPRY-domain protein 1. Drs1, Drs2 and Drs3 were expressed in spermatocytes and spermatids from the early stage of spermatogenesis and Drs4 and Drs5 were expressed in spermatogonia, spermatocytes and spermatids. These genes are useful markers for elucidating the sexualization process.     

Taxonomy and geographical distribution of Dugesia japonica and D. ryukyuensis in the Far East

Dugesia japonica Ichikawa et Kawakatsu, 1964, is a common and polymorphic species of freshwater planarian distributed widely in the Far East. In 1976 the geographic populations were separated into 2 subspecies (D.j.japonica and D.j. ryukyuensis). The taxonomy of this species is reconsidered once again from the morphological, anatomical, histological, and karyological viewpoints. From the result of these studies, D.j. ryukyuensis is elevated to the rank of species: D. ryukyuensis Kawakatsu, 1976. D. japonica (n = 8, 2x = 16, 3x = 24) differs from D. ryukyuensis (n = 7, 2x = 14, 3x = 21) in having an asymmetrical penis papilla without a well-developed valve surrounding its basal part, and a well-developed vagina (distribution: the Japanese Islands, Taiwan, the Korean Peninsula, China, and Primorskiy, Northeast Siberia, in Russia). D. ryukyuensis is characterized by an asymmetrical penis papilla with a well-developed valve surrounding its basal part, and a less-developed vagina (distribution: the Southwest Islands of Japan).     

A review of chromosomal variation in Dugesia japonica and D. ryukyuensis in the Far East

The chromosome numbers of Dugesia japonica Ichikawa et Kawakatsu, 1964, are n = 8, 2x = 16 and 3x = 24; those of Dugesia ryukyuensis Kawakatsu, 1976, are n = 7, 2x = 14 and 3x = 21. The karyotypes of both species include diploid, triploid and mixoploid; aneuploidic and mixoaneuploidic karyotypes may occur. In 785 specimens studied of D. japonica, the occurrence rates of specimens having each karyotype are substantially the same (29–37%). Diploid sexual specimens represented nearly 10% of the total and virtually no triploid or mixoploid sexual specimens were found. The diploid karyotype can be inherited by both sexual and asexual reproduction; the triploid and mixoploid karyotypes will be inherited only by asexual reproduction. In 51 specimens studied of D. ryukyuensis, the different karyotypes are diploid (ca 39%), triploid (ca 57%) and mixoploid (ca 4%). Diploid sexual specimens represented nearly 25% of the total; sexual specimens with tripooidic karyotypes made up nearly 27%. The diploid, triploid and mixoploid karyotypes were also found in juveniles hatched from cocoons. The diploid karytyype is inherited by both sexual and asexual reproductions; the other karyotypes may be inherited by parthenogenesis or self-fertilization (including pseudogamy) and asexual reproduction.     

Regulation of the Feeding Behavior of the Planarian Dugesia (Girardia) tigrina

In planarians, individual components of the feeding behavior are identified. The reaction of approaching the food object is a motor response of the body to chemical or physical signals coming from the object. As a continuation of the first response, the motor reaction of the pharynx appears aimed at capturing the food. Both reactions can occur independently, but an interdependence between them is also revealed. After ablation of the pharynx, the food cannot be captured, but the reaction of approaching it also is inhibited. After removal of the ganglion, the reaction of approaching the food object is inhibited, but the food capture also is partially suppressed. The food digestion occurring at the last stage affects the food-procuring reactions. The described reactions together with the process of digestion are integrated into the single complex forming the planarian feeding behavior.     

恒定磁场对日本三角涡虫无性横裂生殖及2种抗氧化酶活力的影响

目的:探讨恒定磁场对日本三角涡虫(Dugesia japonica)无性横裂生殖及抗氧化酶活力的影响.方法:应用种群累积培养法,观察了恒定磁场对日本三角涡虫的种群增长、无性横裂生殖及涡虫体内超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活力的影响.结果:场强大小在200mT-500mT范围的恒定磁场能促进涡虫的无性横裂生殖,但恒定磁场显著抑制涡虫的SOD及CAT活力.结论:日本三角涡虫对恒定磁场的生理学响应较为敏感,可作为生物磁学研究适宜的实验材料.