What role do annelid neoblasts play? A comparison of the regeneration patterns in a neoblast-bearing and a neoblast-lacking enchytraeid oligochaete

The term 'neoblast' was originally coined for a particular type of cell that had been observed during annelid regeneration, but is now used to describe the pluripotent/totipotent stem cells that are indispensable for planarian regeneration. Despite having the same name, however, planarian and annelid neoblasts are morphologically and functionally distinct, and many annelid species that lack neoblasts can nonetheless substantially regenerate. To further elucidate the functions of the annelid neoblasts, a comparison was made between the regeneration patterns of two enchytraeid oligochaetes, Enchytraeus japonensis and Enchytraeus buchholzi, which possess and lack neoblasts, respectively. In E. japonensis, which can reproduce asexually by fragmentation and subsequent regeneration, neoblasts are present in all segments except for the eight anterior-most segments including the seven head-specific segments, and all body fragments containing neoblasts can regenerate a complete head and a complete tail, irrespective of the region of the body from which they were originally derived. In E. japonensis, therefore, no antero-posterior gradient of regeneration ability exists in the trunk region. However, when amputation was carried out within the head region, where neoblasts are absent, the number of regenerated segments was found to be dependent on the level of amputation along the body axis. In E. buchholzi, which reproduces only sexually and lacks neoblasts in all segments, complete heads were never regenerated and incomplete (hypomeric) heads could be regenerated only from the anterior region of the body. Such an antero-posterior gradient of regeneration ability was observed for both the anterior and posterior regeneration in the whole body of E. buchholzi. These results indicate that the presence of neoblasts correlates with the absence of an antero-posterior gradient of regeneration ability along the body axis, and suggest that the annelid neoblasts are more essential for efficient asexual reproduction than for the regeneration of missing body parts.     

Morphological characterization of the asexual reproduction in the acorn worm Balanoglossus simodensis

The acorn worm Balanoglossus simodensis reproduces asexually by fragmentation and subsequent regeneration from the body fragments. We examined the morphogenesis of its asexual reproduction. At first, we collected asexually reproducing specimens and observed their morphogenesis. Then, we succeeded in inducing the asexual reproduction artificially by cutting the worm at the end of the genital region. The process of morphogenesis is completely the same between naturally collected and artificially induced specimens. The stages during morphogenesis were established on the basis of the external features of the asexually reproducing fragments. The internal features of the fragments were also examined at each stage. In a separate phase of the study, the capacity for regeneration of some body parts was also examined by dividing intact worms into about 10 fragments. Although the capacity for regeneration varied among the different body parts, some fragments regenerated into complete individuals in 1 month. The process of regeneration was the same as that in the asexually produced fragments.     

Bipolar head regeneration induced by artificial amputation in Enchytraeus japonensis (Annelida, Oligochaeta)

The Enchytraeida Oligochaeta Enchytraeus japonensis propagates asexually by spontaneous autotomy. Normally, each of the 5-10 fragments derived from a single worm regenerates a head anteriorly and a tail posteriorly. Occasionally, however, a head is formed posteriorly in addition to the normal anterior head, resulting in a bipolar worm. This phenomenon prompted us to conduct a series of experiments to clarify how the head and the tail are determined during regeneration in this species. The results showed that (1) bipolar head regeneration occurred only after artificial amputation, and not by spontaneous autotomy, (2) anesthesia before amputation raised the frequency of bipolar head regeneration, and (3) an extraordinarily high proportion of artificially amputated head fragments regenerated posterior heads. Close microscopic observation of body segments showed that each trunk segment has one specific autotomic position, while the head segments anterior to the VIIth segment do not. Only the most posterior segment VII in the head has an autotomic position. Examination just after amputation found that the artificial cutting plane did not correspond to the normal autotomic position in most cases. As time passed, however, the proportion of worms whose cutting planes corresponded to the autotomic position increased. It was suspected that the fragments autotomized after the artificial amputation (corrective autotomy). This post-amputation autotomy was probably inhibited by anesthesia. The rate at which amputated fragments did not autotomize corresponded roughly to the rate of bipolar regeneration. It was hypothesized then that the head regenerated posteriorly if a fragment was not amputated at the precise autotomic position from which it regenerated without succeeding in corrective autotomy.     

视黄酸对赤子爱胜蚓再生头尾轴形成的影响

为了探讨视黄酸对蚯蚓再生的影响,用视黄酸处理了从不同部位剪切的蚯蚓体段．观察其存活率、重量和再生长度的变化。结果表明,有头无尾的体段存活率受视黄酸影响较小,而无头有尾的处理受视黄酸影响较大;视黄酸处理后30d,各处理再生长度和存活率均小于对照;视黄酸对蚯蚓再生有明显影响,能延迟和干扰再生,影响头部的形成。视黄酸影响蚯蚓再生的作用方式可能是通过干扰前后体轴的形成,从而影响蚯蚓再生图式形成。     

Asexual Reproduction and Segmental Regeneration, but not Morphallaxis, are Inhibited by Boric Acid in Lumbriculus variegatus (Annelida: Clitellata: Lumbriculidae)

Body fragmentation, in some animal groups, is a mechanism for survival and asexual reproduction. Lumbriculus variegatus (Müller, 1774), an aquatic oligochaete worm, is capable of regenerating into complete individuals from small body fragments following injury and reproduces primarily by asexual reproduction. Few studies have determined the cellular mechanisms that underlie fragmentation, either regenerative or asexual. We utilized boric acid treatment, which blocks regeneration of segments in amputated fragments and blocks architomic fission during asexual reproduction, to investigate mechanistic relationships and differences between these two modes of development. Neural morphallaxis, involving changes in sensory fields and giant fiber conduction, was detected in amputated fragments in the absence of segmental regeneration. Furthermore, neural morphallactic changes occurred as a result of developmental mechanisms of asexual reproduction, even when architomy was prevented. These results show that fragmentation in L. variegatus, during injury or asexual reproduction, employs developmental and morphallactic processes that can be mechanistically dissociated by boric acid exposure. In regeneration following injury, compensatory morphallaxis occurred in response to fragmentation. In contrast, anticipatory morphallaxis was induced in preparation for fragmentation during asexual reproduction. Thus, various forms of regeneration in this lumbriculid worm can be activated independently and in different developmental contexts.     

Pattern Regulation of the Leg Disc of the Fleshfly, Sarcophaga peregeina

A fate map of the hind leg disc of Sarcophaga peregrina was constructed by examining the adult structures of implanted disc fragments. The locations of presumptive adult structures in the disc were similar to those of fore leg disc of Drosophila and Sarcophaga ruficornis . However, the concentric borderlines of the segments could not be ascertained in the present case.
Pattern regulation of disc fragments was studied by culturing them either in adult females for several days or for 3 days in mature larvae placed on wet condition. Cultured disc fragments regenerated or duplicated as in Drosophila , with some exceptions. For instance, the region with a high density of positional values, the upper medial quarter, of the fore leg disc of Drosophila was not found. A characteristic difference in the rate of regeneration or duplication was observed in the implanted fragments, when cultured in larvae or adult hosts. This variable pattern regulation in larval and adult hosts could be due to different compositions of the hemolymph in which would healing of the implanted disc fragments takes place.     

Germline regeneration: the worms' turn

Asexual reproduction in the annelid Enchytraeus japonensis entails the regeneration of primordial germ cells from body parts that lack gonads. New primordial germ cells arise from piwi-expressing germline stem cells that are distinct from somatic stem cells.     

Differential fragment regeneration in Syntrichia caninervis Mitt. from the Gurbantunggut Desert of China

Fragments of the desert moss Syntrichia caninervis Mitt. were grown on the surface of moistened sand to assess their regeneration capacity. The plant material was collected in two different years (2014 and 2015) and divided into five fragment classifications (stem apices, green leaves, yellow-green leaves, brown leaves and stems). All fragments of the stem apices, green leaves and stems regenerated within 10 days of culture while some fragments of yellow-green leaves (two 2014 fragments and one 2015 fragment) and brown leaves (three 2014 fragments and three 2015 fragments) died. Fragments of stem, stem apices and green leaves regenerated more quickly, produced longer protonemata and more shoots as compared to fragments of yellow-green and brown leaves. These differences were statistically significant but there was no difference in regeneration between the fragments from 2014 and 2015. Differential regeneration and proliferation of different plant fragments has important implications for the clonal propagation of S. caninervis in the Gurbantunggut Desert.     

Early segregation of germ and somatic lineages during gonadal regeneration in the annelid Enchytraeus japonensis

Although regeneration studies are useful for understanding how organs renew, little information is available about regeneration of reproductive organs and germ cells. We here describe the behavior of germ-cell precursors during regeneration of the oligochaete annelid worm Enchytraeus japonensis, which has the remarkable feature of undergoing asexual (by fission) and sexual reproduction . We first found that the gonad can regenerate from any body fragment yielded by fission during asexual reproduction. We then examined behavior of germ-cell lineage during this regenerative process, by using a homolog of the Piwi gene (Ej-piwi) as a marker. We found that in asexually growing animals, specialized cells expressing Ej-piwi are distributed widely in the body as single cells. These cells seem to serve as a reservoir of germ-cell precursors because during asexual propagation these cells migrate into the regenerating tissue, where they ultimately settle in the prospective gonads, and give rise to germ cells upon sexualization. These cells are distinct from the neoblasts, thought to be stem cells in other animals. This is the first report to directly show that the germ and somatic lineages are segregated in asexually growing animals and behave differently during regeneration.     

浙江天目山孑遗落叶阔叶树种的种群结构及更新策略

通过对浙江天目山国家级自然保护区内6个孑遗落叶阔叶树种进行群落学调查,分析了不同微地形单元上种群结构、更新途径及更新个体的空间关联性,探讨了不同干扰体系下孑遗落叶阔叶树种的更新策略.结果表明:孑遗落叶阔叶树种具有较强的萌枝能力,种群结构以间歇型为主,部分种类选择长距离扩散,在适宜生境中间歇型更新,如青钱柳、缺萼枫香等;部分种类因生境限制,在不断的地表冲刷干扰下,选择在母树周围进行间歇型更新,如领春木、香果树.在相对稳定的顶坡和坡地上,青钱柳、缺萼枫香、蓝果树、枫香等树种以种子更新占据一次干扰后形成的林窗生境,并通过其固有的萌枝能力增殖树干,形成径级大小相似的多主干植株,迅速占据优势地位.在不稳定的谷床、谷坡和谷头凹地中,香果树、领春木、枫香等树种因频繁干扰导致种子更新受限、植株损伤并常有个体枯死,通过其极强的萌枝能力补充零星更新的幼苗以及干扰造成的个体损伤.基于生境适应、种群更新和竞争策略,将孑遗落叶阔叶树种大致划分为“林窗修复种”和“生境填充种”两个类型,并提出应加强对其特殊生境的保护.     

Asexual reproduction and anterior regeneration under high and low temperatures in the sponge associate Polydora colonia (Polychaeta: Spionidae)

Regeneration in polychaetes is an important process because of its role in recovery after injury and in asexual reproduction via architomy. This study examined architomy and regeneration in the spionid worm, Polydora colonia (Moore 1907) a symbiont of sponges. Based on collections of P. colonia from Long Island, New York, prevalence of architomy was 24% (188 out of 780 worms) with the highest prevalence recorded during the summer and early fall and the lowest prevalence during late fall and winter. Morphogenesis during regeneration of P. colonia was studied with light and scanning electron microscopy at two different temperatures. Worms regenerated faster under high temperatures (24°C), whereas it took more than twice as long to regenerate under low temperatures (14°C). Morphogenesis during anterior regeneration included the formation of a blastema from which a maximum of eight anterior segments regenerated. At high temperatures, palp buds and initial segments were observed to form by day 2 and 1–2 major spines were observed in the fifth segment by day 8. This is the first report of asexual reproduction in the field for the genus Polydora and the results indicate that temperature plays a role in regeneration.     

Caudal regeneration in Tubifex tubifex (Oligochaeta, Tubificidae) following copper exposure

Abstract. The oligochaete, Tubifex tubifex (Oligochaeta, Tubificidae), is capable of the auto-tomization and regeneration of missing body parts. The posterior end of the worm is subject to autotomy (e.g., after predation or after exposure to toxic substances) and can be regenerated thereafter. Despite the ecological importance of T. tubifex , processes of autotomy and regeneration have been little studied. This work describes daily morphological and histological observations of caudal regeneration of this worm. We also compared regeneration processes when autotomy was induced by artificial transsection of the worms (control worms) and by exposure to copper ion, a frequently encountered environmental contaminant. The worms regenerated a functional posterior end (defecation possible) in 7 d as well as a prepygidial zone from which new segments could be regenerated. In T. tubifex , regeneration involved the presence of activated mesoblastocytes in the worm, increased number of neoblasts, and the increased migration of these cells. However, Cu²⁺ did not affect these processes, since no difference could be detected between regeneration of control and Cu-autotomized worms.     

Morphogenesis during asexual reproduction in Pygospio elegans Claparede (Annelida, Polychaeta)

The spionid Pygospio elegans reproduces both asexually and sexually. Using scanning electron and bright field microscopy, we examined morphogenesis following asexual reproduction to determine how "lost" body regions were regenerated after a worm spontaneously divided. Asexual reproduction occurred through transverse fission and divided the parent worm into 2 to 6 fragments (architomy). All fragments retained their original anterior-posterior polarity. Regeneration in all fragments followed a specific series of events: wound healing (day 1); extension of the blastema to generate lost body regions-specifically, the head and thorax for posterior fragments and the tail and pygidium for anterior fragments (days 2-3); segmentation (days 3-6); and differentiation of segment- or region-specific structures (days 4-8). This pattern occurred regardless of where the original division took place. Subsequent growth occurred through addition of terminal setigers anterior to the pygidium followed by differentiation of tail setigers into abdominal setigers, leaving the tail region about 6 to 10 setigers in size. Division rates were compared in worms from three populations in Nova Scotia, Canada. Worms from two populations (Conrad's Beach, Starr's Point) divided more frequently (about 1.2 and 1.3 weeks between divisions, respectively) than worms from Bon Portage Island (3.5 weeks between divisions). Fragments containing the original head (original mouth intact, generally much larger fragment) had a higher survivorship than fragments containing the original tail.     

中华真地鳖的断足再生

报道了中华真地鳖Eupolyphaga sinensis Walker的断足再生特征。研究结果表明,不同虫龄期的若虫都有断足再生能力;足的不同部位断足后均能再生;断掉不同数量的足后,只要能成活均可再生。断足再生后,继续断掉再生足的原位或其他部位也可以再生。再生足的跗节均比正常的少一节,具有再生不完整性。断足后,只要经1～2次蜕皮,均可再生。断掉一对足的腿节后,再生足出现大小不一的现象,小的一般发育不全,断足数量多容易出现再生足发育不全。再生足比正常足要小,但生长速度要快,断掉足的腿节或跗节后的再生足经过2次蜕皮后基本可恢复到正常足大小。     

狐尾藻属狭域种和广布种断枝的生长与再生能力比较研究

对比研究了广布种穗花狐尾藻(Myriophyllum spicatum L.)和狭域种扬子狐尾藻(M. oguraense Mikisubsp. yangtzense Wang)不同长度(5、10 和15 cm)和不同位置(顶枝、中枝和底枝)断枝的生长和再生能力。研究结果显示, 两物种由断枝生长所形成植株的总生物量、分枝生物量、分枝数均随断枝长度的增加而增大;在由不同位置的断枝生长所形成的植株中, 顶枝所形成植株的总生物量最大, 中枝所形成植株的分枝生物量、分枝数和平均分枝长最大。由断枝所形成植株的总生物量、分枝生物量、分枝数、平均分枝长和新分枝所处位置在种间均存在显著差异, 穗花狐尾藻顶枝和中枝的生物量较大, 顶枝所产生的分枝位于断枝基部; 扬子狐尾藻的分枝生物量、分枝数及平均分枝长较大, 新产生的分枝多位于断枝顶端附近。研究结果表明穗花狐尾藻和扬子狐尾藻断枝的再生对策不同, 前者是增加对断枝总生物量的投资, 而后者是增加对断枝产生新分枝的投资。研究结果可为进一步开展水生植物广布种与狭域种的生活史对策研究提供资料, 也为湿地物种多样性保护与管理提供科学指导       

Regeneration in youngBunodactis verrucosa Pennant (actinaria,anthozoa)

Summary YoungBunodactis verrucosa Pennant at the 12 tentacle stage are employed to test the applicability of the polar coordinate model to coelenterate regeneration. The animals are cut along every radius into fragments of 3 to 9 segments. Most fragments are patent 3–4 weeks later, but small fragments have a higher mortality rate than large fragments. Some fragments do not regenerate and occasionally tentacles fuse, thereby reducing the number of segments. Small fragments tend to regenerate more tentacies than large fragments, but large fragments may regenerate great numbers of supernumerary tentacles. Twenty-two percent of the fragments restore the missing number of tentacles, while 76% of all fragments produce an even number of tentacles.Fragments restoring the correct numbers of tentacles show a marked tendency to form the correct tentacles (regulative regeneration). Fragments regenerating two less than the number of tentacles already present show a marked tendency to reproduce tentacles of the types already present (miror image formation). Other fragments produce missing segments (forward regeneration), or those already present (reverse regeneration) at lower frequencies.No fragments beginning or ending with the number 1 directive tentacle fail to regenerate entirely, while first cycle segments maximally remote from segment 1 are associated with the absence of regeneration. No fragments beginning or ending with the number 4 directive tentacle fail to undergo forward regeneration, regulate or produce a mirror image when the appropriate number of segments are regenerated. In contrast, segment 4 is associated with a low frequency of reverse regeneration, and second cycle segments cut away from immediate contact with segment 4 show an increase in the frequency of reverse regeneration. Controls through morphogenic substances rather than polar coordinates seem to explain these results. Such substances would control the number and direction of tentacle regeneration.This work was performed while the author was on sabbatical leave from the University of Pittsburgh at the Stazione Zoologica di Napoli. The author gratefully acknowledges the assistance of Mr. Ciro Gargiulo and of Ms. Gisella Princivalli. This work was supported by a travel grant from the United States Italy Cooperative Science Program of the National Science Foundation. The paper is dedicated to Dr. Alberto Monroy whose generosity made it possible     

Regeneration following duplication in imaginal wing disc fragments of Drosophila melanogaster

Fragments from the imaginal wing disc of Drosophila melanogaster were cultured in vivo for periods up to 28 days. One type of edge fragment first duplicated and then ceased to grow, but others often continued to grow following initial duplication and regenerated structures characteristic of other areas of the disc. After 28 days of culture, about 50% of fragments from the presumptive ventral hinge region of the disc grew extensively and produced regenerated as well as original structures. The regenerated structures in some implants were produced at the line of mirror-image symmetry. Regeneration was associated with fragment growth and in many cases was accompanied by loss of duplicate structures. Fragments which were only duplicated after the culture period could in some cases be stimulated to grow by additional culture in fresh hosts, but the results of coculturing two fragments in each host show that culture conditions alone do not control growth and regulation in the fragments. The large, normally regenerating fragment, complementary to the ventral fragment, did not appear to grow following regeneration and only occasionally produced supernumerary structures during prolonged culture. Intact wing discs cultured under similar conditions never produced supernumerary structures. Our results suggest that a duplicated pattern is less stable than a complete, regenerated pattern, which in turn is less stable than an intact disc. We propose that the growth of duplicated disc fragments is stimulated by polarity reversals present at lines of mirror-image symmetry.     

Effects of nerve injury and segmental regeneration on the cellular correlates of neural morphallaxis

Functional recovery of neural networks after injury requires a series of signaling events similar to the embryonic processes that governed initial network construction. Neural morphallaxis, a form of nervous system regeneration, involves reorganization of adult neural connectivity patterns. Neural morphallaxis in the worm, Lumbriculus variegatus, occurs during asexual reproduction and segmental regeneration, as body fragments acquire new positional identities along the anterior-posterior axis. Ectopic head (EH) formation, induced by ventral nerve cord lesion, generated morphallactic plasticity including the reorganization of interneuronal sensory fields and the induction of a molecular marker of neural morphallaxis. Morphallactic changes occurred only in segments posterior to an EH. Neither EH formation, nor neural morphallaxis was observed after dorsal body lesions, indicating a role for nerve cord injury in morphallaxis induction. Furthermore, a hierarchical system of neurobehavioral control was observed, where anterior heads were dominant and an EH controlled body movements only in the absence of the anterior head. Both suppression of segmental regeneration and blockade of asexual fission, after treatment with boric acid, disrupted the maintenance of neural morphallaxis, but did not block its induction. Therefore, segmental regeneration (i.e., epimorphosis) may not be required for the induction of morphallactic remodeling of neural networks. However, on-going epimorphosis appears necessary for the long-term consolidation of cellular and molecular mechanisms underlying the morphallaxis of neural circuitry.     

Distal transformation in Drosophila leg imaginal disc fragments.

For an appendage to regenerate distal elements, it has been thought that the stump must contain a full set of circumferential positional information. We have shown that this rule is not binding for tarsus regeneration in the male foreleg imaginal disc of Drosophila melanogaster. Distal transformation was not restricted to fragments containing complete proximal segments, but was also observed in pieces with small or even substantial deficiencies that were not regenerated in their proximal segments.     

The regeneration capacity of an earthworm, Eisenia fetida, in relation to the site of amputation along the body

It is well known that parts of earthworms can survive if they are cut off. Our aim was to link the regeneration capacity of an earthworm, Eisenia fetida (Oligochaeta, Annelida) with the site of the amputation, so we amputated earthworms at different body segment locations along the length of the body to examine the different survival rates and regeneration lengths of the anterior, posterior, and medial sections.
The greatest survival rates occurred for earthworms with the most body segments remaining after amputation. The anterior regeneration lengths were of two types. The lengths of regeneration of amputated from body segment 6/7 to further down the body posteriorly increased gradually (Type L_I). However, the regeneration lengths of earthworm which were amputated behind the 23rd segment, with less than a quarter of the total segments remaining, did not increase until the blastema and tail bud formation (Type L_II). These treatments were not completely regeneration. There were significant differences in both survival rates and lengths of regeneration lengths between immature earthworms and clitellate adult earthworms during the early stages of regeneration, but not at later stages of regeneration. The immature earthworms had a greater regeneration potential than clitellate adults amputated at the same segment. The survival rates of earthworms were correlated significantly with the number of body segments remaining after amputation, but not with the position of the amputation. The relationships between the survival rates and the numbers of remaining segments could be described by linear regressions. The anterior regeneration lengths were correlated with the position of the amputation, but not with the number of remaining segments; the posterior regeneration lengths, were not correlated with the number of segments remaining nor the amputation position. The anterior regeneration length was not related to the survival rates for all earthworm amputations after 30 days but was related in this way after 60 days.