Evidence of regenerative ability in Myxicola infundibulum (Annelida,Sabellida): evolutionary and systematic implications

Members of only a few species of annelids are reported as being incapable of regeneration; of these, Myxicola infundibulum is the only example in the family Sabellidae. Interestingly, its congener Myxicola aesthetica exhibits noteworthy regenerative ability. Unambiguously identifying non‐regenerating species is critical to reconstructing how regenerative abilities evolved within the phylum. However, studies designed specifically to assess the regenerative potential of M. infundibulum have never been performed. In this study, we aimed to confirm the lack of regeneration ability of M. infundibulum, reported previously for Atlantic specimens, or to determine the extent to which regeneration occurs. Our results showed that individuals from the Mediterranean Sea (Adriatic Sea) do undergo regeneration of lost body parts, although to a lesser extent than do other sabellids. Therefore, M. infundibulum should no longer be considered a non‐regenerating species. At present, uncertainties regarding phylogenetic relationships of Sabellidae prevent inferences about the polarity of change in M. infundibulum. Since our findings are counter to those of previous studies which describe Atlantic specimens as non‐regenerating, more extensive analysis is required to ascertain if they could actually belong to a different species than Mediterranean M. infundibulum, accounting for these differences in reported regenerative capacity.     

General characterization of regeneration in Aeolosoma viride (Annelida,Aeolosomatidae)

Regeneration has long been the focus of scientific interest for its potential to restore lost, damaged, or aged tissues and organs. A wide range of regenerative studies have been conducted on different vertebrate and invertebrate model organisms. Annelids are known for their regenerative capacities, and because of their relatively complex organ systems, they are an ideal organism for regeneration study. Our present work focused on the freshwater annelid Aeolosoma viride, an asexually reproducing annelid capable of regenerating both anteriorly and posteriorly. Even though regenerative ability has been documented in this animal in previous studies, detailed characterization of the process is still unavailable. The objective of this study was to evaluate the regenerative ability of A. viride. We described the sequential morphological events during the process of regeneration, such as wound healing and the formation of blastema, mouth, and pygidium. In order to clarify the capacity and type of regeneration, we conducted a series of observations and experiments using a cell proliferation assay. Massive proliferation and the absence of cell migration indicated that the animal regenerates primarily through epimorphosis. Our study of the epimorphic regenerative process of A. viride provides a clearer picture of the evolutionary origin of regeneration in annelids.     

Regeneration of posterior segments and terminal structures in the bearded fireworm,Hermodice carunculata (Annelida: Amphinomidae)

Like many other annelids, bearded fireworms, Hermodice carunculata, are capable of regenerating posterior body segments and terminal structures lost to amputation. Although previous research has examined anterior regeneration in other fireworm species, posterior regenerative ability in fireworms remains poorly studied. As the morphology of the anal lobe (a small, fleshy terminal structure of unknown function) has been used to distinguish East and West Atlantic H. carunculata populations, there is a more imminent need to understand the morphology and organization of tissues in specimens undergoing posterior regeneration, and the timeframe in which significant developmental changes occur. To further investigate this phenomenon, we amputated the posterior segments of living H. carunculata specimens collected from the Gulf of Mexico and monitored posterior regeneration over a 6‐month study period. Although many aspects of posterior regeneration in H. carunculata are consistent with the findings of other annelid regeneration studies, histological analysis revealed that once formed, anal lobe morphology remains relatively unchanged at all stages of posterior regeneration; East Atlantic morphotypes were not observed in the West Atlantic specimens studied here. Additionally, we found that the ventral nerve chord, which is partially responsible for the regeneration of lost body parts in polychaete annelids, terminates within the anal lobe, suggesting that this structure may play a role in the formation of new segments. J. Morphol. 275:1103–1112, 2014. © 2014 Wiley Periodicals, Inc.     

Plant regeneration capacity of mesophyll protoplasts from Brassica napus and related species

Protoplasts from a total of thirty-six genotypes of Brassica species – B. napus, B. campestris (syn. B. rapa), B. juncea, and three distant relatives, Orychophragmus violaceus, Isatis indigotica and Xinjiang wild rape – were analysed for shoot regeneration using a feeder culture system. With the exception of B. campestris and Xinjiang wild rape, some genotypes of all the species could regenerate plants with high efficiency (above 20% of isolated calli initiating shoots). Several genotypes with high regeneration ability were elite breeding lines. Culture conditions as well as genotype had a significant impact on shoot regeneration frequency. In particular, silver nitrate added to the regeneration medium at doses of 6 and 30 μM improved shoot regeneration frequency to 25.4% and 52.2% of isolated calli, respectively, compared to 7.3% percent shoot regeneration without silver nitrate in seven responsive genotypes. Addition of silver nitrate to the regeneration medium also induced shoot regeneration in non-responsive genotypes. Intact plants could be obtained within three months from protoplast isolation in the regenerative genotypes using the current culture system. Advantages of mesophyll protoplasts as compared to protoplasts isolated from hypocotyls for genetic manipulation in Brassica species are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Investigating the developmental onset of regenerative potential in the annelid Capitella teleta

An animal's ability to regrow lost tissues or structures can vary greatly during its life cycle. The annelid Capitella teleta exhibits posterior, but not anterior, regeneration as juveniles and adults. In contrast, embryos display only limited replacement of specific tissues. To investigate when during development individuals of C. teleta become capable of regeneration, we assessed the extent to which larvae can regenerate. We hypothesized that larvae exhibit intermediate regeneration potential and demonstrate some features of juvenile regeneration, but do not successfully replace all lost structures. Both anterior and posterior regeneration potential of larvae were evaluated following amputation. We used several methods to analyze wound sites: EdU incorporation to assess cell proliferation; in situ hybridization to assess stem cell and differentiation marker expression; immunohistochemistry and phalloidin staining to determine presence of neurites and muscle fibers, respectively; and observation to assess re-epithelialization and determine regrowth of structures. Wound healing occurred within 6 h of amputation for both anterior and posterior amputations. Cell proliferation at both wound sites was observed for up to 7 days following amputation. In addition, the stem cell marker vasa was expressed at anterior and posterior wound sites. However, growth of new tissue was observed only in posterior amputations. Neurites from the ventral nerve cord were also observed at posterior wound sites. De novo ash expression in the ectoderm of anterior wound sites indicated neuronal cell specification, although the absence of elav expression indicated an inability to progress to neuronal differentiation. In rare instances, cilia and eyes re-formed. Both amputations induced expanded expression of the myogenesis gene MyoD in preexisting tissues. Our results indicate that amputated larvae complete early, but not late, stages of regeneration, which indicates a gradual acquisition of regenerative ability in C. teleta. Furthermore, amputated larvae can metamorphose into burrowing juveniles, including those missing brain and anterior sensory structures. To our knowledge, this is the first study to assess regenerative potential of annelid larvae.     

Survey of the differences between regenerative and non-regenerative animals

To investigate the boundaries between regenerative and non-regenerative animals, we first survey regenerative ability across animal phyla from sponges to chordates (including mammals). There are both regenerative and non-regenerative animals in each phylum. The cells participating in regeneration also vary among different species. Thus, it is hard to find clear rules concerning regeneration ability across the animal kingdom, suggesting that it is not useful to compare the difference of regenerative ability across phyla to seek the boundary between regenerative and non-regenerative animals. Instead, if we carefully compare the differences of regenerative ability between closely related species within each phylum and accumulate these differences at the cellular molecular levels, we may be able to clarify the boundary between regenerative and non-regenerative animals. Here we introduce our comparative analysis of cellular events after amputation of lower jaws between frogs and newts. Then we propose that such comparative analyses using closely related species within the same phylum should be accumulated to understand the boundary between regenerative and non-regenerative animals in order to apply this understanding for realizing regenerative medicine in the future.     

Effect of storage time on the physiological characteristics and vegetative regeneration of desiccation‐tolerant mosses on the Loess Plateau,China

The artificial cultivation of moss biocrusts can accelerate the recovery of degraded arid lands and is closely related to moss productivity. Understanding the properties of inoculation materials on the regenerative capacity of mosses, in particular the effect of time limits on storage, has the potential to benefit the cultivation of artificial moss biocrusts. We investigated the vegetative regeneration and physiological characteristics of three desiccation‐tolerant mosses (Barbula unguiculata, Didymodon vinealis, and Didymodon tectorum) upon rehydration after periods of desiccation storage for 40, 89, 127, and 197 days. Regenerative capacity, represented by gametophyte vigor index, decreased with increased storage time. The greatest change in vegetative regeneration among storage times was observed in B. unguiculata, where the gametophyte vigor index decreased by 95.74% after 197 days of storage. Over the same period, there were smaller decreases in gametophyte vigor index of D. vinealis and D. tectorum of 42.17% and 13.30%, respectively. Malondialdehyde and soluble sugar increased with longer periods of storage time, while soluble protein content first increased, then decreased. Oxidation levels are important factors influencing the recovery of desiccation‐tolerant mosses. All three moss species regenerated after 197 days of storage, but regenerative capacity is dependent on species. For land managers, biocrust restoration can be facilitated by careful screening of suitable moss species, based on their capacity to regenerate new growth after extended periods of storage and selection of species based on variations in physiological characteristics.     

Characteristics of the reparative regeneration of fins in the polypterid fish (Polypteridae,Actinopterygii)

Epimorphic regeneration of fins was studied in different ray-finned fishes (Actinopterygii), but species representing the phylogenetically basal lineages of the taxon have remained outside the attention of researchers. Information on the regenerative abilities of these groups is important both for understanding the evolutionary origins of the epimorphic regeneration phenomenon and for assessing the universality of regenerative potencies in Actinopterygii. Addressing this problem, we studied for the first time fin regeneration in two members of the archaic family Polypteridae: the ropefish (Erpetoichthys calabaricus) and the Senegal bichir (Polypterus senegalus). Along with the ability to regenerate the bony rays of fins, widespread among Actinopterygii, polypterids show the ability to effectively regenerate the endoskeleton and musculature of their fins. This unusual feature allows us to suggest polypterids as new model organisms for the study of the mechanisms of vertebrate limb regeneration.     

High-frequency direct shoot regeneration and continuous production of rapid-cycling Brassica oleracea in vitro

Summary An in vitro method was developed for high-frequency shool regeneration and continuous production of rapid-cycling B. oleracea in large numbers. The high regenerative capacity was tissue-dependent. Developmental polarity (apical and basal ends) of the explants appeared to play a regulatory role in shoot morphogenesis in this system. High-frequency shoot regeneration was obtained with N⁶-benzyladenine or thidiazuron-supplemented media. Delayed and reduced regenerative ability of cultures in air-tight vessels and the dramatic suppression of shoot regeneration in internodal explants by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid implicate a possible involvement of ethylene in shoot morphogenesis in this species. Rotting of regenerated shoots of B. oleracea occurred readily on α-naphthaleneacetic acid-supplemented media. Rooted plantlets were successfully established in soil and developed normal fertile flowers and viable seeds.     

Biology of Pseudopotamilla reniformis (Müller 1771) in the White Sea,with description of asexual reproduction

The tube-dwelling polychaete Pseudopotamilla reniformis (Sabellidae) forms dense and complex aggregations of flexible tubes on hard substrates in the subtidal zone of the White Sea. No sexual reproduction was observed in this study and recruitment appeared to be due to asexual reproduction by architomy in winter, from October to March. The posterior part of the abdomen undergoes spontaneous fission into from 2 to 4 fragments and depending on their position, the fragments regenerate their anterior ends or both anterior and posterior ends. Regeneration in P. reniformis takes place via a combination of epimorphosis (replacement of missing parts by cell proliferation and the growth of new tissue) and morphallaxis (the remodelling of pre-existing structures without cell proliferation). The morphogenetic events during regenerative restoration include de novo formation of branchial crown, formation of thoracic segments and restoration of the posterior end. Asexual reproduction appears to play a crucial role for formation of P. reniformis aggregations and is very important for the population in the White Sea, at the margin of the species’ range.     

Imaginal discs: Renaissance of a model for regenerative biology

Many animals display a capacity to regenerate tissues or even a complete body. One of the main goals of regenerative biology is to identify the genes and genetic networks necessary for this process. Drosophila offers an ideal model system for such studies. The wide range of genetic and genomic approaches available for use in flies has helped in initiating the deciphering of the mechanisms underlying regeneration, and the results may be applicable to other organisms, including mammals. Moreover, most models of regeneration require experimental manipulation, whereas in Drosophila discrete domains can be ablated by genetically induced methods. Here, we present a summary of current research into imaginal disc regeneration and discuss the power of this tissue as a tool for understanding the genetics of regeneration.     

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.     

Sponge cell aggregation: checkpoints in development indicate a high level of organismal complexity

Studies of regeneration provide insight across many scales of animal biology from the processes of cellular communication to the ecology of whole populations. Sponges are highly regenerative animals, with studies showing adults can both recover large portions of their body after predation or damage due to storms, and even reform whole individuals, via an aggregation stage, from dissociated tissues. While sponges are clearly highly regenerative, few studies actually show dissociated cells forming functional individuals. As sponges often serve as model organisms for studying the development and function of traits in metazoans, determining the universality and mechanics of their regeneration potential is important. We tested the capacity of members of seven sponge species from temperate freshwater and marine environments, from a range of taxonomic positions, and with different habits, to form functional sponges after dissociation. Development to a functional sponge progressed through a series of checkpoints: the sorting of cells and removal of debris; adhesion to a substrate and differentiation of cells; organization of cells into tissues; and regionalization of tissues. Two of the seven species tested, Spongilla lacustris and Haliclona cf. permollis, progressed through all four checkpoints, while the remaining five species progressed to various levels of development before aggregates disintegrated. Our findings highlight three important conclusions: (1) The ability of aggregates to differentiate into functional sponges is not as widespread as previously thought; (2) The species‐specific ability of aggregates to develop to functional sponges appears to be an adaptive trait; and (3) The progression of development in aggregates through checkpoints, which in later development involves formation of tissues and regionalization of tissues, highlights the complexity of the sponge body plan and suggests fundamental rules in development shared across metazoans.     

Regeneration of the nervous and muscular system after caudal amputation in the polychaete <Emphasis Type="Italic">Alitta virens</Emphasis> (Annelida: Nereididae)

Regenerating segments in polychaetes offer a vivid example of epimorphic recovery of the lost organs and tissues. It is also a promising object for studying positional information and the mechanisms maintaining the body integrity. With the aim to develop a convenient standardized model, we described the dynamics of recovery of the major anatomical structures and created a staging system for the caudal regeneration in Alitta virens. In average the normal organization of the posterior body end is restored within 10 days after amputation (dpa). The whole regenerative process was divided into 5 stages: (1) wound healing (0–1 dpa), (2) blastema formation (1–2 dpa), (3) patterning and growth of the blastema (2–3 dpa), (4) differentiation of the first regenerated segment (3–5 dpa), (5) formation and differentiation of the subsequent 5–6 segments (5–10 dpa). The regeneration is carried out mainly by epimorphosis, although the elements of intercalary growth as well as the morphallactic transformation of the stump have been noted. Terminal structures of the pygidium (muscles of the anal sphincter, pygidial cavity, pygidial ring nerve, pygidial cirri) appear at stages 1–3, and then (from stage 3) the formation of new metameres begins in front of the pygidium. Differentiation of the first newborn segment is associated with the tissue remodeling in the last old segment. Formation of the next segments resembles accelerated postlarval growth. The neural elements of the regenerative bud are developing faster than the surrounding muscles. The neurites extending from the CNS and PNS come to the surface of the wound epithelium at stage 1. Later, nerve fibers from the CNS lengthen and thicken along with the growth of the regenerative bud. Ganglion, parapodial nerves, oblique muscles and coeloms of the first segment are detected at stage 4. Longitudinal muscles regenerate in anterior to posterior progression, being constantly in contact with the corresponding fibers of the old tissues. All other muscles differentiate from blastemal cells in isolation from the old musculature of the stump. Our data promote the further using of the posterior body end regeneration in A. virens as an experimental model for resolving crucial problems of developmental biology.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

von Willebrand factor D and EGF domains is an evolutionarily conserved and required feature of blastemas capable of multitissue appendage regeneration

Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema—a transient structure that houses a pool of progenitor cells that can regenerate the missing tissue. We have identified the expression of von Willebrand factor D and EGF domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species, including axolotl (Ambystoma mexicanum), lungfish (Lepidosiren paradoxa), and Polpyterus (Polypterus senegalus). Further, vwde expression is tightly linked to the ability to regenerate appendages in Xenopus laevis. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent growth factor in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily informed approach for identifying conserved genetic components of regeneration.     

Inheritance of somatic embryogenesis in red clover (Trifolium pratense L.)

 Red clover genotypes capable of regenerating plantlets in vitro from non-meristematic tissue-derived callus are rare. Selection for genotypes capable of somatic embryogenesis identified a clone comprised of a group of plantlets regenerated from a hypocotyl-derived callus culture on L2-based media and another group of plantlets originating from crown divisions of the epicotyl-derived plant. The callus-derived plants of this clone were highly regenerative when reintroduced to callus culture, but the epicotyl-derived plants produced nonregenerative callus cultures. F₁, F₂ and BC₁ populations were evaluated to determine the mode of inheritance of the regeneration trait. Reciprocal crosses did not differ, indicating a lack of maternal effects. Results were compatible with genetic control of regeneration by two complementary genes. We propose the genotype Rn1-Rn2- for regenerative plants. Three petiole segment explants were sufficient to evaluate regenerative ability in seedlings. Regenerative ability was often associated with abnormal leaf morphology in a few to several leaves. Received: 17 February 1998 / Accepted: 7 April 1998     

Tools for conservation of Balsamorhiza deltoidea and Balsamorhiza sagittata: Karrikin and thidiazuron-induced growth

Wildfires are having both devastating and regenerative impacts on the ecosystems in the Pacific Northwest of North America. Balsamorhiza sagittata and B. deltoidea (balsamroot) are ecologically important species in this region, and B. sagittata populations are increasing, while B. deltoidea is critically imperiled. The aim of this research was to establish in vitro protocols for germination and regeneration of Balsamorhiza spp. to enable conservation efforts. It was hypothesized that karrikins, which are plant growth regulators released from burning plants during wildfires, would induce seed germination in Balsamorhiza spp. Three karrikins (KAR₁, KAR₂, and KAR₁₁) were tested for the ability to enhance germination in these species at two levels (5 or 10 μM). KAR₂ had the strongest positive effect on germination and induced 47% and 60% germination, respectively, in B. sagittata seeds compared to 14% germination obtained in the control (water agar media). In B. deltoidea, KAR₂ treatment resulted in a germination rate of 73.1% and 100%, compared to 69% in the control. A germplasm collection of seedlings of both species was established for conservation and regeneration experiments. Thidiazuron treatment (10 μM) induced formation of embryo-like structures in seedlings of both B. sagittata and B. deltoidea, with regenerants originating from the crown of seedlings. The present study provides in vitro methods for conservation and mass propagation of Balsamorhiza species.

     

Effects of sediment deposition on the seaweed Sargassum on a fringing coral reef

 Coral reef degradation may involve shifts from coral to algal dominance and may be caused in part by increased sediment loads. Inshore fringing reef flats in the central Great Barrier Reef region are often subjected to periods of high sedimentation and are often dominated by macroalgae such as Sargassum. Experiments reported here examine the impacts of sediments on the recruitment, growth, survival, degeneration and vegetative regeneration of Sargassum microphyllum on a fringing coral reef flat in the central Great Barrier Reef. Comparison of three levels of sediment deposition (experimental addition, control (ambient condition) and experimental removal) showed that increased amounts of sediment significantly decreased rates of recruitment, growth, survival and vegetative regeneration, but not degeneration of S. microphyllum. In addition, the regenerative ability of S. microphyllum thalli with short, persistent erect branches (untreated) was compared with that of thalli experimentally cut back to the holdfast. This experimental damage significantly reduced regeneration. Accepted: 6 October 1997