Free-living flatworms under the knife : past and present Traditionally , regeneration research has been closely tied to flatworm research , as flatworms -LRB- Plathelminthes -RRB- were among the first animals where the phenomenon of regeneration was discovered . Since then , the main focus of flatworm regeneration research was on triclads , for which various phenomena were observed and a number of theories developed . However , free-living flatworms encompass a number of other taxa where regeneration was found to be possible . This review aims to display and to compare regeneration in all major free-living flatworm taxa , with special focus on a new player in the field of regeneration , Macrostomum lignano -LRB- Macrostomorpha -RRB- . Findings on the regeneration capacity of this organism provide clues for links between regeneration and -LRB- post - -RRB- embryonic development , starvation , and asexual reproduction . The role of the nervous system and especially the brain for regeneration is discussed , and similarities as well as particularities in regeneration among free-living flatworms are pointed out . Introduction Regeneration research in flatworms has a long standing history , but was focussed on triclads since its beginning in 1774 , when Pallas -LRB- Pallas 1774 -RRB- described the regeneration of the triclads Dendrocoelum lacteum and Bdellocephala punctata -LRB- from Brøndsted 1969 -RRB- . Since then , a vast amount of studies about regeneration in flatworms has appeared -LRB- for recent reviews , see Agata and Watanabe 1999 ; Sánchez Alvarado 2000 , 2003 , 2004 , 2006 ; Saló and Baguñà 2002 ; Newmark and Sánchez Alvarado 2002 ; Agata 2003 ; Agata et al. 2003 ; Reddien and Sánchez Alvarado 2004 ; Reuter and Kreshchenko 2004 ; Sánchez Alvarado and Kang 2005 ; Saló 2006 -RRB- , but many of the old questions and many of the most intriguing phenomena that have been discovered can not be explained today . Why can some animals regenerate , while others can not ? How is the duplication of heads or tails -LRB- heteromorphoses -RRB- possible in adult organisms ? What conditions are sufficient and what are necessary for successful regeneration ? Is regeneration a side-product of asexual reproduction , or the other way round ? Is regeneration recapitulating pathways used in embryonic and postembryonic development ? How are stem cells controlled and directed in regeneration ? Are stem cells in adult flatworms totipotent , and can they be likened to embryonic blastomeres ? This review provides a brief synopsis on what is known about the regeneration capacity in free-living flatworms , and addresses open questions about regeneration , with special emphasis on an emerging model organism , Macrostomum lignano -LRB- Egger et al. 2006a , b -RRB- . A comparison of the regeneration capacity of the macrostomorph flatworm M. lignano with other free-living flatworms necessarily involves a look at their phylogenetic relationship . The Macrostomorpha belong to the largest taxon Rhabditophora , which also encompasses the Polycladida , Lecithoepitheliata , Prolecithophora , Proseriata , Bothrioplanida , Tricladida , Rhabdocoela , and the parasitic Neodermata -LRB- Ehlers 1985 ; Rieger 1996 ; Tyler et al. 2006 -RRB- . Macrostomorpha are considered to be the most basal taxon within the Rhabditophora due to a simple pharynx , entolecithal eggs , and the lack of vitellaria -LRB- Ehlers 1985 -RRB- . Referring to their small size -LRB- millimeter range -RRB- , macrostomorphans are members of the `` microturbellarians '' , a nonsystematic denomination . Polyclads and triclads are the only taxa belonging to the `` macroturbellaria '' , with forms generally in the centimeter range . Triclads , as more derived rhabditophorans , have vitellaria , a rather complicated embryonic development -LRB- e.g. , Cardona et al. 2005 -RRB- and an often very pronounced regeneration capacity -LRB- see Brøndsted 1969 -RRB- . The status of the acoels While many molecular -LRB- e.g. , Baguñà and Riutort 2004a , b -RRB- and also some morphological studies -LRB- e.g. , Reuter et al. 2001 , summed up in Egger and Ishida 2005 -RRB- have placed the acoels outside the Plathelminthes , a number of characteristics -LRB- apomorphies -RRB- speak in favor of acoels being flatworms -LRB- Ehlers 1985 -RRB- , most notably the stem cell system and the special mode of epidermal replacement -LRB- Smith et al. 1986 ; Tyler and Hooge 2004 -RRB- . In this review , acoels and nemertodermatids are considered members of the Plathelminthes . What is a planarian ? In flatworm regeneration research , other groups than triclads are often neglected and can easily be overlooked . This may in part be due to an ambiguous use of the term `` planarian '' , which is most often referring merely to triclads -LRB- more specifically , to paludicolans , e.g. , Agata 2003 ; Reddien and Sánchez Alvarado 2004 -RRB- , but is also commonly used as a synonym for all free-living flatworms -LRB- e.g. , Baguñà 1998 ; Saló and Baguñà 2002 -RRB- or even used interchangeably for free-living flatworms and triclads alike -LRB- e.g. , Newmark and Sánchez Alvarado 2001 -RRB- . The term `` freshwater planarian '' , while somewhat more specific , is still ambiguous , as a number of nontriclad flatworms , e.g. , Stenostomum and Microstomum , are freshwater dwellers as well . In publications dealing with several flatworm taxa , it is often unclear what the term planarian signifies . It seems therefore desirable to agree on using less ambiguous denominations in scientific contexts , such as unequivocal scientific names , and to get rid of the term planarian . Overview of regeneration capacity in free-living flatworm groups All major free-living flatworm taxa with their tentative general regeneration capacities are given in Fig. 1 . Asexually reproducing forms are only known for acoels , catenulids , macrostomorphans , and triclads . Within free-living flatworms , either catenulids -LRB- Ehlers 1985 -RRB- or acoelomorphs -LRB- acoels and nemertodermatids , Rieger 1996 -RRB- are considered as the basal-most taxon . All other flatworms are encompassed by the rhabditophorans , with macrostomorphans and polyclads at the base -LRB- Ehlers 1985 ; Rieger 1996 -RRB- . Fig. 1The regeneration capacity of the major free-living flatworm taxa . In all groups with asexual reproduction , species with a pronounced regeneration capacity can be found -LRB- green shades -RRB- . Not all species in the listed taxa are necessarily showing the same regeneration capacity ; only a broad classification of regeneration capacities is given . The major taxa were compiled after Ehlers -LRB- 1985 -RRB- , Rieger -LRB- 1996 -RRB- , and Tyler et al. -LRB- 2006 -RRB- Catenulida The regeneration capacity of two catenulid genera , namely , Stenostomum and Catenula , has been extensively investigated . Probably linked with asexual reproduction -LRB- paratomy -RRB- , excellent regeneration capacity has been reported after artificial amputation -LRB- Ritter and Congdon 1900 ; Child 1903a , b ; Ruhl 1927a ; van Cleave 1929 ; and Palmberg 1990 for Stenostomum ; Moraczewski 1977 for Catenula -RRB- . Regeneration from the so-called organ primordia was found to be possible for all organ systems . Acoela Based on experiments mainly with Polychoerus caudatus -LRB- Stevens and Boring 1905 ; Child 1907 ; Keil 1929 -RRB- and Paramecynostomum diversicolor -LRB- Peebles 1913 -RRB- , Hyman notes that acoels `` have limited powers of regeneration '' -LRB- Hyman 1951 , p 182 -RRB- . The studied species were not able to regenerate ganglia -LRB- brain -RRB- or the statocyst . Later studies with Amphiscolops langerhansi -LRB- Steinböck 1954 , 1955 , 1963a , b -RRB- and Hofstenia giselae -LRB- Steinböck 1966 , 1967 -RRB- illustrated a substantial regeneration capacity in these acoels , where H. giselae was found to even regenerate the statocyst after amputation -LRB- Steinböck 1966 -RRB- . Several species can regenerate their eyes -LRB- Hanson 1967 for A. langerhansi and Pseudohaplogonaria sutcliffei , Yamasu 1991 for Praesagittifera naikaiensis , and Åkesson et al. 2001 for Convolutriloba longifissura -RRB- . Acoels are now known to show all main modes of asexual reproduction , that is architomy -LRB- e.g. , C. longifissura , Åkesson et al. 2001 -RRB- , paratomy -LRB- e.g. , Paratomella unichaeta , Ax and Schulz 1959 -RRB- , and budding -LRB- e.g. , C. retrogemma , Hendelberg and Åkesson 1988 -RRB- . Architomy is characterized by fission before formation of new organs , paratomy by formation of new organs before fission . Budding refers to a special case of paratomy , where the axis of the new piece is not corresponding to the axis of the old piece -- in C. retrogemma , the axis of the buds is tilted by 180 ° in relation to the main animal -LRB- Hendelberg and Åkesson 1988 , 1991 -RRB- . Nemertodermatida No account of regeneration potential or asexual reproduction has been given for animals from this taxon . Macrostomorpha As already observed in catenulids and acoels , the regeneration capacity of macrostomorphans is closely associated with the mode of reproduction . Thus , in the asexually reproducing taxa Microstomum lineare and Alaurina -LRB- paratomy -RRB- , the regeneration capacity is most pronounced and all organs can be regenerated -LRB- von Graff 1908 ; Ruhl 1927a ; Palmberg 1986 , 1990 , 1991 -RRB- . Regeneration research with the exclusive sexually reproducing genus Macrostomum started with Ruhl -LRB- 1927a -RRB- , who found that Macrostomum tuba can not regenerate the head -LRB- brain , eyes , and pharynx -RRB- and requires at least a quarter of the gut in the anterior piece to be able to regenerate posterior parts , including copulatory organs . More recently , Ladurner et al. -LRB- 1997 -RRB- noted that in Macrostomum hystricinum marinum and Macrostomum pusillum the anterior part can fully regenerate the posterior part , while the posterior part can not regenerate the anterior part . Similar findings were reported for M. lignano : cut in midbody , only the anterior part was found to regenerate the missing part -LRB- Rieger et al. 1999 -RRB- . Salvenmoser et al. -LRB- 2001 -RRB- described the regeneration of the musculature in M. lignano after the removal of the posterior end . Using the same species , Egger et al. -LRB- 2006a , b -RRB- characterized the regeneration capacity at various cutting levels . Anterior regeneration is possible if the animals are cut in front of the brain ; posterior regeneration can be successful from cutting levels starting with the posterior end of the pharynx . A distinct regeneration blastema occurs in M. lignano , but not in M. lineare , where organ primordia are formed without conspicuous accumulation of neoblasts at the wound site -LRB- Palmberg 1986 -RRB- . Polycladida The best-studied polyclad genus regarding regeneration is Leptoplana . Leptoplana alcynoe -LRB- now Letoplana alcinoi -RRB- and Leptoplana velutinus , as well as Thysanozoon brocchi and Cryptocelis alba , were shown to regenerate posterior parts by Monti -LRB- 1900a -RRB- . Using Leptoplana atomata , Schultz -LRB- 1901 , 1902 -RRB- observed the regeneration of the posterior part , including gonads and copulatory organs , whereas head and pharynx were not regenerated . Similar results were obtained with Leptoplana tremellaris and L. alcinoi , although the pharynx was found to regenerate here -LRB- Child 1904a -- c , 1905a ; von Levetzow 1939 -RRB- . L. tremellaris can regenerate both anterior and posterior parts if only about half the brain is removed by transversal amputation . At amputation levels posterior of the brain , no complete anterior regeneration is possible -LRB- Child 1904a -- c -RRB- . Notoplana humilis shows a comparable regeneration capacity -LRB- Ishida 1998 -RRB- . Besides more posterior parts , T. brocchi is also able to regenerate the main tentacles , gut , and pharynx -LRB- Monti 1900a ; von Levetzow 1939 -RRB- . Studying regeneration in Leptoplana saxicola , Pucelis litoricola , and Planocera californica , Olmsted -LRB- 1922a -RRB- reports that these species follow the `` polyclad rule for regeneration '' , that is , regeneration of all amputated parts -LRB- including eyes -RRB- is possible if the brain remains intact . Several studies were dedicated to find out whether polyclads can regenerate the brain -LRB- Morgan 1905 for Leptoplana littoralis and von Levetzow 1939 for T. brocchi -RRB- , and how the behavior changes after excision of the brain -LRB- Morgan 1905 ; Olmsted 1922b ; von Levetzow 1939 ; Koopowitz et al. 1975 , 1976 -RRB- or after severance of the main lateral nerve cords -LRB- Olmsted 1922b ; Koopowitz et al. 1975 , 1976 -RRB- . Severed nerve cords and brain halves can fuse and resume their functions -LRB- Olmsted 1922b ; Faisst et al. 1980 -RRB- , but the brain is not regenerated . Full anterior regeneration , including the brain , after complete removal of the brain was only observed in Cestoplana , provided the amputation level is a short distance behind the brain -LRB- Child 1905b -RRB- . All mentioned species , except T. brocchi , are members of the taxon Acotylea -LRB- Tyler et al. 2006 -RRB- . From what is known , both Cotylea and Acotylea seem to share a comparable regeneration capacity . Lecithoepitheliata Only scarce reports about regeneration are available for this neoophoran taxon . For Prorhynchus stagnalis , Ruhl -LRB- 1927a -RRB- remarks that no regeneration capacity whatsoever could be detected at anterior , posterior , and midbody cutting levels . The posterior pieces survived for 30 days . When cutting Geocentrophora baltica a short distance behind the pharynx , no regeneration could be observed -LRB- Steinböck 1927 -RRB- . All pieces died at the latest after 17 days . A closer examination of the regeneration capacity of Geocentrophora sphyrocephala revealed that these animals can approximately rebuild the mouth opening anteriorly in 3 -- 5 weeks but can not regenerate the brain . Both parts die after 40 days if the animals are cut just behind the pharynx . Cut in midbody or more posteriorly , the anterior part can regenerate the tail with adhesive organs in 2 -- 3 weeks , although the animals were never observed to reach the original body length . The posterior part dies without regeneration after 1 day -LRB- Hagleithner 1946 -RRB- . Prolecithophora According to Pechlaner -LRB- 1957 -RRB- , Plagiostomum lemani is reported to have a low regeneration capacity -LRB- Keller 1894 -RRB- , while Plagiostomum girardi can regenerate all amputated parts -LRB- Monti 1900b -RRB- . Proseriata von Graff -LRB- 1882 -RRB- describes amputation experiments with Monocelis fusca , where posterior pieces resumed movement after 3 -- 5 days . In M. fusca , Monocelis lineata and other monocelidids -LRB- Coelogynopora , Archilopsis -RRB- , and also in otoplanids -LRB- Itaspiella , Bothriomolus -RRB- , Giesa -LRB- 1966 -RRB- observed posterior regeneration of parts containing the brain in 3 -- 4 days . Even pieces amputated just behind the brain can regenerate . The brain does not regenerate , though . In Otomesostoma auditivum , Pechlaner -LRB- 1957 -RRB- found promising regeneration potential , severely limited by a short life span of 9 -- 10 months and low living temperatures -LRB- about 4 °C -RRB- . Gut , part of the gonads , and sensory organs can regenerate . First signs of pharynx , eye , and brain regeneration were observed after 9 -- 15 weeks , but the animals died before regeneration was complete . The statocyst was not regenerated . Bothrioplanida While no extensive regeneration studies have been undertaken , Sekera -LRB- 1911 -RRB- found that Bothrioplana semperi is able to regenerate the pharynx posteriorly . Tricladida Dalyell -LRB- 1814 -RRB- noted that Planaria nigra -LRB- today : Polycelis nigra -RRB- could `` almost be called immortal under the edge of the knife '' -LRB- in Brøndsted 1969 -RRB- . However , not all triclad species are equally powerful regarding regeneration . Ŝivickis -LRB- 1930 -RRB- distinguished five groups of triclads according to their ability to regenerate a head . This classification was subsequently refined into eight types by Teshirogi et al. -LRB- 1977 -RRB- . For the Phagocata velata type , the Dugesia dorotocephala type , the Polycelis auriculata type , and the Dendrocoelopsis lactea type 2 -LRB- Sapporo population -RRB- , regeneration of a head is possible from any part of the body , but with varying probability of success . Animals of the Dendrocoelopsis lactea type 1 -LRB- Kuroishi population -RRB- fail to regenerate a head from tail pieces up to the male gonopore . The Dendrocoelopsis ezensis type and the Dendrocoelum lacteum type 1 are very similar in that they are generally only able to regenerate a head if amputated anterior to the pharynx , whereas the Bdelloura type -LRB- e.g. , Bdelloura candida -RRB- completely lacks the ability to regenerate a head . While a vast amount of literature on regeneration in triclads is available , recent publications are not so much concerned with studying the regeneration capacities of different triclads , but more with unraveling cellular and molecular mechanisms of regeneration in only a few model triclads , such as Dugesia japonica and Schmidtea mediterranea . Rhabdocoela The typhloplanoid flatworm Mesostoma ehrenbergi can not regenerate any organs , only approximately restore the original shape posteriorly -LRB- Steinmann and Bresslau 1913 -RRB- . Regeneration experiments with Mesostoma productum , Mesostoma lingua , and Mesostoma punctatum revealed that anterior regeneration of parts in front of the brain is possible , although often done imperfectly . The tail can regenerate ; the more posterior the cut is made , the better is the regeneration . Brain and pharynx can not be regenerated -LRB- Fulinski 1922 -RRB- . In Phaenocora unipunctata and Phaenocora megalops -LRB- also typhloplanoids -RRB- , no regeneration capacity was found -LRB- Fulinski 1922 -RRB- . For the the dalyelliid Dalyellia millportiana and the kalyptorhynch Gyratrix hermaphroditus , Ruhl -LRB- 1927a -RRB- remarks curtly that all animals died within 1 day after amputation and no regeneration being observable . Dalyellia viridis and Typhloplana viridata survive the amputation of the caudal-most part of the tail , but show no signs of regeneration , not even of the adhesive organs . In Rhynchomesostoma rostratum , another typhloplanoid , the amputated tail tip is regenerated , or at least a tail-like shape is restored -LRB- Hein 1928 -RRB- . Comparison of the regeneration capacity between free-living flatworm taxa While the experimental evidence for some groups is still patchy , we find three main types of regeneration capacity in free-living flatworms . Some taxa can not regenerate at all , such as most rhabdocoels and some lecithoepitheliates . On the other end of the scope are species that can regenerate all organs , such as the acoel Convolutriloba , the catenulid Stenostomum , the macrostomorphan Microstomum , the polyclad Cestoplana , and many triclads . Most taxa however lie in between these extremes , lacking the ability to regenerate brain , eyes , pharynx , or statocyst -LRB- many acoels -RRB- , but are able to regenerate gonads , copulatory organs , sucker , duo-gland adhesive systems , gut , and the anterior-most part in front of the brain . The regeneration capacity of M. lignano can be likened most closely to triclads of the Bdelloura group , which can not regenerate a head -LRB- brain or eyes -RRB- . Most polyclads , although often able to regenerate a pharynx , are also roughly comparable to Macrostomum regarding their regeneration capacity . Members of other diverse taxa like lecithoepitheliates and rhabdocoels are bearing resemblance with regeneration in Macrostomum as well . Such similarities can be found crossing the boundaries of the major flatworm taxa , making the regeneration capacity a feature of no immediate phylogenetic value . Close relatives like Microstomum and Alaurina -LRB- both macrostomids -RRB- surpass Macrostomum 's regeneration capacity by far . Asexual reproduction and regeneration Not surprisingly , the ability to regenerate is often linked to asexual reproduction -LRB- Fig. 1 , Brøndsted 1969 ; Sánchez Alvarado 2000 -RRB- . In taxa lacking asexual reproduction , the regeneration capacity is generally less pronounced than in taxa with asexual reproduction , where regeneration is necessarily involved -LRB- Brøndsted 1969 ; Reuter and Kreshchenko 2004 -RRB- . As described in the section `` Acoela '' , in architomy , regeneration events follow fission , while in paratomy and budding , the term `` pregeneration '' seems to be more accurate , as organs are duplicated in the presence of the old organs in the new animal -LRB- zooid -RRB- before fission occurs . The notable difference between regeneration and pregeneration is the presence of a brain in the latter , which is regarded as a decisive organ for regeneration in many species -LRB- Henley 1974 ; Kreshchenko et al. 2001 -RRB- . It is interesting to note that the catenulid Stenostomum leucops , reproducing asexually by paratomy , often fails to regenerate a head if amputated artificially -LRB- van Cleave 1929 -RRB- . This finding hints at an actual difference between paratomy and architomy regarding the influence on regeneration capacity . Asexual reproduction in flatworms occurs in more basal taxa -LRB- Acoela , Catenulida , and Macrostomorpha -RRB- as well as in more derived taxa -LRB- Tricladida and Neodermata -RRB- . Triclads , like acoels , show paratomy -LRB- Dugesia fissipara and Dugesia paramensis , Hyman 1951 -RRB- as well as architomy -LRB- e.g. , Dugesia tahitiensis , Peter et al. 2001 -RRB- . Just like regeneration capacity , asexual reproduction is too dispersed among diverse taxa to provide insight into phylogenetic relationships . Why can some flatworm species regenerate while others can not ? If a profound regeneration capacity is a plesiomorphic feature , there might have been a selection pressure against regeneration during evolution -LRB- Goss 1991 ; in Sánchez Alvarado 2000 -RRB- . Why would such a useful feature be reduced or abolished ? Because regeneration is dangerous , as a plethora of evidence of double-headed or double-tailed flatworms testify . Such aberrant forms are easily produced in species that can regenerate -LRB- see section `` Heteromorphoses '' -RRB- , but are not known from flatworms lacking regeneration capabilities . Other than as a side effect of asexual reproduction , the ability to regenerate is seemingly not useful enough to outweigh the inherent dangers . What are the chances that a predator spares a tail fragment that can regenerate to a complete animal in a couple of days if left alone ? What are the chances that a disease only affects a part of the animal , but spares the rest to readily regenerate the damaged organs ? A totipotent stem cell system out of control might be too high a risk for comparably little reward . This could be the reason why regeneration capabilities decreased in species that no longer propagate through asexual reproduction . In this light , it seems more likely that in evolutionary terms , asexual reproduction is a primitive and not a derived feature -LRB- Rieger 1986 ; Gremigni 1992 ; Reuter and Kreshchenko 2004 -RRB- , and that regeneration capability results from asexual reproduction as an added bonus . Regeneration can be seen as a form of asexual reproduction , only triggered by different stimuli . What is the decisive factor that determines the regeneration capacity of a species ? Citing regeneration experiments with Dendrocoelum and Bdellocephala , Brøndsted -LRB- 1969 -RRB- comes to the conclusion `` that the posterior part of the body in these two species simply lacks the potential to respond to head inducing substances '' . This may also hold true for M. lignano , considering that in transversal amputations a head was never regenerated , but in longitudinal incisions a second head -- in the presence of the original head -- could be formed -LRB- Fig. 2c , d , Egger et al. 2006a , b -RRB- . This experiment suggests that the ability to induce head formation , although present in adult M. lignano , is restricted to the head region only . Serotonin and possibly other substances related to the brain or nervous system might be key factors deciding on the regeneration capacity of flatworms . Fig. 2Heteromorphoses resulting from embryogenesis and regeneration . a Twin embryo of M. lignano , 8-cell stage . The two embryos are not separated by a dividing wall . In some cases , such embryos may grow to siamese twins . Diameter of individual embryos is about 150 μm . b Different animal than a found in culture dishes . The juvenile animals are connected at the level of the pharynx , possibly the result of two embryos growing together . c Adult M. lignano , 1 week after longitudinal incision -LRB- arrowhead -RRB- . Only a small indentation is visible . d Same specimen as in c 29 days after longitudinal incision . A second head with brain , a single eye , and pharynx has emerged . Scale bars are 100 μm . c , d Same scale bar It is interesting to note that double-headed specimens of M. lignano can sometimes be observed in untreated culture dishes . These aberrant forms might rather be the result of twin embryos -LRB- Fig. 2a -RRB- that have grown together during embryonic development , than adults that were accidentally incised longitudinally -LRB- Sekera 1911 ; Steinmann and Bresslau 1913 ; Seilern-Aspang 1958 -RRB- . Ovaries restricting regeneration ? The possible evolutionary origin and function of regeneration aside , what genetical , physiological , developmental , or environmental factors are responsible to determine to what extent an organism can regenerate ? Even close relatives may differ vastly in regeneration capacity -LRB- e.g. , Microstomum -- Macrostomum -RRB- , and also the same species in different developmental stages . Stenostomum alternates seasonally between asexual and sexual reproduction -LRB- protandric hermaphroditism -RRB- . Asexual and male specimens of Stenostomum grande can regenerate all organs , but lose the ability to regenerate a head after the appearance of ovaries -LRB- van Cleave 1929 -RRB- . While the presence of ovaries and the loss of regeneration capacity might be coincidental , it appears possible that in S. grande ovaries are producing substances that inhibit regeneration . Could sexualization be directly affecting a general decrease of regeneration potential ? At present , the molecular nature of such an inhibitory substance remains unclear . Vasa-related genes are expressed strongly both in gonads and in the early blastema in D. japonica and in M. lignano -LRB- Shibata et al. 1999 ; Pfister and Ladurner 2005 -RRB- , making an inhibitory function of vasa during regeneration improbable , at least in these two species . Also , contrary to the findings in Stenostomum , several triclads are known to regenerate all organs in the presence of gonads -LRB- e.g. , S. mediterranea , Sánchez Alvarado 2003 -RRB- . Two possibilities may explain differences in the regeneration capacity in the presence of ovaries : either the ovaries in Stenostomum are producing a unique inhibitory substance that is not synthesized in Schmidtea , or Schmidtea is able to counteract the effects of such an inhibitory agent . Regeneration in juveniles If gonads , specifically ovaries , are indeed hindering regeneration in some species , then sexually immature juveniles could possibly be more potent regenerates than adults . In the rhabdocoel Mesostoma , young animals -LRB- the author does not specify their age -RRB- are reported to regenerate better than adults -LRB- Fulinski 1922 -RRB- . Contrastingly , in the acoel species H. giselae , no regeneration even of large juveniles -LRB- 2.5 mm long -RRB- takes place -LRB- Steinböck 1967 -RRB- . In most cases , however , juveniles regenerate as well -LRB- or bad -RRB- as adults . An explanation is that gene expression for the building of gonads is already occurring in immature animals , thus diminishing possible differences in the regeneration capacity between juveniles and adults . Regeneration in juvenile triclads has been studied in B. punctata by Brøndsted , who found that animals recently hatched from cocoons were able to regenerate the head at the same rate as mature adults -LRB- Brøndsted 1942 , 1969 -RRB- . Pechlaner -LRB- 1957 -RRB- studied the regeneration of freshly hatched animals , juveniles and mature adults in the proseriate O. auditivum . He did not observe significant differences in the regeneration capacity between these age groups . Neither juvenile nor adult dalyellids were found to regenerate -LRB- Hein 1928 -RRB- . In the lecithoepitheliate G. sphyrocephala , the regeneration capacity of juveniles corresponds to that of adults -LRB- Hagleithner 1946 -RRB- . The results for M. lignano support these findings and show that even very young animals -LRB- 1 day after hatching -RRB- are capable of regeneration , indicating that the neoblast stem cell system is already present and functional as found in adults . Accordingly , BrdU labeling of 1-day-old hatchlings revealed a similar pattern of S-phase cell distribution as in adults , with the majority of labeled cells along the sides of the animal , and no labeled cells in the rostrum . Just like adults , juveniles were not able to regenerate a head -LRB- Egger et al. 2006a , b -RRB- . Similarities between postembryonic development and regeneration However similar , regeneration has some characteristics not shared with postembryonic development . Unlike postembryonic development , regeneration in flatworms is triggered by a disruptive event like fission or amputation -LRB- but not by chemical destruction of tissue , burning , or high voltage ; see Brøndsted 1969 -RRB- . As in other flatworms , in M. lignano , the wound is soon closed after amputation by contraction of the ring musculature minimizing the wound surface , and by flattening of the surrounding epidermal cells -LRB- Salvenmoser et al. 2001 -RRB- . In the next days , undifferentiated cells -LRB- neoblasts -RRB- accumulate at the wound site , forming a distinct blastema , covered by new and old epidermal cells . Subsequently , differentiation of microorgans and organs becomes prevalent within the blastema , superseding the blastema proper -LRB- Egger et al. 2005 , 2006a , b -RRB- . Epidermal cells are not undergoing mitoses , as new epidermal cells and blastema cells are derived from neoblasts located in the mesodermal space . Different to triclads , neoblasts are proliferating also within the blastema , not only at the blastema border -LRB- Egger et al. 2005 -RRB- . While wound closure and blastema formation are regeneration-specific events , similarities between regeneration and postembryonic development become prevalent starting with differentiation in the -LRB- former -RRB- blastema . In extreme cases , where only a small piece remains for regeneration -LRB- e.g. , in pharynx-level regenerates , Egger et al. 2006a , b -RRB- , resemblance with postembryonic development is particularly striking -LRB- Fig. 3 -RRB- . The remaining organs in these regenerates undergo extensive morphallactic changes to accommodate to the new body proportions -LRB- Fig. 3n -- o in Egger et al. 2006a -RRB- . Fig. 3Comparison of M. lignano hatchlings with posterior regenerates amputated at the pharynx level . b and d are depicting the same individual . a Hatchling up to 24 h old . b Pharynx level regenerate 7 days after amputation . c 7-day-old hatchling . d Pharynx level regenerate 14 days after amputation . Scale bar for all subpanels is 100 μm One week after amputation , pharynx-level regenerates of M. lignano are confronted with similar challenges as freshly hatched juveniles : both have to build gonads and the male and female genital organs , grow a full set of duo-gland adhesive glands , and increase their size by five or six times -LRB- Figs. 3a , b , and 4 -RRB- . It seems plausible , that after wound healing and restoration of the basic functionality of the gut , pharynx-level regenerates are making use of similar or same developmental pathways that are used in hatchlings -LRB- Fig. 4 -RRB- -LRB- cooption , Sánchez Alvarado 2000 -RRB- . This view is supported by the ability of hatchlings and juveniles to regenerate just like adults , and by the similarity of the stem cell system in hatchlings , juveniles , and adults -LRB- Ladurner et al. 2000 ; Egger et al. 2006a , b -RRB- . Vasa gene expression is similar in hatchlings and in gonad-recovering regenerates as well -LRB- Pfister and Ladurner 2005 -RRB- . Also , the time needed for developing to a full-grown adult in M. lignano is about the same for hatchlings -LRB- Morris et al. 2004 -RRB- and 1-week-old pharynx-level regenerates , that is about 2 weeks -LRB- Fig. 3 and Egger et al. 2006a , b -RRB- . In the proseriate M. fusca , the regeneration of small anterior pieces also goes through stages similar to postembryonic development -LRB- Giesa 1966 -RRB- , which is observed in triclads as well -LRB- Baguñà 1998 -RRB- . Starved adults , reducing their size and breaking down the gonads -LRB- Nimeth et al. 2004 -RRB- , are also likely recapitulating their postembryonic development while growing back to full size -LRB- Fig. 4 -RRB- . Not only is regeneration akin to postembryonic development , amputated animals can even be rejuvenated by regeneration -LRB- Fig. 4 and Haranghy and Balázs 1964 for triclads , Egger et al. 2006a , b for M. lignano -RRB- . While untreated animals were found to live for about 10 months in culture conditions , they were outlived by animals amputated 45 times over a period of 20 months , effectively doubling their life span so far -LRB- Egger et al. 2006a , b ; Egger , unpublished observations -RRB- . At the time of writing , many of these repeatedly amputated animals are still alive . Fig. 4Similarities between -LRB- post - -RRB- embryonic development , regeneration , and starvation considering as example M. lignano . Successive developmental stages from left to right , from embryonic to postembryonic development -LRB- formation of gonads and genital organs -RRB- until adulthood . Regeneration If an adult animal is amputated just posterior of the pharynx , the anterior piece will pass stages similar to early juveniles and subadults before regeneration to a normal adult is completed . The same animal can be amputated repeatedly , each time seemingly repeating parts of its postembryonic development . Repeated regeneration was also shown to have a rejuvenation effect . A connection between the formation of organ primordia during embryonic development and the organ differentiation in regenerating animals is possible , but has not been shown yet for M. lignano . Some developmental pathways are likely shared between late embryos and regenerates as well , e.g. , the initial building of microorgans like the duo-gland adhesive systems . Both in embryos and posterior regenerates , these organs are built from scratch . Starvation Animals starved for 30 days or longer break down their gonads and genital organs shrink , but can recover and regain sexual prowess after feeding . Starvation leading to an extended life span was shown for triclads and is also likely to occur in M. lignano A fountain-of-youth effect through starvation was shown to occur in triclads -LRB- Haranghy and Balázs 1964 -RRB- , but was not yet proven for M. lignano -LRB- Fig. 4 -RRB- . Embryonic development and regeneration Early embryogenesis is vastly different between diverse free-living flatworm taxa -LRB- for recent publications , see Jondelius 2004 for nemertodermatids ; Ramachandra et al. 2002 for acoels ; Morris et al. 2004 for macrostomorphans , Younossi-Hartenstein and Hartenstein 2000 for polyclads , Cardona et al. 2005 for triclads , and Younossi-Hartenstein and Hartenstein 2001 for rhabdocoels -RRB- . Even comparably close relatives show significant differences in their early embryonic development . Lacking vitellaria and therefore producing entolecithal eggs , Macrostomorpha and Polycladida are the only archoophoran Rhabditophora , placing them at the basal-most positions in this largest flatworm taxon -LRB- Ehlers 1985 ; Rieger 1996 -RRB- . Nonetheless , their early embryonic development starts to deviate after about the third cleavage -LRB- Fig. 5 -RRB- . If developmental pathways from the embryonic development are reused in regeneration , it is most likely from the formation of organ primordia and later stages -LRB- Fig. 4 -RRB- . Cardona et al. -LRB- 2005 -RRB- point out the similarities between embryogenesis and early regeneration in triclads concerning the building of a provisional epidermis and the development of muscles and nerves , suggesting that not only postembryonic , but also embryonic developmental pathways are utilized during regeneration , a viewpoint shared with Vannini -LRB- 1966 -RRB- . A comparison of gene expression patterns between regenerates -LRB- see Reddien et al. 2005 for triclads -RRB- , late embryos and hatchlings seem to be a worthwhile task . Fig. 5Early embryonic development of two basal rhabditophorans . a The 12-cell stage of the polyclad Pseudostylochus intermedius . Egg without eggshell . b 12-cell stage of the macrostomorphan M. lignano . Different to the clear spiral cleavage of polyclads , M. lignano forms the so-called hull cells after the third cleavage to enclose the rest of the embryo . The diameter of the embryos is about 150 μm Early blastomere ablation in polyclads has revealed a determinative development as in other spiralians -LRB- Boyer 1986 -RRB- , the fate of blastomeres depending on micromere -- macromere interaction -LRB- Boyer 1989 -RRB- . Polyclads do not have an invariant cell lineage , however -LRB- Boyer 1992 -RRB- . Polyclad embryos can be obtained devoid of an eggshell -LRB- Fig. 5a -RRB- and still develop normally in a culture dish treated with antibiotics . Regeneration experiments with polyclad embryos may thus be relatively easy to carry out and could provide additional clues about the requirements for regeneration in flatworm embryos . Also , the regeneration capacity of polyclad larvae , as compared to hatchlings of directly developing polyclads , would be interesting to know . A connection between regulation in early embryonic development and regeneration capacity does not seem to exist . This notion is supported by regeneration experiments with embryos of the triclad P. nigra . Regeneration does not take place before organ systems , such as the nervous system , reach a certain degree of differentiation in the embryo , so that stage five embryos are unable to regenerate , while stage six embryos with a more differentiated nervous system readily regenerate lost parts -LRB- Le Moigne 1966 -RRB- . This author held that the nervous system plays a critical role in the formation of a blastema during regeneration . Nervous system and role of the brain for regeneration The nervous system , especially the brain , has long been considered to play a key role in regeneration -LRB- Kreshchenko et al. 2001 -RRB- . In many free-living flatworms , posterior parts can be regenerated , while the brain can not . In anterior regenerates of M. lignano missing the brain , a blastema-like structure was observed , which was unable to give rise to all missing organs . The more posterior the animals were amputated , the less pronounced was this abortive anterior blastema -LRB- Egger et al. 2006a , b -RRB- . In Macrostomum , are the brain and the nerve cell clusters associated with the pharynx -LRB- Fig. 6 -RRB- required to release factors needed for the regeneration of brain , eyes , and pharynx ? The study of gene expression in head-amputated regenerates of the triclads D. japonica and S. mediterranea revealed necessary genes for regeneration of the brain -LRB- Cebrià et al. 2002a ; Koinuma et al. 2003 ; Cebrià and Newmark 2005 -RRB- . Also , with nou-darake a factor was identified that restricts brain tissue to the head region of D. japonica . If nou-darake is interrupted by RNA interference , brains appear in all body regions -LRB- Cebrià et al. 2002b -RRB- . In species that are not able to regenerate a brain , nou-darake or a similar factor might be expressed not only in the brain region but also throughout the body , thus preventing the formation of a new brain even if the old brain was lost . Fig. 6Immunocytochemical stainings of the nervous system of M. lignano . Confocal images . a GYIRFamidergic immunoreactivity . Clusters of immunoreactive cells are found lateral to the pharynx and in front of the eyes . b , c Serotonergic immunoreactivity in the head region . b Full stack of confocal images . Note the stained cell clusters lateral to the pharynx and in front of the eyes . c Subset of image stack in b . In this focal plane , stained cells anterior to the eyes become apparent . Arrowheads denote the level of the eyes In M. hystricinum marinum and M. pusillum , large clusters of serotonergic cells have been found besides the posterior part of the pharynx -LRB- Ladurner et al. 1997 -RRB- . These results were corroborated for M. lignano by staining of the serotonergic and the GYIRFamidergic nervous system -LRB- Fig. 6 -RRB- . Amputees cut in the middle of the pharynx lose this cluster of nerve cells , as well as a large part of the pharynx , which is known to be a critical structure for regeneration in some triclads -LRB- Brøndsted 1969 -RRB- and polyclads -LRB- Ishida 1998 -RRB- . Ladurner et al. -LRB- 1997 -RRB- held that the distribution of serotonergic nerve cells is correlated with the regeneration capacity . In species where serotonergic neurons are restricted to the head area , the posterior part of the animals can not regenerate the head . In the asexually reproducing species M. lineare and S. leucops , serotonergic nerve cells are found over the whole length of the main nerve cords . The inability of M. lignano to regenerate eyes after transversal amputation or oblique amputation between the eyes , is possibly linked with a severe brain damage caused by these amputations , and the loss of strongly immunoreactive cells in front of the eyes -LRB- Fig. 6c -RRB- . In two cases of longitudinal incisions , lost eyes were regenerated or supernumerary eyes were built -- but in these cases , the brain was only slightly injured . In a series of experiments , Lender -LRB- 1950 , 1951a -- c -RRB- and Wolff and Lender -LRB- 1950a , b -RRB- stress the importance of the head ganglia for the regeneration of eyes in P. nigra . If the brain was repeatedly excised or X-irradiated , no regeneration of eyes took place : an intact brain was obviously required to induce eye regeneration . These results were corroborated for Dugesia -LRB- now Schmidtea -RRB- lugubris by Török -LRB- 1958 -RRB- . However , L. littoralis , L. saxicola , and P. litoricola can regenerate eyes even if the brain was excised -LRB- Morgan 1905 ; Olmsted 1922a -RRB- . Vannini -LRB- 1965 -RRB- observed in S. lugubris and P. nigra that the testes do not regenerate in the absence of a brain . Ghirardelli -LRB- 1965 -RRB- found that if a two-headed Dugesia was decapitated on one side , the testes on the same side degenerated . Contrastingly , the gonads in M. lignano are viable for an extended period of time after decapitation or oblique amputation between the eyes , and are only reduced if the pharynx is lost as well , causing malnourishment and starvation -LRB- Egger et al. 2006a , b -RRB- . This may be an indication that the building of the gonads is not under the primary control of the brain in M. lignano , just like the tail plate can be built without the presence of a head . The regeneration of a tail in headless regenerates is possible in M. lignano as well as in triclads of the Dendrocoelum group -LRB- Brøndsted 1939 , 1969 -RRB- , in the polyclads L. littoralis , L. saxicola , and P. litoricola -LRB- Morgan 1905 ; Olmsted 1922a -RRB- , and also in the rhabdocoel Mesostoma -LRB- Fulinski 1922 -RRB- . Therefore , the head in all of these organisms is not required to control the regeneration of the tail . In Cestoplana and T. brocchi , the pharynx can regenerate in the absence of the brain -LRB- Child 1905c ; von Levetzow 1939 -RRB- ; in Cestoplana often a secondary pharynx is built in the presence of the old pharynx -LRB- Child 1905c -RRB- . Also , the regulation of tissue breakdown -LRB- e.g. , gonads -RRB- and body size reduction during starvation is possible in decapitated pieces , so the brain is not responsible for this regulation as well . Headless M. lignano with intact pharynx are able to feed , in accordance with the polyclads Notoplana acticola and Planocera gilchristi -LRB- Koopowitz et al. 1976 -RRB- . These animals are able to identify and devour food in the absence of a brain , while Enchiridium punctatum , a polyclad possessing a tubular instead of a plicate pharynx , can not feed without the brain -LRB- Koopowitz et al. 1976 -RRB- . Similarly , some triclads , also equipped with a tubular pharynx , are not able to feed if the brain was amputated -LRB- Hyman 1951 -RRB- . Brainless M. ehrenbergi can not feed but can move normally -LRB- Steinmann and Bresslau 1913 -RRB- , whereas brainless Phaenocera is reported to behave similarly to normal animals -LRB- Fulinski 1922 -RRB- . G. sphyrocephala , different to M. lignano , can not adhere to the substrate after decerebration -LRB- Hagleithner 1946 -RRB- . The main function of the brain in M. lignano is probably to be found in directed motion -LRB- e.g. , light avoidance , predator avoidance ; see also Koopowitz et al. 1976 and literature therein -RRB- , satiation reflex -LRB- Koopowitz et al. 1976 -RRB- , mating behavior -LRB- Scharer et al. 2004 -RRB- , and chemical analyzation of the medium . The totipotency of neoblasts Pharynx-level regenerates of M. lignano have shown that the regeneration of gonads is possible after removal of all gonadal tissue . The viability of the regenerated gonads has been demonstrated by breeding experiments with fully regenerated animals . Besides the gonads , also the gut and the copulatory organs have been rebuilt de novo in pharynx-level regenerates -LRB- Egger et al. 2006a , b -RRB- . In similar experiments , Morgan -LRB- 1902 -RRB- removed all gonads in Schmidtea lugubris , and the animals regenerated to fully functional sexual organisms . It is still possible that the remaining neoblasts in the amputee are only pluripotent and not totipotent , different types of neoblasts being competent for different -LRB- groups of -RRB- tissues . A convincing experiment to prove the totipotency of neoblasts would be the injection of a single neoblast into a lethally X-irradiated animal that is to be amputated some weeks after the injection . In case the animal survives , and is able to restore all removed organs , then either redifferentiation of cells would have taken place , or -LRB- at least some -RRB- adult neoblasts would be totipotent . A similar experiment , with the injection of 20,000 -- 24,000 cells enriched in neoblasts -LRB- 30 -- 88 % neoblasts -RRB- into X-irradiated S. mediterranea , was performed by Baguñà et al. -LRB- 1989 -RRB- . This experiment was not replicated in the literature , but the results made highly probable that neoblasts as a whole are totipotent in routine cell turnover , leaving open the questions if this holds also true for regeneration and if this totipotency applies to one single cell type or to several different types of stem cells . Are neoblasts in species with limited regeneration potential not totipotent , or are they just not receiving the right stimulus or guidance for regeneration ? Supporting the latter notion is the ability of Macrostomum to duplicate heads after longitudinal incision -LRB- Fig. 2d and Egger et al. 2006a , b -RRB- , while decapitated animals were never observed to regenerate a head -LRB- Egger et al. 2006a , b -RRB- . In this species , it seems that stimuli from the brain are required to duplicate or regenerate the brain . Is there a specific subset of predetermined `` brain '' neoblasts in species that can not regenerate a brain ? These neoblasts could be located in the vicinity of the brain and therefore lost by amputation of the brain . Brain excision experiments in polyclads , where only the encapsuled brain was removed -LRB- von Levetzow 1939 ; Koopowitz et al. 1976 -RRB- , do not support the existence of such brain-specific neoblasts , as these animals were not able to regenerate the brain . Also , amputation experiments in M. lignano , where only brain and the neoblast-free rostrum were amputated , showed no brain regeneration -LRB- Egger et al. 2006a , b -RRB- . While neoblasts are required for regeneration in general , they do not determine whether a species is able to regenerate a specific organ such as the head , as was found with grafting experiments in triclads -LRB- Brøndsted 1969 -RRB- . Can a single neoblast from an adult develop to a whole organism in an adequate environment , just like a zygote ? For the parasitic flatworm Taenia crassiceps , it was shown that single cells of trypsinated cysticerci are able to produce complete cysticerci in the mouse host -LRB- Toledo et al. 1997 -RRB- . So in these cestodes , a single totipotent cell , other than a zygote , is able to recover a whole animal . No such experiment is known for free-living flatworms . Besides the difficulty in providing a suitable culture medium , distinct differences between a single neoblast and a zygote are yolk content or surrounding yolk cells and maternal determinants that are present in eggs , but not in neoblasts . Do -LRB- all or some -RRB- neoblasts have embryonic characteristics , as some authors held -LRB- e.g. , Keller 1894 -RRB- ? When and where do neoblasts origin in the embryo ? Polyclads with their clear and traceable embryonic development appear to be suitable flatworms for investigating these questions . Heteromorphoses Hetermorphoses , i.e. , the occurrence of supernumerary heads or tails , in species that can not regenerate an amputated head , have shown that the regeneration potential of neoblasts is not to be equated with the regeneration capacity of the species . Heteromorphoses are also a placative warning sign for the dangers coming with the ability to regenerate -LRB- all -RRB- organs . Last but not the least , hetermorphoses in free-living flatworms show that the duplication of axes is not only possible during embryonic development -LRB- Fig. 2b , siamese twins -RRB- , but also in adults , even after comparably minor surgery . The M. lignano specimen depicted in Fig. 2c , d shows that a longitudinal incision in the anterior part of the animal can establish a second anteroposterior body axis parallel to the original axis . The new anteroposterior axis reaches from the rostrum tip to the caudal tip and duplicates all organs including head , pharynx , female genital opening , and male copulatory organ . Even the breadth of the animal approximates that of two single specimens . Over time , however , the animal degenerated in both halves losing all eyes , and was not able to recover to a viable state -LRB- Egger et al. 2006a , b -RRB- . A decreased viability in animals with heteromorphoses was also observed in Macrostomumappendiculatum and Bothrioplana bohemica -LRB- Sekera 1911 -RRB- , as well as in S. leucops -LRB- Ruhl 1927b -RRB- . In triclads , duplicated or even multiple heads -LRB- e.g. , 10-headed D. lacteum , Lus 1924 -RRB- and tails have been generated and studied by a great number of researchers -LRB- see Brøndsted 1969 -RRB- . Randolph -LRB- 1897 -RRB- made longitudinal and lateral incisions in Planaria maculata -LSB- today : Dugesia -LRB- Girardia -RRB- tigrina -RSB- leading to additional eyes and pharynges . It was found that when a duplicated head or tail was amputated close enough to the second main axis , regeneration was inhibited by the presence of the remaining head or tail -LRB- Rand and Mildred 1926 -RRB- . In the polyclad Leptoplana , double-heads and double-tails were experimentally produced by longitudinal incision -LRB- Child 1905a -RRB- . With the same method , tails , but not heads , could be duplicated in the rhabdocoel Mesostoma -LRB- Fulinski 1922 -RRB- and in the acoel P. caudatus -LRB- Keil 1929 -RRB- . Constrastingly , heads , but not tails could be duplicated by incision in the catenulid S. leucops -LRB- Ruhl 1927b -RRB- . Another type of heteromorphoses is created by provoking a body axis reversed to the original body axis . This was found in M. lignano after longitudinal incision in the head , that induced the creation of a tail plate appendage in the anterior body region -LRB- Egger et al. 2006a , b -RRB- . Agata et al. -LRB- 2003 -RRB- suggested that one of the main functions of a blastema in triclads is producing positional signals and inducing intercalary regeneration between stump and blastema . An incision may cause the building of a very small blastema at the wound site , which provides anterior or posterior positional signals . Depending on the signal the blastema produces , either a head or a tail plate is subsequently formed . Incisions may also have a long-range effect , as was observed by Randolph -LRB- 1897 -RRB- in triclads , where outgrowths appeared at body regions not affected by incision . A similar phenomenon as in M. lignano was observed by Steinböck -LRB- 1967 -RRB- in H. giselae , where an animal was divided into 10 pieces , one of which developed a tail-like structure at the anterior end besides the head . Just as was observed in M. lignano -LRB- Egger et al. 2006a , b -RRB- , the anterior tail in H. giselae was gradually moving along the side of the animal towards the posterior end , when after 36 days the additional tail was located at the posterior tip of the animal . Conclusion A pronounced regeneration capacity and the existence of possibly totipotent stem cells in adults are two key features of many flatworms not found in this combination in any other bilaterian taxa . It could be shown that regeneration follows similar principles in many different taxa of free-living flatworms , even if the regeneration capacity can vastly differ . Such comparisons make it possible to recognize unique patterns , and also to form general ideas how , why , and when regeneration works . For this reason , it appears to be valuable to establish several flatworm model organisms from various taxa , to address old and upcoming questions regarding regeneration and the neoblast stem cell system . One of these comparably new model flatworms is M. lignano , a small but versatile worm that facilitates regeneration studies on the cellular and molecular level .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

planarian acoela rhabditophora catenulida