The nervous system of <Emphasis Type="Italic">Convolutriloba</Emphasis> (Acoela) and its patterning during regeneration after asexual reproduction

We describe the serotonergic and cholinergic nervous system of the asexually reproducing acoel Convolutriloba longifissura Bartolomaeus & Balzer, 1997 by means of immunohistochemistry, conventional histochemistry and transmission electron microscopy. Immunocytochemical staining for serotonin revealed neurons in the brain, in a pair of ventral main longitudinal cords, in two pairs of smaller dorsal longitudinal nerve cords, and in a submuscular nerve net. The brain comprises a ventral-anterior commissure and a less intensely stained dorsal commissure joined together by connectives into a three-ringed scaffold from which the longitudinal nerves extend. We followed the regeneration of the serotonergic part of the nervous system up to the second day after fission. Within this time period, the offspring reestablished bilateral symmetry in the nervous system and developed full motor control. The presence of aminergic cell bodies associated with the main lateral nerve cords of C. longifissura shows that the acoelan nervous system is more similar to that of other platyhelminths (triclads, rhabditophorans) than previously assumed. The presence of serotonergic cell bodies along the main nerve cord correlates with the capacity for asexual reproduction via fissioning. We also describe the single fission mode of C. hastifera Winsor 1990, which brings the modes of asexual reproduction employed by members of the Convolutrilobinae to three.     

Fission in Convolutriloba longifissura: asexual reproduction in acoelous turbellarians revisited

Studies of the asexual reproduction of Convolutriloba longifissura (Acoela, Acoelomorpha) revealed that there is no longitudinal fission of the whole animal as has been described by Bartolomaeus and Balzer (1997) . Instead, the first step is a transverse fission. This results in the detachment of the caudal fourth of the mother animal. The detached part forms what we call the butterfly stage, which initially has no mouth and no eye fields. This stage gives rise to two new individuals by a longitudinal fission. Within 2–3 days the eye fields and a mouth develop in each of the two progenies formed in this way. In the meantime the mother individual grows and develops the three typical caudal lobes. The mother animal can repeat this process resulting in three individuals every fourth day. The finding of this new pattern of reproduction in the Acoela has prompted us to review the various ways by which asexual reproduction occurs in the group. The peculiar combination of few cases but high diversity of asexual reproduction in the Acoela is discussed from an evolutionary point of view.     

Unique patterns of longitudinal body-wall musculature in the Acoela (Plathelminthes): the ventral musculature of Convolutriloba longifissura

The ventral musculature of Convolutriloba longifissura (Acoela) has been studied using electron microscopy and fluorescently labeled whole mounts to demonstrate filamentous actin. Attention was directed to the reorganization and renewal of musculature during asexual reproduction and the adaptation of muscle sets for special predatory behavior. Three ventral subepidermal muscle layers could be distinguished in adult C. longifissura: (1) outer circular muscles that encircle the body, (2) intermediate modified longitudinal muscles with concentric pattern around the mouth and V-shaped orientation in the posterior part of the animal, and (3) inner special pore muscles with radial alignment fanning out from the mouth. Additionally, a few very fragile muscles were found at the anterior margin of the animal. The anterior ventral muscle system built a funnel with the mouth opening as organizing center. The special radial muscles and the antagonistically concentric muscles are perfectly adapted to catch prey in such a way that the funnel is put over the prey to press it through the mouth into the digestive syncytium. Convolutriloba longifissura shows a unique way of asexual reproduction by a two-step fission which results in three individuals. Immediately after separation from the mother animal, daughter individuals are missing the concentric and the radial muscle sets around the mouth completely, but within 30 h these sets are renewed for the most part. Two to three days after separation, the mouth opening is visible and the animals move for capturing prey. The peculiar course of longitudinal muscles in C. longifissura with concentric rings anteriorly and a V-shape muscle layer posteriorly shows that the pattern of body-wall musculature in such basal Plathelminthes as the Acoela may be highly modified from the original pattern of longitudinal and circular muscles.     

Evolution of regeneration and fission in annelids: insights from engrailed- and orthodenticle-class gene expression

The recent explosion of information on the role of regulatory genes in embryogenesis provides an excellent opportunity to study how these genes participate in post-embryonic developmental processes. We present a detailed comparison of regulatory gene expression during regeneration and asexual reproduction (by fission) in the segmented worm Pristina leidyi (Annelida: Oligochaeta). We isolated three genes from Pristina, one homolog of engrailed and two homologs of orthodenticle, and characterized their expression in different developmental contexts. In situ hybridization studies on worms undergoing normal growth, regeneration and fission demonstrate that in all three processes, Pl-en is expressed primarily in the developing nervous system, and Pl-Otx1 and Pl-Otx2 are expressed primarily in the anterior body wall, foregut and developing nervous system. Our data reveal extensive similarities between expression during regeneration and fission, consistent with the idea that similar developmental processes underlie these two types of development. Thus, we argue that in these annelids fission may have evolved by recruitment of regenerative processes. Furthermore, by comparing our data to existing data from leech embryos, we find evidence that embryonic processes are re-deployed during regeneration and fission.     

Conularia sp. from the Pyrénées: further support for scyphozoan affinities of the Conularida

McKinney, F.K, Delvolve, 7.-J. & Sobieraj, J. 1995 11 30: Conularia sp. from the Pyrénées: further support for scyphozoan affinities of the Conularida.
Several specimens of Conularia from the mid-Carboniferous Culm Formation of the Pyrénées occur as clusters with a large central specimen and several smaller divergent individuals that may be buds from the larger central specimen. Other individual and clustered specimens exhibit complex polygonal cross-sections that are inferred to have resulted from longitudinal fission of a precursor pyramid into two or more descendant individuals. Apparent budding and transverse fission (strobilation) of conulariids have been reported previously, but we have found no earlier record of longitudinal fission. The existence of this additional mode of asexual increase in conulariids further supports their affinities with the scyphozoan Cnidaria. Conularida, Scypho-zoa, fission, Carboniferous, Pyrénées .     

Making heads from tails: Development of a reversed anterior-posterior axis during budding in an acoel

The anterior-posterior axis is a key feature of the bilaterian body plan. Although axis specification during embryogenesis has been studied extensively, virtually nothing is known about how this axis can be established post-embryonically, as occurs in budding animals. We investigated bud formation in the acoel Convolutriloba retrogemma, which reproduces by a remarkable process involving the formation of animals with linked but completely opposite body axes. Reverse axes are established anew during each round of budding and manifestations of the bud's new axis develop gradually, with regionalization of axial patterning genes (Hox and otx) and the establishment of organized musculature occurring secondarily, after bud initiation. A swath of tissue at the parent-bud boundary has no regenerative potential and appears devoid of inherent axial polarity. GSK-3 inhibitor trials suggest that Wnt/β-catenin or Hedgehog signalling may mediate the establishment of this unpolarized zone. Formation of unpolarized tissue may provide a buffer between opposing polarity cues and be a general mechanism by which budding animals establish and maintain linked body axes. In addition to elucidating the developmental basis of budding in a bilaterian, this study provides insight into convergence in animal budding mechanisms, redeployment of embryonic gene expression during budding, and Hox gene evolution.     

Phylogenetic distribution of regeneration and asexual reproduction in Annelida: regeneration is ancestral and fission evolves in regenerative clades

Regeneration, the ability to replace lost body structures, and agametic asexual reproduction, such as fission and budding, are post‐embryonic developmental capabilities widely distributed yet highly variable across animals. Regeneration capabilities vary dramatically both within and across phyla, but the evolution of regeneration ability has rarely been reconstructed in an explicitly phylogenetic context. Agametic reproduction appears strongly associated with high regenerative abilities, and there are also extensive developmental similarities between these two processes, suggesting that the two are evolutionarily related. However, the directionality leading to this relationship remains unclear: while it has been proposed that regeneration precedes asexual reproduction, the reverse hypothesis has also been put forward. Here, we use phylogenetically explicit methods to reconstruct broad patterns of regeneration evolution and formally test these hypotheses about the evolution of fission in the phylum Annelida (segmented worms). We compiled from the literature a large dataset of information on anterior regeneration, posterior regeneration, and fission abilities for 401 species and mapped this information onto a phylogenetic tree based on recent molecular studies. We used Markovian maximum likelihood and Bayesian MCMC methods to evaluate different models for the evolution of regeneration and fission and to estimate the likelihood of each of these traits being present at each node of the tree. Our results strongly support anterior and posterior regeneration ability being present at the basal node of the annelid tree and being lost 18 and 5 times, respectively, but never regained. By contrast, the ability to fission is reconstructed as being absent at the basal node and being gained at least 19 times, with several possible losses. Models assuming independent evolution of regeneration and fission yield significantly lower likelihoods. Our findings suggest that anterior and posterior regeneration are ancestral for Annelida and are consistent with the hypothesis that regenerative ability is required to evolve fission.     

Anomalous Cases of Conjugation and Spontaneous Autogamy in Stylonychia mytilus: A Note on the Asexuality of Early Conjugants

Four types of anomalous conjugation were documented in Stylonychia mytilus. Type I pairs were formed between mates of different sizes. These pairs exhibited an abnormal site of fusion in at least one of the mates, and the mates might face each other ventrally throughout conjugation instead of the normal side-by-side position. Type I pairs underwent sexual nuclear development and proceeded with the first cortical reorganization as in normal conjugants. Type II involved pairing at the anterior ends of mates with ventral surfaces facing the same direction. These pairs also underwent sexual nuclear development. Hence, aberrant orientation of the mates, and also ectopic sites of cytoplasmic fusion, if extensive, would permit sexual development. Type III pairs were united ventral-to-ventral with their anterior-left sides at the adoral zone of membranelles, and remained as such throughout conjugation. In these pairs, nuclear and cortical events were typical of the asexual development of physiological reorganization. In Type IV pairs, one mate of the pair possessed a fission furrow and developed two sets of ciliature typical of binary fission, while the other mate might undergo physiological reorganization or binary fission. Type III and Type IV pairs thus reveal the asexual state of early conjugants, which can pursue either one of the two modes of asexual cortical reorganization; these cases reinforce the notion of overlap of asexual and sexual cycles during conjugation of hypotrichs. Spontaneous autogamy was documented for the first time for this genus. The autogamonts proceeded with nuclear development and with the first cortical reorganization. Some probably underwent second and third reorganizations, as in conjugants, but accompanied by abnormalities, particularly in the stages beyond fertilization. Post-autogamous clones were nonviable except for one dubious case.     

Interference of asexual and sexual reproduction in the green hydra

The green hydra, Hydra viridissima, has three sexes: hermaphrodite, male, and female. I investigated the reproductive strategies of the green hydra and the relationship between asexual budding and sexual reproduction. The proportion of mature individuals in the asexually reproducing population increased with increasing temperature. Sexual reproduction did not interrupt asexual budding in hermaphrodites or males; sexual reproduction did interrupt asexual budding in females. Sexual reproduction also resulted in exponential population growth during the reproductive season. The number of asexual buds on each parental individual was positively correlated with the parental individual size in asexual individuals and in males. The same positive correlation was found between the number of testicles and the size of males. These correlations reflect a common tendency in asexual and sexual reproduction: larger parental individuals have a greater number of propagules or gametes. No correlation was found between size and buds or size and gonads in hermaphrodites; hermaphrodites had at most one asexual bud and were significantly larger than males, females, and asexual individuals. The larger size of hermaphrodites supports the hypothesis that producing both female and male gonads is more energetically costly than producing only one type of gamete (gonochorism).     

A basic helix-loop-helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei

Members of the APSES protein group are basic helix-loop-helix (bHLH) proteins that regulate processes such as mating, asexual sporulation and dimorphic growth in fungi. Penicillium marneffei is a human pathogen and is the only member of its genus to display a dimorphic growth transition. At 25 degrees C, P. marneffei grows with a filamentous morphology and produces asexual spores from multicellular con-idiophores. At 37 degrees C, the filamentous morphology is replaced by yeast cells that reproduce by fission. We have cloned and characterized an APSES protein-encoding gene from P. marneffei that has a high degree of similarity to Aspergillus nidulans stuA. Deletion of stuA in P. marneffei showed that it is required for metula and phialide formation during conidiation but is not required for dimorphic growth. This suggests that APSES proteins may control processes that require budding (formation of the metulae and phialides, pseudohyphal growth in Saccharomyces cerevisiae and dimorphic growth in Candida albicans) but not those that require fission (dimorphic growth in P. marneffei). The A. nidulans DeltastuA mutant has defects in both conidiation and mating. The P. marneffei stuA gene was capable of complementing the conidiation defect but could only inefficiently complement the sexual defects of the A. nidulans mutant. This suggests that the P. marneffei gene, which comes from an asexual species, has diverged significantly from the A. nidulans gene with respect to sexual but not asexual development.     

Genetic control of fungal differentiation: The three sporulation pathways of Neurospora crassa

Sporulation in the mold Neurospora crussa can proceed along three very different pathways, leading to the production of three types of spores. Two asexual sporulation pathways that lead to the formation of macroconidia and microconidia involve budding from hyphae by two different mechanisms. A much more complex sexual reproductive pathway involves the formation of a fruiting body called a perithecium, in which meiosis takes place and ascospores are formed in sac-like cells called asci. Numerous mutations exist that affect these developmental pathways, and genes have been isolated that are expressed preferentially during sporulation. The Neurospora sporulation pathways offer a simple system with which to study mechanisms and regulation of development that are usually obscured by complex cell-cell interactions involved in animal and plant development.     

Endogenous Bud Formation in Histriculus vorax, a New Asexual Reproductive Process In the Hypotrichida

SYNOPSIS. Histriculus vorax (Stokes) Corliss, a hypotrichous ciliate, has been separated from activated sludge and cultured monoxenically in the laboratory. The asexual life cycle has been observed and the stages of development photographed. There are large variations in shape and size of cells within clonal cultures. Both large (190–250 μ long) and small (70–140 μ long) cells are capable of normal asexual binary fission but only the large cells are able to grow endogenous buds, which, when mature, are extruded through the body wall of the mother cell. Newly deposited buds can either develop directly into an embryonic form, or, if unfavorable conditions prevail, first encyst. Similar embryonic stages bearing caudal and frontal cirri are produced both by direct development and by excystment of the encysted bud. Embryos develop rapidly into trophic forms which finally grow into small adults capable of asexual binary fission. During binary fission the nuclei behave as described previously for other members of the Oxytrichidae. A reorganization band forms at the outer end of each macronucleus, and these bands move along the macronuclei towards each other, finally disappearing at the inner ends. A fusion nucleus is then formed which splits into 4 pieces, 2 of which pass to the proter and 2 to the opisthe. During bud formation the reorganisation bands form and move as in binary fission. No fusion nucleus is formed, but the macronuclei divide immediately after reorganization, and the anterior macronucleus of each pair so produced migrates to the budding region. The infraciliature of H. vorax resembles that of Opisthotricha monspessulana. 'Erratic' kinetosomes, which are present in the adult, divide during binary fission and so produce the infraciliature of the proter and opisthe. During bud formation the budding region receives a number of these 'erratic' kinetosomes from the mother cell, and these divide to form the infraciliature of the bud.     

Longitudinal axon guidance

Our knowledge about molecular mechanisms underlying axon guidance along the antero-posterior axis in contrast to the dorso-ventral axis of the developing nervous system is very limited. During the past two years in vitro and in vivo studies have indicated that morphogens have a role in longitudinal axon guidance. Morphogens are secreted proteins that act in a concentration-dependent manner on susceptible groups of precursor cells and induce their differentiation to a specific cell fate. Thus, gradients of morphogens are responsible for the appropriate patterning of the nervous system during early phases of neural development. Therefore, it was surprising to find that gradients of two of these morphogens, Wnt4 and Shh, can be re-used for longitudinal axon guidance during later stages of nervous system development.     

Sex or Sanctuary: How do Asexual Worms Survive the Winter?

The common and geographically widespread freshwater worm Stylaria lacustris (Linnaeus, 1767) (Oligochaeta: Naididae) typically reproduces asexually through transverse paratomic fission during the spring, summer, and autumn. With the onset of shorter days and colder conditions, S. lacustris becomes a sexually mature simultaneous hermaphrodite and produces resting eggs that are capable of overwintering. However, like many naidid species, S. lacustris shows widespread variation in reproductive mode with some populations never attaining sexual maturity and others apparently exhibiting both sexual and obligately asexual genotypes. How then do obligately asexual genotypes and populations survive the harsh winter conditions? Extensive winter sampling of two, largely obligately, asexual populations of S. lacustris in Oxfordshire, UK, demonstrate that adult individuals can survive over the winter, but at densities way below that normally detected by standard sampling procedures. Laboratory experiments confirm that asexual individuals can survive cold water conditions but not freezing (unlike sexually produced cocoons). The proposed advantage of this seemingly risky reproductive strategy is that naidids like Stylaria, with their remarkably fast asexual reproductive rate, can respond instantly to favourable change in conditions.     

A new mode of colony multiplication by modified budding in the ascidian Clavelina gemmae n. sp. (Clavelinidae)

Abstract. A new mechanism of asexual multiplication of colonies is described in a colonial ascidian of the genus Clavelina (Aplousobranchiata). The mechanism consisted of the production of star-shaped buds that originate from a basal vessel that bends anteriorly and extends along the dorsal region of the zooids. Once they are well developed, the buds detached easily and were dispersed by water movement. Analysis of the fine structure of the buds revealed that they were a modification of the stolonic budding common in this genus. Time-lapse video recordings showed that released buds required several days to develop, allowing for a potentially significant dispersal range. The buds underwent organogenesis during which the central part gave rise to a new blastozooid with a defined polarity; the arms of the star gave rise to stolons. A new species is defined based on the presence of this type of budding and on other morphological features. The significance of these findings, which adds to the known mechanisms of asexual reproduction in ascidians, is discussed in relation to the biology and distribution of the species.     

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

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

Androgenesis: a review through the study of the selfish shellfish Corbicula spp

Among the asexual reproductive modes, androgenesis is probably one of the most astonishing and least studied mechanisms. In this 'paternal monopolization', the maternal nuclear genome fails to participate in zygote development and offspring are paternal nuclear clones. Obligate androgenesis is known in only a few organisms, including multiple species of clam in the genus Corbicula. Corbicula is a good system to review the evolutionary consequences of this 'all-male asexuality' because the cytological mechanisms of androgenetic reproduction have been described. In Corbicula, sperm are unreduced and, after fertilization, the maternal nuclear chromosomes are extruded as two polar bodies. Hermaphroditic lineages of Corbicula have a worldwide distribution and seem to reproduce through androgenesis, whereas their sexual relatives have restricted ranges. The invasive success of these androgenetic Corbicula lineages may be linked to their asexual mode of reproduction. We review the phenomenon of androgenesis, focusing on evolutionary perspectives, using the genus Corbicula as an exemplar system.     

Comparative larval myogenesis and adult myoanatomy of the rhynchonelliform (articulate) brachiopods Argyrotheca cordata, A. cistellula, and Terebratalia transversa

Background

Primary agametic-asexual reproduction mechanisms such as budding and fission are present in all non-bilaterian and many bilaterian animal taxa and are likely to be metazoan ground pattern characters. Cnidarians display highly organized and regulated budding processes. In contrast, budding in poriferans was thought to be less specific and related to the general ability of this group to reorganize their tissues. Here we test the hypothesis of morphological pattern formation during sponge budding.

Results

We investigated the budding process in Tethya wilhelma (Demospongiae) by applying 3D morphometrics to high resolution synchrotron radiation-based x-ray microtomography (SR-μCT) image data. We followed the morphogenesis of characteristic body structures and identified distinct morphological states which indeed reveal characteristic spatiotemporal morphological patterns in sponge bud development. We discovered the distribution of skeletal elements, canal system and sponge tissue to be based on a sequential series of distinct morphological states. Based on morphometric data we defined four typical bud stages. Once they have reached the final stage buds are released as fully functional juvenile sponges which are morphologically and functionally equivalent to adult specimens.

Conclusion

Our results demonstrate that budding in demosponges is considerably more highly organized and regulated than previously assumed. Morphological pattern formation in asexual reproduction with underlying genetic regulation seems to have evolved early in metazoans and was likely part of the developmental program of the last common ancestor of all Metazoa (LCAM).     

Life cycle of the multiarmed sea star Coscinasterias acutispina (Stimpson, 1862) in laboratory culture: sexual and asexual reproductive pathways

The multiarmed sea star Coscinasterias acutispina generally has 7-10 arms and 2-5 madreporites. It is known to be able to reproduce by asexual fission, and we have previously observed that this species also has the ability to reproduce sexually; however, there has been no report until now of spawning in this species. We succeeded in establishing a long-term culture of juveniles produced by artificial fertilization. Twelve months after the completion of metamorphosis, three individuals had six arms of the same length and a madreporite. At this time, fission occurred in two of these individuals, while the remaining individual underwent fission four months later. Each sea star divided into two halves, provided with three arms each. Thereafter, four or five new arms and two or four madreporites were formed anew in each of the six daughter sea-stars, so that by 30 days after the first fission the number of arms and madreporites in each was similar to that in adults. A second fission occurred in four of these six individuals, four or five months after the first fission, and in three of them the plane of division was the same as that of the first fission. The original three individuals eventually proliferated to 12 by undergoing fission. All individuals had fully developed gonads by 1-3 months after the second fission. Some of them eventually spawned under laboratory culture, and the resulting larvae metamorphosed into juveniles. Our observations demonstrate that individuals of C. acutispina possess the potential for both sexual and asexual reproduction.