Quantifying Cell Proliferation During Regeneration of Aquatic Worms.

Many species of aquatic worms, including members of the phyla Nemertea, Annelida, Platyhelminthes, and Xenacoelomorpha, can regenerate large parts of their body after amputation. In most species, cell proliferation plays key roles in the reconstruction of lost tissues. For example, in annelids and flatworms, inhibition of cell proliferation by irradiation or chemicals prevents regeneration. Cell proliferation also plays crucial roles in growth, body patterning (e.g., segmentation) and asexual reproduction in many groups of aquatic worms. Cell proliferation dynamics in these organisms can be studied using immunohistochemical detection of proteins expressed during proliferation-associated processes or by incorporation and labeling of thymidine analogues during DNA replication. In this chapter, we present protocols for labeling and quantifying cell proliferation by (a) antibody-based detection of either phosphorylated histone H3 during mitosis or proliferating cell nuclear antigen (PCNA) during S-phase, and (b) incorporation of two thymidine analogues, 5'-bromo-2'-deoxyuridine (BrdU) and 5'-ethynyl-2'-deoxyuridine (EdU), detected by immunohistochemistry or inorganic "click" chemistry, respectively. Although these protocols have been developed for whole mounts of small (<2 cm) marine and freshwater worms, they can also be adapted for use in larger specimens or tissue sections.

Monogenean parasites from the stomach of Oreochromis mossambicus from South Africa: two new species of Enterogyrus (Dactylogyridae: Ancyrocephalinae).

The study provides descriptions of two new species of Enterogyrus coexisting in the stomach of the Mozambique tilapia, Oreochromis mossambicus (Peters). Enterogyrus multispiralis n. sp. and Enterogyrus mashegoi n. sp. were collected from hosts sampled at Nwanedi-Luphephe Dam, Limpopo River System, South Africa. The two new species can be differentiated from other members of the genus based on the spirality characteristics of the cirrus. Enterogyrus multispiralis n. sp. has an unique 8/9-2-4 cirrus spiral formula. The length of the cirrus and its spriral formula 5-2-3 make E. mashegoi n. sp. morphometrically unique from other Enterogyrus spp. Sequences of the nuclear ribosomal DNA partial 18S and internal transcribed spacer (ITS1) and 28S were obtained and compared with available sequences of Enterogyrus in GenBank. This is the first record of data on 18S rDNA region of Enterogyrus spp. A phylogenetic comparison was conducted, which included all data available for Enterogyrus spp., but this was limited to 28S data. The closest species to both new species of the present study was an undescribed Enterogyrus sp. 2 from Sarotherodon galilaeus (Linnaeus) described from Senegal (i.e. 2.1 and 2.9% uncorrected pairwise genetic distance to E. multispiralis n. sp. and E. mashegoi n. sp., respectively). These species represent new records for Africa resulting in a total number of 12 described valid species.

Planarian EGF repeat-containing genes megf6 and hemicentin are required to restrict the stem cell compartment.

The extracellular matrix (ECM) is important for maintaining the boundaries between tissues. This role is particularly critical in the stem cell niche, as pre-neoplastic or cancerous stem cells must pass these boundaries in order to invade into the surrounding tissue. Here, we examine the role of the ECM as a regulator of the stem cell compartment in the planarian Schmidtea mediterranea, a highly regenerative, long-lived organism with a large population of adult stem cells. We identify two EGF repeat-containing genes, megf6 and hemicentin, with identical knockdown phenotypes. We find that megf6 and hemicentin are needed to maintain the structure of the basal lamina, and in the absence of either gene, pluripotent stem cells migrate ectopically outside of their compartment and hyper-proliferate, causing lesions in the body wall muscle. These muscle lesions and ectopic stem cells are also associated with ectopic gut branches, which protrude from the normal gut towards the dorsal side of the animal. Interestingly, both megf6 and hemicentin knockdown worms are capable of regenerating tissue free of both muscle lesions and ectopic cells, indicating that these genes are dispensable for regeneration. These results provide insight into the role of planarian ECM in restricting the stem cell compartment, and suggest that signals within the compartment may act to suppress stem cell hyperproliferation.

Morphologic and molecular characterization of Gyrodactylus cyclopteri Scyborskaja, 1948, from Cyclopterus lumpus L., 1758.

Interest and use of the lumpfish Cyclopterus lumpus L., 1758, as a cleaner fish in salmon aquaculture has grown significantly over the past 10 years. This has resulted in an explosion of new hatcheries to supply juveniles to the salmon industry. Until recently, these hatcheries have utilized a significant amount of wild broodstock to source the eggs required. Importation of wild fish into aquaculture systems brings an inherent risk of introducing pathogens into the culture systems. Gyrodactylus cyclopteri Scyborskaja, 1948, was found on local wild collected lumpfish that were brought in to start a captive lumpfish aquaculture program in Maine. Little information on the identification or description of G. cyclopteri was available. A re-description of the parasite, supplemented with molecular data, was undertaken to facilitate future identification and support research on this parasite of an emerging, economically significant new aquaculture species.

A mechanism for temporary bioadhesion.

The flatworm Macrostomum lignano features a duo-gland adhesive system that allows it to repeatedly attach to and release from substrates in seawater within a minute. However, little is known about the molecules involved in this temporary adhesion. In this study, we show that the attachment of M. lignano relies on the secretion of two large adhesive proteins, M. lignano adhesion protein 1 (Mlig-ap1) and Mlig-ap2. We revealed that both proteins are expressed in the adhesive gland cells and that their distribution within the adhesive footprints was spatially restricted. RNA interference knockdown experiments demonstrated the essential function of these two proteins in flatworm adhesion. Negatively charged modified sugars in the surrounding water inhibited flatworm attachment, while positively charged molecules impeded detachment. In addition, we found that M. lignano could not adhere to strongly hydrated surfaces. We propose an attachment-release model where Mlig-ap2 attaches to the substrate and Mlig-ap1 exhibits a cohesive function. A small negatively charged molecule is secreted that interferes with Mlig-ap1, inducing detachment. These findings are of relevance for fundamental adhesion science and efforts to mitigate biofouling. Further, this model of flatworm temporary adhesion may serve as the starting point for the development of synthetic reversible adhesion systems for medicinal and industrial applications.

Discovery of Eurytrema Eggs in Sediment from a Colonial Period Latrine in Taiwan.

In this study we take a closer look at the diseases that afflicted Japanese police officers who were stationed in a remote mountainous region of Taiwan from 1921 to 1944. Samples were taken from the latrine at the Huabanuo police outpost, and analyzed for the eggs of intestinal parasites, using microscopy and ELISA. The eggs of Eurytrema sp., (possibly E. pancreaticum), whipworm and roundworm were shown to be present. True infection with Eurytrema would indicate that the policemen ate uncooked grasshoppers and crickets infected with the parasite. However, false parasitism might also occur if the policemen ate the uncooked intestines of infected cattle, and the Eurytrema eggs passed through the human intestines. These findings provide an insight into the diet and health of the Japanese colonists in Taiwan nearly a century ago.

Flatworm-specific transcriptional regulators promote the specification of tegumental progenitors in Schistosoma mansoni.

Schistosomes infect more than 200 million people. These parasitic flatworms rely on a syncytial outer coat called the tegument to survive within the vasculature of their host. Although the tegument is pivotal for their survival, little is known about maintenance of this tissue during the decades schistosomes survive in the bloodstream. Here, we demonstrate that the tegument relies on stem cells (neoblasts) to specify fusogenic progenitors that replace tegumental cells lost to turnover. Molecular characterization of neoblasts and tegumental progenitors led to the discovery of two flatworm-specific zinc finger proteins that are essential for tegumental cell specification. These proteins are homologous to a protein essential for neoblast-driven epidermal maintenance in free-living flatworms. Therefore, we speculate that related parasites (i.e., tapeworms and flukes) employ similar strategies to control tegumental maintenance. Since parasitic flatworms infect every vertebrate species, understanding neoblast-driven tegumental maintenance could identify broad-spectrum therapeutics to fight diseases caused by these parasites.

No evidence for strong cytonuclear conflict over sex allocation in a simultaneously hermaphroditic flatworm.

BACKGROUND: Cytoplasmic sex allocation distorters, which arise from cytonuclear conflict over the optimal investment into male versus female reproductive function, are some of the best-researched examples for genomic conflict. Among hermaphrodites, many such distorters have been found in plants, while, to our knowledge, none have been clearly documented in animals. METHODS: Here we provide a quantitative test for cytonuclear conflict over sex allocation in the simultaneously hermaphroditic flatworm Macrostomum lignano. We used a quantitative genetic breeding design, employing pair-wise crosses of 2 × 15 independent inbred lines, to partition the phenotypic variance in several traits (including sex allocation) into its nuclear and cytoplasmic components. RESULTS: Although the nuclear genetic background had a significant effect on all traits analyzed, we found significant cytoplasmic genetic variation only for ovary size, there explaining just 4.1% of the variance. A subsequent statistical power analysis showed that the experimental design had considerable power to detect cytonuclear interactions. CONCLUSION: We conclude that there were no strong effects of cytonuclear conflict in the studied populations, possibly because the usually compact mitochondrial genomes in animals have a lower evolvability than the large mitochondrial genomes in plants or because the sampled populations currently do not harbor variation at putative distorter and/or the restorer loci.

Transcriptional signatures of somatic neoblasts and germline cells in Macrostomum lignano.

The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea. However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39, Mlig-rrm1, Mlig-rpa3, Mlig-cdk1, and Mlig-h2a, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.

Rapid identification of nine species of diphyllobothriidean tapeworms by pyrosequencing.

The identification of diphyllobothriidean tapeworms (Cestoda: Diphyllobothriidea) that infect humans and intermediate/paratenic hosts is extremely difficult due to their morphological similarities, particularly in the case of Diphyllobothrium and Spirometra species. A pyrosequencing method for the molecular identification of pathogenic agents has recently been developed, but as of yet there have been no reports of pyrosequencing approaches that are able to discriminate among diphyllobothriidean species. This study, therefore, set out to establish a pyrosequencing method for differentiating among nine diphyllobothriidean species, Diphyllobothrium dendriticum, Diphyllobothrium ditremum, Diphyllobothrium latum, Diphyllobothrium nihonkaiense, Diphyllobothrium stemmacephalum, Diplogonoporus balaenopterae, Adenocephalus pacificus, Spirometra decipiens and Sparganum proliferum, based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene as a molecular marker. A region of 41 nucleotides in the cox1 gene served as a target, and variations in this region were used for identification using PCR plus pyrosequencing. This region contains nucleotide variations at 12 positions, which is enough for the identification of the selected nine species of diphyllobothriidean tapeworms. This method was found to be a reliable tool not only for species identification of diphyllobothriids, but also for epidemiological studies of cestodiasis caused by diphyllobothriidean tapeworms at public health units in endemic areas.

Morphological and molecular identification of Gyrodactylus bubyri Osmanov, 1965 (Monogenea: Gyrodactylidae) from Caucasian dwarf goby, Knipowitschia caucasica (Berg) (Actinopterygii: Gobionellidae) from a Black Sea lagoon.

Gyrodactylus bubyri Osmanov, 1965, a monogenean parasite of Caucasian dwarf goby Knipowitschia caucasica (Berg) described from Aral Sea and subsequently reported from the same host from Strymon River, Greece, is recorded from Atanasovsko Lake, Bulgarian Black Sea coast (the first record of G. bubyri from the Black Sea basin). The species is redescribed by light and scanning electron microscopy as well as the ITS rDNA sequence is obtained. As comparative materials, specimens of G. bubyri from K. caucasica (Strymon River), G. charon Vanhove and Huyse in Vanhove et al., 2014 from Knipowitschia milleri (Acheron Delta, Greece) and G. micropsi Gläser, 1974 from Potamoschistus microps from North Sea (Belgium) are studied. Comparative morphology and molecular data demonstrate that G. micropsi is a junior synonym of G. bubyri (new synonymy). The validity of G. charon is questioned, pending examination of additional materials in order to prove it as a distinct species or as a junior synonym of G. bubyri.

Toxic effects of cadmium on flatworm stem cell dynamics: A transcriptomic and ultrastructural elucidation of underlying mechanisms.

Stem cells or undifferentiated cells can cope more easily with external stresses. To evaluate the impact of toxic compounds on stem cell dynamics in vivo, in relation to other biological responses, we use the carcinogenic element cadmium and the regenerating model organism Macrostomum lignano. Through both BrdU and anti-histone H3 immunostainings, cadmium-induced effects were investigated at different stages of the stem cell cycle. A 24-h exposure to 100 and 250 µM CdCl2 significantly decreased the number of stem cells (neoblasts) in mitosis, whereas the number of cells in the S phase remained unchanged. After this short-term exposure, the ultrastructure of the neoblasts was minimally affected in contrast to the epidermal tissues. These results were supported by gene expression data: transcripts of cdc2 and pig3 were significantly upregulated during all treatments. Both genes are involved in the cell cycle progression and are transcribed in the gonadal region, where stem cells are highly represented. Based on a substantial increase in gene expression of heat shock proteins (HSP) and their high activity in the gonadal region, we hypothesize that these proteins are key players in the protection of stem cells against external stresses. Apart from the strong HSP induction, other protective processes including cell division, apoptosis and anti-oxidative defence, were also activated. We, therefore, conclude that the protection of stem cells against external stressors may be based on the interplay between stem cell maintenance, i.e. repair and recovery through division, on one hand and apoptosis on the other hand. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1217-1228, 2016.

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano.

The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell-fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ∼75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.

The phylogenetic position of the Loimoidae Price, 1936 (Monogenoidea: Monocotylidea) based on analyses of partial rDNA sequences and morphological data.

Phylogenetic analyses of partial sequences of 18S and 28S rDNA of some monogenoids, including monocotylids and a specimen of Loimosina sp. collected from a hammerhead shark off Brazil, indicated that the Loimoidae (as represented by the specimen of Loimosina sp.) represents an in-group taxon of the Monocotylidae. In all analyses, the Loimoidae fell within a major monocotylid clade including species of the Heterocotylinae, Decacotylinae, and Monocotylinae. The Loimoidae formed a terminal clade with two heterocotyline species, Troglocephalus rhinobatidis and Neoheterocotyle rhinobatis, for which it represented the sister taxon. The following morphological characters supported the clade comprising the Loimoidae, Heterocotylinae, Decacotylinae and Monocotylinae: single vagina present, presence of a narrow deep anchor root, and presence of a marginal haptoral membrane. The presence of cephalic pits was identified as a putative synapomorphy for the clade (Loimoidae (T. rhinobatidis, N. rhinobatis)). Although rDNA sequence data support the rejection of the Loimoidae and incorporating its species into the Monocotylidae, this action was not recommended pending a full phylogenetic analysis of morphological data.

Meiosis-I in Mesostoma ehrenbergii spermatocytes includes distance segregation and inter-polar movements of univalents, and vigorous oscillations of bivalents.

In this article, we describe meiosis-I in spermatocytes of the free-living freshwater flatworm Mesostoma ehrenbergii. The original observations of Oakley (1983, 1985) and Fuge (Eur J Cell Biol 44:294-298, 1987, Cell Motil Cytoskeleton 13:212-220, 1989, Protoplasma 160:39-48, 1991), the first to describe these cells, challenge our understanding of cell division, and we have expanded on these descriptions with the aim of laying the framework for further experimental work. These cells contain three bivalents and four univalent chromosomes (two pairs). Bivalent kinetochores oscillate vigorously and regularly throughout prometaphase, for up to several hours, until anaphase. Anaphase onset usually begins in the middle of the kinetochore oscillation cycle. Precocious cleavage furrows form at the start of prometaphase, ingress and then remain arrested until the end of anaphase. The four univalents do not pair, yet by anaphase there is one of each kind at each pole, an example of "distance segregation" (Hughes-Schrader in Chromosoma 27:109-129, 1969). Until proper segregation is achieved, univalents move between spindle poles up to seven times in an individual cell; they move with velocities averaging 9 µm/min, which is faster than the oscillatory motions of the bivalent kinetochores (5-6 µm/min), and much faster than the anaphase movements of the segregating half-bivalents (1 µm/min). Bipolar bivalents periodically reorient, most often resulting in the partner kinetochores exchanging poles. We suggest that the large numbers of inter-polar movements of univalents, and the reorientations of bivalents that lead to partners exchanging poles, might be because there is non-random segregation of chromosomes, as in some other cell types.

Methods for rearing Mesostoma ehrenbergii in the laboratory for cell biology experiments, including identification of factors that influence production of different egg types.

Mesostoma ehrenbergii spermatocytes are uniquely useful to study various aspects of cell division. Their chromosomes are large in size and few in number, with only three bivalent and four univalent chromosomes. During prometaphase, bipolar bivalents oscillate regularly to and from the poles for 1-2 hours. The univalents remain at the poles but occasionally move from one pole to the other. In addition, a precocious cleavage furrow forms during prometaphase and remains partially constricted until anaphase. Attempts to rear these animals indefinitely in laboratory conditions, however, have been mostly unsuccessful because of their reproductive strategy. M. ehrenbergii are hermaphroditic flatworms that can produce viviparous offspring (termed S eggs) and/or diapausing eggs (termed D eggs) and they follow either one of two reproductive patterns: (1) they first form S eggs and following the delivery of these eggs produce D eggs, or (2) they only produce D eggs. When only D eggs are formed, which is common under laboratory conditions, the stocks die out until the D eggs hatch, which is irregular and creates unpredictable wait times. Consequently, in order to maintain M. ehrenbergii stocks to study their spermatocytes, we examined various factors that might influence egg-type production. Feeding them daily and keeping them at 25°C favours S egg production. Currently, our cultures have reached the 53rd generation. We herein describe our rearing and dissection methods, and some experiments which led to our present rearing methods.

Mesostoma ehrenbergii spermatocytes--a unique and advantageous cell for studying meiosis.

Mesostoma ehrenbergii have a unique male meiosis: their spermatocytes have three large bivalents that oscillate for 1-2 h before entering into anaphase without having formed a metaphase plate, have a precocious ('pre-anaphase') cleavage furrow, and have four univalents that segregate between spindle poles without physical interaction between them, that is via 'distance segregation'. These unique and unconventional features make Mesostoma spermatocytes an ideal organism for studying the force produced by the spindle to move chromosomes, and to study cleavage furrow control and 'distance segregation'. We review the literature on meiosis in Mesostoma spermatocytes and describe our current research with Mesostoma spermatocytes, rearing the animals in the laboratory using methods that described in our companion article [Hoang et al. (2013); Cell Biol Int].

The Hippo pathway regulates stem cells during homeostasis and regeneration of the flatworm Macrostomum lignano.

The Hippo pathway orchestrates activity of stem cells during development and tissue regeneration and is crucial for controlling organ size. However, roles of the Hippo pathway in highly regenerative organisms, such as flatworms, are unknown. Here we show that knockdown of the Hippo pathway core genes in the flatworm Macrostomum lignano affects tissue homeostasis and causes formation of outgrowths through hyperproliferation of stem cells (neoblasts), and leads to disruption of allometric scaling during regeneration and increased size of regenerated parts. We further show that Yap, the downstream effector of the Hippo pathway, is a potential neoblast marker gene, as it is expressed in dividing cells in M. lignano and is essential for neoblast self-renewal. The phenotypes we observe in M. lignano upon knockdown of the Hippo pathway core genes and Yap are consistent with the known functions of the pathway in other model organisms and demonstrate that the Hippo pathway is functionally conserved between flatworms and mammals. This work establishes M. lignano as a productive model for investigation of the Hippo pathway.

Measurement of S-phase duration of adult stem cells in the flatworm Macrostomum lignano by double replication labelling and quantitative colocalization analysis.

Platyhelminthes are highly attractive models for addressing fundamental aspects of stem cell biology in vivo. These organisms possess a unique stem cell system comprised of neoblasts that are the only proliferating cells during adulthood. We have investigated Ts (S-phase duration) of neoblasts during homoeostasis and regeneration in the flatworm, Macrostomum lignano. A double immunohistochemical technique was used, performing sequential pulses with the thymidine analogues CldU (chlorodeoxyuridine) and IdU (iododeoxyuridine), separated by variable chase times in the presence of colchicine. Owing to the localized nature of the fluorescent signals (cell nuclei) and variable levels of autofluorescence, standard intensity-based colocalization analyses could not be applied to accurately determine the colocalization. Therefore, an object-based colocalization approach was devised to score the relative number of double-positive cells. Using this approach, Ts (S-phase duration) in the main population of neoblasts was ∼13 h. During early regeneration, no significant change in Ts was observed.

Proliferation pattern during rostrum regeneration of the symbiotic flatworm Paracatenula galateia: a pulse-chase-pulse analysis.

The remarkable totipotent stem-cell-based regeneration capacities of the Platyhelminthes have brought them into the focus of stem cell and regeneration research. Although selected platyhelminth groups are among the best-studied invertebrates, our data provide new insights into regenerative processes in the most basally branching group of the Platyhelminthes, the Catenulida. The mouth- and gutless free-living catenulid flatworm Paracatenula galateia harbors intracellular bacterial symbionts in its posterior body region, the trophosome region, accounting for up to 50% of the volume. Following decapitation of this flatworm, we have analyzed the behavior of the amputated fragments and any anterior and posterior regeneration. Using an EdU-pulse-chase/BrdU-pulse thymidine analog double-labeling approach combined with immunohistochemistry, we show that neoblasts are the main drivers of the regeneration processes. During anterior (rostrum) regeneration, EdU-pulse-chase-labeled cells aggregate inside the regenerating rostrum, whereas BrdU pulse-labeling before fixation indicates clusters of S-phase neoblasts at the same position. In parallel, serotonergic nerves reorganize and the brain regenerates. In completely regenerated animals, the original condition with S-phase neoblasts being restricted to the body region posterior to the brain is restored. In contrast, no posterior regeneration or growth of the trophosome region in anterior fragments cut a short distance posterior to the brain has been observed. Our data thus reveal interesting aspects of the cellular processes underlying the regeneration of the emerging catenulid-bacteria symbiosis model P. galateia and show that a neoblast stem cell system is indeed a plesiomorphic feature of basal platyhelminths.