Freshwater Triclads Research Articles

Planarian Mucus: A Novel Source of Pleiotropic Cytotoxic and Cytostatic Agents against Cancer Cells.

Biological evolution has generated a vast array of natural compounds produced by organisms across all domains. Among these, secondary metabolites, selected to enhance an organism's competitiveness in its natural environment, make them a reservoir for discovering new compounds with cytotoxic activity, potentially useful as novel anticancer agents. Slime secretions, the first barrier between epithelial surfaces and the surrounding environment, frequently contain cytotoxic molecules to limit the growth of parasitic organisms. Planarians, freshwater Triclads, continuously secrete a viscous mucus with multiple physiological functions. The chemical composition of planarian mucus has been only partially elucidated, and there are no studies reporting its cytotoxic or cytostatic effects. In this study, we developed a protocol for collecting mucus from Dugesia japonica specimens and we demonstrated that it inhibits the growth of cancer cells by activating cytostatic and ROS-dependent cytotoxic mechanisms inducing lipid droplet accumulation and mitochondrial membrane reorganization. Although further research is needed to identify the specific chemicals responsible for the anticancer activity of planarian mucus, this work opens up numerous research avenues aimed at better understanding the mechanisms of action of this product for potential therapeutic applications.

Current state of knowledge on freshwater planarians (Platyhelminthes, Tricladida, Dugesiidae) from Chile

The unique geography of Chile encompasses a wide diversity of ecosystems and a rich biodiversity. However, the platyhelminth fauna has been poorly studied. The aim of this work is to compile the historical record of freshwater planarians described for this country. We accessed worldwide databases and published articles to provide a comprehensive review of their discovery history, morphological characteristics and their localities. Freshwater planarians have been collected mainly in central and southern Chile, while in the northern region a single species has been described. The discovery of new species of freshwater triclads has the potential to reveal novel animal models to study regeneration and/or biological adaptations, as some species are suitable for culture in the laboratory. We discuss the many reasons why further research is needed for this animal group, which should include genomic and molecular genetic studies.

Under threat: Two new troglobitic species of Girardia (Platyhelminthes: Tricladida) from sandstone and limestone caves in Brazil

The diversity of freshwater triclads of the suborder Continenticola in the Neotropical region is poorly known, with few studies in South America. In this work, we describe two new cave-dwelling species of Girardia: G. corumbataiensis sp. nov. is described from a single sandstone cave located in an area covered by semi-deciduous vegetation, southeastern Brazil; G. nobresis sp. nov. is described from a single flooded limestone cave located in an area of Cerrado vegetation, central-western Brazil. Specimens of G. corumbataiensis sp. nov. have a slight pigmented dorsal surface with highly triangular head and two eyes, whereas specimens of G. nobresis sp. nov. lack body pigmentation and eyes and have pointed auricles. Girardia corumbataiensis sp. nov. shows a pear-shaped bulbar cavity with forked portions turned dorsally and a slightly bent bursal canal. Girardia nobresis sp. nov. has an ample bulbar cavity with numerous folds and an angled bursal canal. The two new species are categorized as troglobites and easily recognized by a unique combination of characters of their external morphology and copulatory apparatus. G. corumbataiensis sp. nov. occurs in a cave inserted in a protected area, but no effective action for preservation has been provided. This species is abundant in a single stream crossing the cave, which is subject to intensive visitation and control of bat populations, besides Eucalyptus plantations nearby. The type-locality of G. nobresis sp. nov., a single flooded cave near a municipal road, has no legal protection. There are impacts, such as intensive visitation for fishing and deforestation in its surroundings.

Enhancing the still scattered knowledge on the taxonomic diversity of freshwater triclads (Platyhelminthes: Dugesiidae) in caves from two Brazilian Biomes

ABSTRACT Recent studies have recorded new species of freshwater planarians in caves of the biomes Amazonia, Atlantic Forest, Cerrado, and Caatinga. Herein we contribute to enhancing this knowledge by describing three new cave-dwelling species of Girardia from three different cave systems situated in two biomes (Atlantic Forest and Cerrado) in eastern Brazil. Girardia spelaea sp. n., from a limestone cave, is eyeless and shows a whitish body. The other two species, G. asymmetrica sp. n. and G. ibitipoca sp. n., from limestone and quartzite caves, show pigmented bodies and eyes. The three species are characterized by dorsal and/or dorsoventral testes and a bulbar cavity with forked ental portions, varying from round or ovoid to elongate. Each new species is easily recognized by a unique combination of characters of their external morphology and reproductive system. Girardia spelaea is probably a troglobitic species and the two other species may be trogrophiles, all of them with a restricted known distribution. urn:lsid:zoobank.org:act:AC4F9F3C-EF6D-4BB8-ACBA-EB5812036314 urn:lsid:zoobank.org:act:6BD22CDA-99E7-46DC-98D6-8FD1ECB46320 urn:lsid:zoobank.org:act:C5D97034-9F1A-4020-9635-DE76202A85C1

Efficient depletion of ribosomal RNA for RNA sequencing in planarians

BackgroundThe astounding regenerative abilities of planarian flatworms prompt steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed.ResultsIn this study, we describe a workflow for the efficient depletion of rRNA in the planarian model species S. mediterranea. Our protocol is based on subtractive hybridization using organism-specific probes. Importantly, the designed probes also deplete rRNA of other freshwater triclad families, a fact that considerably broadens the applicability of our protocol. We tested our approach on total RNA isolated from stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consistent with poly(A) libraries. However, ribodepleted libraries revealed higher transcript levels for transposable elements and histone mRNAs that remained underrepresented in poly(A) libraries. As neoblasts experience high transposon activity this suggests that ribodepleted libraries better reflect the transcriptional dynamics of planarian stem cells. Furthermore, the presented ribodepletion procedure was successfully expanded to the removal of ribosomal RNA from the gram-negative bacterium Salmonella typhimurium.ConclusionsThe ribodepletion protocol presented here ensures the efficient rRNA removal from low input total planarian RNA, which can be further processed for RNA-seq applications. Resulting libraries contain less than 2% rRNA. Moreover, for a cost-effective and efficient removal of rRNA prior to sequencing applications our procedure might be adapted to any prokaryotic or eukaryotic species of choice.

Flatworms like it round: nematode consumption by Planaria torva (Müller 1774) and Polycelis tenuis (Ijima 1884)

The aim of this study was to enhance current knowledge of thus far largely neglected meiofaunal–macrofaunal trophic channels in freshwater ecosystems. The strength and shape (functional response) of the predator–prey interaction between two freshwater triclad flatworm species (Polycelis tenuis and Planaria torva) and individuals of the nematode species Caenorhabditis elegans were measured in a set of laboratory experiments. We hypothesized that feeding on adult nematodes results in a hyperbolic type II, whereas juvenile prey led to sigmoidal type III functional response and that different habitat textures would affect predation success by providing refuge for prey. However, our results revealed that both flatworm species exhibited type III sigmoidal functional response curves and the smallest predator consistently ingested larger amounts of nematodes. Generally, our results suggest the existence of a strong predator–prey interaction, given that both flatworms daily ingested a significant proportion of their biomass by feeding only on nematodes (up to 30% for P. tenuis). However, P. torva was unable to process nematodes in sandy sediment, and the ingestion rates of both flatworm species were reduced in a complex litter habitat.

Comparative and Transcriptome Analyses Uncover Key Aspects of Coding- and Long Noncoding RNAs in Flatworm Mitochondrial Genomes.

Exploiting the conservation of various features of mitochondrial genomes has been instrumental in resolving phylogenetic relationships. Despite extensive sequence evidence, it has not previously been possible to conclusively resolve some key aspects of flatworm mitochondrial genomes, including generally conserved traits, such as start codons, noncoding regions, the full complement of tRNAs, and whether ATP8 is, or is not, encoded by this extranuclear genome. In an effort to address these difficulties, we sought to determine the mitochondrial transcriptomes and genomes of sexual and asexual taxa of freshwater triclads, a group previously poorly represented in flatworm mitogenomic studies. We have discovered evidence for an alternative start codon, an extended cox1 gene, a previously undescribed conserved open reading frame, long noncoding RNAs, and a highly conserved gene order across the large evolutionary distances represented within the triclads. Our findings contribute to the expansion and refinement of mitogenomics to address evolutionary issues in this diverse group of animals.

Two new species of freshwater flatworms (Platyhelminthes: Tricladida:Continenticola) from South American caves.

The diversity of freshwater triclads in the Neotropical region is considered to be low, but extensive areas of South America remain almost unexplored. Herein we describe two cave-dwelling, new species of Girardia, one from a transition zone of the Cerrado and Caatinga phytophysiognomies and the other from the Cerrado phytophysiognomy. The species from the Cerrado-Caatinga transition is a troglobite, eyeless and whitish; the species from the Cerrado area is possibly a troglophile, since it shows heavily pigmented body and eyes. Each species is easily recognized by a unique combination of features in its external morphology and copulatory apparatus. The two new species of Girardia show a restricted distribution, even the troglophile, and occur in caves without legal protection. Therefore, they must be considered as vulnerable organisms in a conservation context.

A key to the freshwater triclads (Platyhelminthes, Tricladida) of Herzegovina watercourses

The aim of this paper is to describe morphological characteristics of freshwater triclads in Herzegovina and to provide a key for their identification based mainly on their external morphology. Our research is based on the freshwater triclads collected at 26 sites in Herzegovina, including 10 rivers, 6 springs and 1 lake. Triclads were collected by hand and with the bait jar with a lid bearing many small perforations. Specimens were identified immediately after being collected or we transported them to the laboratory in termal containers with ice. The morphological features used for species identification in this key are: body coloration (color of the dorsal and ventral side), the size of the pharynx, the presence of tentacles and their location, number and position of the eyes in respect to each other and the body margin, and the shape of the head. Specimens were fixed using Steinmann’s fluid and conserved in 70 % alcohol. Inner morphological feature, shape of a penis, was used in determination of Polycelis tenuis and Polycelis nigra. We collected a total of 11 species, belonging to 8 genera (Polycelis Ehrenberg, 1831, Crenobia Kenk, 1930, Phagocata Leidy, 1847, Planaria Müller, 1776, Dendrocoelum Örsted, 1844, Schmidtea Ball, 1974, Dugesia Girard, 1850, Girardia Ball, 1974) and 3 families. The present study forms a baseline for future studies on the diversity and biogeography of Herzegovinian freshwater triclads.

A key to the freshwater triclads (Platyhelminthes, Tricladida) of Herzegovina watercourses

The aim of this paper is to describe morphological characteristics of freshwater triclads in Herzegovina and to provide a key for their identification based mainly on their external morphology. Our research is based on the freshwater triclads collected at 26 sites in Herzegovina, including 10 rivers, 6 springs and 1 lake. Triclads were collected by hand and with the bait jar with a lid bearing many small perforations. Specimens were identified immediately after being collected or we transported them to the laboratory in termal containers with ice. The morphological features used for species identification in this key are: body coloration (color of the dorsal and ventral side), the size of the pharynx, the presence of tentacles and their location, number and position of the eyes in respect to each other and the body margin, and the shape of the head. Specimens were fixed using Steinmann’s fluid and conserved in 70 % alcohol. Inner morphological feature, shape of a penis, was used in determination of Polycelis tenuis and Polycelis nigra. We collected a total of 11 species, belonging to 8 genera (Polycelis Ehrenberg, 1831, Crenobia Kenk, 1930, Phagocata Leidy, 1847, Planaria Muller, 1776, Dendrocoelum Orsted, 1844, Schmidtea Ball, 1974, Dugesia Girard, 1850, Girardia Ball, 1974) and 3 families. The present study forms a baseline for future studies on the diversity and biogeography of Herzegovinian freshwater triclads.

Integrative taxonomy of a new species of planarian from the Lake Ohrid basin, including an analysis of biogeographical patterns in freshwater triclads from the Ohrid region (Platyhelminthes, Tricladida, Dugesiidae)

A new species of the genus Dugesia is described from the Lake Ohrid region in the western part of the Balkan Peninsula, forming the first fully documented species description for this genus in the Ohrid area. The morphological species delimitation is supported by complementary molecular, karyological, and cytogenetic data available from the literature. Therefore, species delineation is based on a truly integrative approach. Further, a short account on the degree of freshwater planarian endemicity in the Ohrid region is provided.

NEW PLANARIANS (TURBELLARIA, TRICLADIDA) FROM ISRAEL AND LEBANON

ABSTRACT Two new species of freshwater triclads belonging to the Dugesia gonocephala species group are described, D. golanica n.sp. from northern Israel and D. libanica n.sp. from Becharre in northern Lebanon. D. golanica is characterized by a large, solid, bilobed adenodactyl positioned dorsally and to the right of the pointed penis papilla, while D. libanica has a large, solid, conical-shaped adenodactyl situated dorsally and to the right of the asymmetrical, finger-shaped penis papilla. These two species are clearly related to each other and to D. iranica (Livanov) from Iran and Turkey.

A new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from the Afromontane forest in South Africa, with an overview of freshwater planarians from the African continent

A new species of the genus Dugesia from the Amatola Mountains in the Eastern Province of South Africa is described,including a karyological account and notes on its life cycle and reproductive modes. The new species differs from itscongeners in a unique combination of morphological characters of the copulatory apparatus, in particular the centralcourse of the ejaculatory duct with its terminal opening at the tip of the penis papilla, the elongated seminal vesicle, theasymmetrical openings of the oviducts into the bursal canal, and the openings of vasa deferentia at about halfway alongthe seminal vesicle. In addition, an overview is provided of all freshwater triclads reported from the African continent including karyological information and notes on reproductive modes.

Ultrastructure of spermatogenesis and mature spermatozoon of the freshwater planarian Schmidtea mediterranea (Platyhelminthes, Paludicola)

The aim of this study was to characterize for the first time spermatogenesis and spermiogenesis in the freshwater planarian Schmidtea mediterranea using both light and electron microscopy. Starting from the border towards the testis lumen we found types I and II spermatogonia, clusters of primary and secondary spermatocytes, spermatids and free spermatozoa. Light microscope observations show that type I spermatogonia have a large and pale nucleus whereas type II spermatogonia are significantly smaller than the one of type I, and show a darker and central bulky nucleus. At the ultrastructure level, both type I and type II spermatogonia are characterized by a wide nucleus with scanty cytoplasm containing free ribosomes, mitochondria and a dense chromatoid body whereas endoplasmic reticulum and Golgi complex were not observed. The cytoplasm of primary and secondary spermatocytes displays numerous free ribosomes and many endoplasmic reticulum cisternae and Golgi complexes, suggesting that the development of these organelles during spermatogenesis might contribute to the synthesis of hormones and proteins such as testosterone, transcription factors and tubulin. Mature spermatozoa structure closely resembles those of other freshwater triclads with a nucleus, a single fused mitochondrion, a row of cortical microtubules and a pair of flagella conforming to the 9+’1′ microtubule pattern described for other Platyhelminthes.

Freshwater planarians (Platyhelminthes, Tricladida) from the Iberian Peninsula and Greece: diversity and notes on ecology

Few studies have examined the diversity of freshwater planarians in the Iberian Peninsula and Greece. We have searched extensively for specimens, mainly in the Iberian Peninsula, and have gathered information on their taxonomy and biogeography. Two new species of Dendrocoelum and six new species of Phagocata are described. We also review the status of five other species and present distribution maps that summarize records of freshwater planarians in the Iberian Peninsula. The diversity of three ecological groups of freshwater planarians in this area is discussed and we show that it has a rich, stream-dwelling freshwater triclad fauna, contrary to the findings of earlier studies. But our findings support the traditional viewpoint that European lake-dwelling species are scarce in Spain. The distribution pattern of Girardia tigrina is also discussed. We conclude that the critical status of Schmidtea mediterranea in this area is partly due to habitat alteration through human interference.

Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola)

The embryonic development of freshwater triclads is mainly known from studies of species of Dendrocoelum, Planaria, Polycelis, and, more recently, Schmidtea. The present study characterizes the development of Girardia tigrina (Girard, 1850) by means of optical microcopy using glycol methacrylate semi-thin sections. 94 cocoons were collected in the period from laying to hatching, with intervals of up to twenty-four hours. The sequence of morphological changes occurring in the embryo permitted the identification of nine embryonic stages. At the time of cocoon laying, numerous embryos were dispersed among many yolk cells, with a rigid capsule covering the entire cocoon. In the first stage (approx. up to 6 hours after cocoon laying), yolk cells and embryonic cells showed random distribution. Stage II (between 12 and 24 hours after cocoon laying) is characterized by aggregates of blastomeres, which later aggregate forming an enteroblastula. Approximately 2 days after cocoon laying (stage III), formation of the embryonic epidermis and embryonic digestive system took place, the latter degenerating during the subsequent stage. Stage V (until the fourth day) is characterized by the formation of the definitive epidermis. Between 4 and 6 days after laying, organogenesis of the definitive inner organs starts (stage VI). Approximately 14 days after laying (stage IX), formation of the nervous system is completed. At this stage, the embryo shows similar characteristics to those of newly hatched juveniles. The hatching of Girardia tigrina occurs in the period between twelve to twenty-two days after cocoon laying.

Freshwater Triclad Planarians (Turbellaria) from Plummers Island, Maryland

Five species of freshwater triclad planarians were found on Plummers Island and the adjacent mainland property, Montgomery County, Maryland. One species (Dugesia [G.] tigrina) occupying the Potomac River and a tributary, is tolerant of degraded habitat. The other four occupy vernal pools (Hymanella retenuova and Phagocata velata) or spring-seeps (Phagocata morgani and Paraplanaria dactyligera) and appear to be indicators of high quality aquatic habitat. These five species represent 36% of the total known Maryland fauna of 14 species.

Chromosome polymorphism and complements in populations of Girardia species (Platyhelminthes, Tricladida, Paludicola) from southern Brazil

The karyotypes of four species of freshwater triclads of the genus Girardia (Platyhelminthes), i.e. G. schubarti, G. tigrina, G. anderlani, and G. biapertura, from populations of different localities of the Rio Grande do Sul State, in southern Brazil, were analyzed. The karyotype of G. biapertura is presented for the first time. Three basic complements of 4, 8, and 9 chromosomes were found. Diploids, triploids, or mixoploids (2n/3n) specimens were frequently detected in these populations. The basic chromosomal complement of n=9 was verified in two different species (G. biapertura and G. anderlani), presenting a large acrocentric chromosome which is rare in the family Dugesiidae. An intra and interspecific chromosomal variability was also detected and its evolutionary implications are discussed.

Ultrastructural and cytochemical aspects of the female gonad of Geoplana burmeisteri (Platyhelminthes, Tricladida, Terricola)

The ultrastructure of the female gonad of the land planarian Geoplana burmeisteri was investigated by means of electron microscopy and cytochemical techniques. It consists of two small germaria located ventral to the intestine and of two irregular, lateral rows of vitelline follicles, both enveloped by a tunica composed of an extracellular lamina and an inner sheath of accessory cells. Accessory cell projections completely surround developing oocytes and vitellocytes. The main feature of oocyte maturation is the appearance of chromatoid bodies and the development of the rough endoplasmic reticulum (RER) and Golgi complexes. These organelles appear to be correlated with the production of egg inclusions of medium electron density, about 1.5-1.8 microm in diameter, which remain scattered in the ooplasm of mature oocytes. On the basis of cytochemical tests demonstrating their glycoprotein composition, these inclusions were interpreted as residual yolk globules. Vitellocytes are typical secretory cells with well-developed RER and Golgi complexes that are mainly involved in the production of yolk globules and eggshell globules, respectively. Eggshell globules appear to arise from repeated coalescence of small Golgi-derived vesicles and, at an intermediate stage of maturation, show a multigranular pattern. Later, after vesicle fusion, they reach a diameter of 1.3-1.6 microm when completely mature and show a meandering/concentric pattern, as is typical of the situation seen in most Proseriata and Tricladida. The content of yolk globules is completely digested by pronase, while the content of eggshell globules is unaffected. Mature vitellocytes contain, in addition, a large quantity of glycogen and lipid droplets as further reserve material. On the basis of the ultrastructural characteristics of the female gonad described above and in relation to the current literature, we conclude that G. burmeisteri appears to be more closely related to the freshwater triclads, in particular to members of the Dugesiidae, than to the marine triclads.

Histological and histochemical aspects of the penial glands of Girardia biapertura Sluys, 1997 (Platyhelminthes, Tricladida, Paludicola).

Girardia biapertura was described with sperm ducts penetrating the penis bulb, subsequently opening separately at the tip of the penis papilla and receiving the abundant secretion of penial glands. In the present work, the penial glands of this species have been histologically and histochemically analysed, and four types of secretory cells are distinguished. The openings of the penial glands into the intrabulbar and intrapapillar sperm ducts, designated here as intrapenial ducts, allow for the distinction between three histologically differentiated regions. The most proximal region possibly corresponds to the bulbar cavity of other freshwater triclads whereas the median and distal portions correspond to the ejaculatory duct. The proximal region of the intrapenial ducts receives mainly the openings of a secretory cell type (type I) that produces a proteinaceous secretion. A second type of secretory cell (type II) that secretes neutral mucopolyssacharides opens into the median region of the intrapenial ducts. The distal portion of the ducts receives two types of secretory cells (types III and IV) which secret glycoprotein and glycosaminoglycans, respectively. Types III and IV open also directly into the male atrium through the epithelium of the penis papilla. A comparison with the results presented here and those of other authors for species of Girardia is provided and the importance of the study of the penial glands for taxonomic characterisation of freshwater triclads is emphasised.