Freshwater Planarian Species Research Articles

Two New Species of Freshwater Planarian (Platyhelminthes, Tricladida, Dugesiidae, Dugesia) from Southern China Exhibit Unusual Karyotypes, with a Discussion on Reproduction in Aneuploid Species

Two new species of the genus Dugesia from Southern China are described by applying an integrative approach, including morphological, karyological, histological, and molecular information. In the molecular phylogenetic tree, the two new species, Dugesia pendula Chen & Dong, sp. nov. and Dugesia musculosa Chen & Dong, sp. nov., fall into an Eastern Palearctic/Oriental clade and an Oriental/Australasian clade, respectively, while sharing only a rather distant relationship. The separate specific status of the two new species is supported also by their genetic distances. Dugesia pendula is characterized by the following features: symmetrical openings of the oviducts into the bursal canal, a duct between seminal vesicle and diaphragm, small diaphragm, dorsally located seminal vesicle, a penis papilla suspended from the dorsal wall of the male atrium, and mixoploid karyotype with diploid complements of 2n=2x=14+0−1 B-chromosome and triploid complements of 2n=3x=21+0−1 B-chromosome, with all chromosomes being metacentric. Dugesia musculosa is characterized by the following features: asymmetrical openings of the oviducts into the bursal canal; a ventrally displaced ejaculatory duct with a terminal opening; two diaphragms; a bursal canal provided with a strong, thick layer of circular muscle, which extends from the copulatory bursa to the common atrium and gonoduct; the left vas deferens opening at the midlateral wall of the seminal vesicle, while the right sperm duct opens at the dorsolateral wall of the seminal vesicle; and karyotype consisting of complicated diploid and aneuploid mosaicism, with diploid complements of 2n=2x=16 and 2n=2x=16−17th-18th, with all chromosomes being metacentric. The uncommon karyotypes, combined with the asexual reproduction of aneuploid animals, are evaluated in the context of the relationship between ploidy levels and reproductive modalities in the genus Dugesia.

PLANARIANS (PLATYHELMINTHES: TRICLADIDA) OF THE SIKHOTEALIN NATURE RESERVE (PRIMORSKY KRAI)

An annotated list of the one terrestrial and three freshwater planarian species from the Sikhote-Alin Nature Reserve is given. The family Geoplanidae, two genera, Polycelis Ehrenberg, 1831 and Diversibipalium Kawakatsu, Ogren, Froehlich et Sasaki, 2002, and three species, Phagocata sibirica (Zabusov, 1903), Polycelis schmidti (Zabusov, 1916) and Diversibipalium sp. are recorded from reserve for the first time. The finding of the terrestrial hammerhead planarians genus Diversibipalium in the Sikhote-Alin Nature Reserve is most north record of the East-Asian subfamily Bipaliinae (Geoplanidae).

The complete mitochondrial genome of a planarian flatworm Girardia tigrina (Tricladida: Dugesiidae)

Girardia tigrina, a freshwater planarian species native to America and introduced to other continents, has been usually used as model organism in many research fields of biology. In this study, we determined the complete mitochondrial genome of G. tigrina using next-generation sequencing (NGS). The complete mitogenome was 15,938 bp in length, with 36 genes, including 12 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNAs, and ATP8 was absent in the mitogenome of G. tigrina as in the mitogenomes of some other flatworms. The maximum-likelihood phylogenetic tree suggested that G. tigrina was closely related to genus Dugesia in the clade of Tricladida.

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

Dugesia hepta and Dugesia benazzii (Platyhelminthes: Tricladida): two sympatric species with occasional sex?

Dugesia hepta Pala, Casu & Vacca, 1981 and Dugesia benazzii Lepori, 1951 are two freshwater planarian species from the islands of Corsica and Sardinia. Dugesia hepta is endemic of Sardinia and distributed in four northern hydrographic basins where it co-occurs with D. benazzii, which has a wider Tyrrhenian distribution. Although these species have been broadly studied—especially D. benazzii—as regards to their variety of reproductive patterns as well as for their karyological diversity, little is known about them from a molecular phylogenetic perspective. For the first time, we present a molecular phylogenetic tree of the two species and their populations based on two molecular markers—one mitochondrial, Cox1, and one nuclear, Dunuc12. Our results not only confirm that both species are molecularly distinct but also show that D. benazzii’s Corsican and Sardinian populations could belong to separate species. Furthermore, we present the first demonstration of a natural hybridization between different species in the genus Dugesia on the basis of molecular data.

First record of freshwater planarian Girardia tigrina (Platyhelminthes, Tricladida, Continenticola) in Eastern Europe.

The genus Girardia Ball, 1974 houses freshwater planarian species and is the second after Dugesia Girard, 1850 within Dugesiidae regarding the number of species. It contains 45 species (Tyler et al. 2006-2019) including three recently described: one from China-Girardia sinensis Chen Wang 2015 (Chen et al. 2015), and two cave-dwelling planarians from Brasil-Girardia desiderensis Souza Leal-Zanchet 2016 and Girardia pierremartini Souza Leal-Zanchet 2016 (Souza et al. 2016).

Two new species of marine flatworm from southern China facilitate determination of the phylogenetic position of the genus Nerpa Marcus, 1948 and the histochemical structure of the nervous system in the genus Paucumara Sluys, 1989 (Platyhelminthes, Tricladida, Maricola).

Two new species of flatworm, collected from a beach at eastern Shenzhen, China, were studied through an integrative approach by combining morphological, histological, histochemical (acetylcholinesterase, AChE), and molecular (18S r- DNA) data. These species belong to two genera of marine triclads, previously unrecorded from China, viz. Nerpa Marcus, 1948 and Paucumara Sluys, 1989. Nerpa fistulata Wang Chen, sp. nov. is characterized by: transparent body; principally pentamerous intestine with three distinct commissures; two very large, prepharyngeal testis follicles; a semi-circular lens in each eye cup; a penis papilla provided with a chitinized, pointed stylet; lateral bursae communicating with the oviduct and opening ventrally to the exterior via a duct. Phylogenetically N. fistulata groups with one member of the family Bdellouridae. This new, Chinese species of Nerpa introduces a major geographic disjunction, as the type species N. evelinae was described from the bay of Santos, Brazil, so that the genus is now known from both Atlantic as well as Pacific coasts. The species Paucumara falcata Wang Li, sp. nov. is characterized by: three distinct pale yellow transverse pigmentation bands on its dorsal side, between which some snowflake-like specks are randomly distributed, and a brown transverse band anteriorly to the eyes; 8-11 testicular follicles on either side of the body, the follicles extending from immediately behind the ovaries to half-way along the pharyngeal pocket; a musculo-parenchymatic organ with a sclerotic, curved tip projecting from the anterior wall of the male atrium, ventrally to the root of the penis papilla. Phylogenetically P. falcata groups with its congener P. trigonocephala, with the genus Paucumara forming the sister taxon of the genus Ectoplana. Comparison of the nerve structure of P. falcata, as revealed by AChE histochemistry, with that of eight other species of triclad suggested that the nervous system of marine planarians is simpler than that of species of freshwater planarians, but revealed also that the nerve structure is rather variable among species. The copulatory position exhibited by two partners in Paucumara falcata is remarkable in that they intertwine, with their heads pointing downwards and the tails pointing upwards, the entire process lasting about 10 min. Such a copulatory position has never before been reported for triclad flatworms.

An intriguing, new planarian species from Tasmania, with a discussion on protandry in triclad flatworms (Platyhelminthes, Tricladida)

AbstractAn account is given of an unusual new species of freshwater planarian from the Hartz Mountains in Tasmania, Australia, Romankenkius flaccidus Sluys, sp. nov. The species is characterized, among other features, by an asymmetrical penis papilla, an extremely large, elongated copulatory bursa, and by the absence of testes in animals with fully developed male and female copulatory apparatus. Instigated by the sexual cycle of this new species, the study opportunely reviews whether planarian flatworms generally are simultaneous or sequential—in particular protandrous—hermaphrodites. It is concluded that real protandry does not occur in triclads and that even less extreme cases of sexual segregation, such as complete reduction of the testes or the more or less complete separation of male and female functionality as present in some species, have only sparsely and convergently evolved within the group of the triclad flatworms.

Quantification of Neurotransmitters from Intact and Regenerating Planarians Using UHPLC-MS/SRM Method.

Freshwater planarian species S. mediterranea is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. It is one of the best model systems available to address the basic biological mechanisms in the regeneration processes. Absolute quantification of metabolites from planarians is imperative to understand their role in the regeneration processes. Here we describe a stable isotope dilution ultrahigh performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC-MS/SRM) assay for a sensitive and quantitative assessment of neurotransmitters (NTs) in planaria. We used this method for the simultaneous quantification of 16 NTs from both intact and regenerating planarians.

Coordination of size-control, reproduction and generational memory in freshwater planarians

Uncovering the mechanisms that control size, growth, and division rates of organisms reproducing through binary division means understanding basic principles of their life cycle. Recent work has focused on how division rates are regulated in bacteria and yeast, but this question has not yet been addressed in more complex, multicellular organisms. We have, over the course of several years, assembled a unique large-scale data set on the growth and asexual reproduction of two freshwater planarian species, Dugesia japonica and Girardia tigrina, which reproduce by transverse fission and succeeding regeneration of head and tail pieces into new planarians. We show that generation-dependent memory effects in planarian reproduction need to be taken into account to accurately capture the experimental data. To achieve this, we developed a new additive model that mixes multiple size control strategies based on planarian size, growth, and time between divisions. Our model quantifies the proportions of each strategy in the mixed dynamics, revealing the ability of the two planarian species to utilize different strategies in a coordinated manner for size control. Additionally, we found that head and tail offspring of both species employ different mechanisms to monitor and trigger their reproduction cycles. Thus, we find a diversity of strategies not only between species but between heads and tails within species. Our additive model provides two advantages over existing 2D models that fit a multivariable splitting rate function to the data for size control: firstly, it can be fit to relatively small data sets and can thus be applied to systems where available data is limited. Secondly, it enables new biological insights because it explicitly shows the contributions of different size control strategies for each offspring type.

Investigation of the ultrastructure of Dendrocoelum constrictum (Platyhelminthes, Tricladida) spermatogenesis and mature spermatozoa

To add to our understanding of dendrocoelid spermatozoa and to describe additional phylogenetic characters, the ultrastructure of the testis was investigated in the subterranean freshwater planarian Dendrocoelum constrictum. This is the first study investigating spermatogenesis and spermatozoon ultrastructure in a subterranean freshwater planarian species. We found that the basic structure of spermatozoa in D. constrictum is similar to that of other Tricladida that have been studied previously. In fact, D. constrictum spermatozoa possess an elongated nucleus, one giant mitochondrion, and two subterminal flagella with a 9+‘1’ pattern. The flagella emerge together from one side of the spermatozoon. However, D. constrictum has some characteristics that have not yet been described for other freshwater planarians. In fact, the number of cortical microtubules reaches the maximum number in the anterior and middle part of region I, and then decrease until they disappear towards the posterior extremity of the spermatozoon. The extreme tip of the anterior region of the spermatozoon exhibits a specific external ornamentation of the plasma membrane.

A new and aberrant species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Madagascar.

In this paper we report a new species of Dugesia of the family Dugesiidae from Madagascar, representing the fourth species of freshwater planarian known from this global biodiversity hotspot. In some respects the new species is aberrant, when compared with its congeners, being characterized by a head with smoothly rounded auricles, a peculiar course of the oviducts, including the presence of a common posterior extension, and by the asymmetrical openings of the vasa deferentia at about halfway along the seminal vesicle. Further, it is characterized by a ventral course of the ejaculatory duct with a terminal opening, very long spermiducal vesicles and unstalked cocoons. Its diploid chromosome complement with 18 chromosomes represents an uncommon feature among fissiparous species of Dugesia.

The first subterranean freshwater planarians from North Africa, with an analysis of adenodactyl structure in the genus Dendrocoelum (Platyhelminthes, Tricladida, Dendrocoelidae)

The paper describes the first species of freshwater planarians collected from subterranean localities in northern Africa, represented by three new species of Dendrocoelum Orsted, 1844 from Tunisian springs. Each of the new species possesses a well-developed adenodactyl, resembling similar structures in other species of Dendrocoelum, notably those from southeastern Europe. Comparative studies revealed previously unreported details and variability in the anatomy of these structures, particularly in the composition of the musculature. An account of this variability is provided, and it is argued that the anatomical structure of adenodactyls may provide useful taxonomic information.

Regeneration in two freshwater planarian species exposed to methylmercury compounds

The regeneration rate was studied by the morphometric method in planarians Polycelis tenuis and Dugesia lugubris after different periods of exposure on food substrates with high (0.30-0.50 mg/kg wet weight) and low (0.02-0.07 mg/kg) concentration of methylmercury compounds. The planarian growth and the ratio of regenerating tissue area to the total fragment area after transverse cutting were evaluated. The rate of size increment was lower in animals with high level than with low level of methylmercury than with low level of methylmercury. The highest relative and absolute body area increment after amputation was observed in D. lugubris and P. tenius, respectively. Thus, natural methylmercury compounds were shown to inhibit tissue regeneration in planarians in a dose-dependent manner.

Planarian neoblast micronucleus assay for evaluating genotoxicity

Planarian neoblasts are somatic stem cells that have the potential to be used in genotoxicity assays due to their proliferative nature, sensitivity to genotoxic agents, and experimental accessibility. Two freshwater planarian species, Girardia tigrina and Girardia schubarti, were used to develop a neoblast-based micronucleus (MN) assay to assess genotoxicity. Intact or regenerating planarians were exposed to γ-rays, methyl methanesulphonate (MMS), or cyclophosphamide (CP), and neoblast MN frequency was measured. Exposure to the clastogens had no detectable effect on the MN frequency of intact planarian neoblasts. However, for regenerating individuals, active neoblast proliferation was induced by decapitation, and γ-ray doses as low as 0.5 Gy, and MMS and CP concentrations as low as 0.8 μM and 100 mM, respectively, induced a significant increase in MN frequency. Exposure to higher doses of γ-rays consistently resulted in detectable increases in MN frequency. For MMS and CP, concentrations of up to 1.6 μM and 200 mM, respectively, resulted in significant increases in MN frequency, but exposures to higher concentrations led to a decrease to non-significant levels, possibly due to cytotoxic effects of MMS and CP. After completion of regeneration, the MN frequencies returned to those of non-exposed controls, indicating that the neoblast MN assay for regenerating G. tigrina or G. schubarti reflects chromosomal damage caused by acute exposure to clastogenic agents. Upon standardization, this assay may represent an interesting alternative that allows damage caused to freshwater organisms by potentially genotoxic environmental pollutants to be monitored.

Identification of Reissner's fiber-like glycoproteins in two species of freshwater planarians (Tricladida), by use of specific polyclonal and monoclonal antibodies.

By using one polyclonal antiserum raised against bovine Reissner's fiber and seven monoclonal antibodies raised against bovine Reissner's fiber and against immunopurified bovine subcommissural organ glycoproteins, we have investigated two freshwater planarian species (Girardia tigrina, Schmidtea mediterranea) by light- and electron-microscopic immunocytochemistry. ELISA probes showed that the monoclonal antibodies recognized different, nonoverlapping, unrepeated, proteinaceous epitopes present in the same compounds of bovine Reissner's fiber. Cells immunoreactive to the polyclonal and monoclonal antibodies were found in the dorsal and ventral integument of both planarian species. Labeled cuboid epidermal cells bore cilia and displayed several types of secretory granules; they were covered by a film of immunoreactive material. Studies on adjacent thin and semithin sections revealed coexistence of label in the same regions and in the same cells when two different monoclonal antibodies were used. These results indicate that a secretory substance immunologically similar to the secretion of the vertebrate subcommissural organ is present in primitive tripoblasts such as planarians, suggesting that these secretions are ancient and well conserved in phylogeny.

A new species of freshwater planarian belonging to the genus Dugesia (Platyhelminthes, Tricladida) from Sardinia

Dugesia leporii sp. n. from SW Sardinia is described. It is characterised by a comparatively small copulatory apparatus, the shape of the penial papilla, and the course and opening of the ejaculatory duct. The penial papilla, which has a penial valve, protrudes into an undivided atrium and is not provided with accessory structures. The taxonomic position of the new species within the D. gonocephala group is discussed.

Karyology and biogeography of Dugesia japonica and Dugesia ryukyuensis in Kyûshû, southern Japan

In southern Japan, two dugesiid species of freshwater planarians are known: Dugesia japonica Ichikawa et Kawakatsu, 1964, and Dugesia ryukyuensis Kawakatsu, 1976. D. japonica is a common and polymorphic species widely distributed in the Far East (karyotypes: n=8, 2x=16, 3x=24). D. ryukyuensis is a species recorded only from the Southwest Islands of Japan (Nansei Shoto) (karyotypes: n=7, 2x=14, 3x=21). Recently, small populations of D. ryukyuensis were found in the lowland areas in Kyushu on the East China Sea (the Nishisonogi Peninsula, the Shimabara Peninsula, the Goto Islands, the Satsuma Peninsula, and the Osumi Peninsula). The current geographical distribution of D. japonica and D. ryukyuensis in southern Japan can be explained from geological and faunal viewpoints, as follows: (1) two separate inversions by the ancestor of D. japonica, one in the Miocene and one after early Quaternary; (2) only one expansion of its domain by the ancestor of D. ryukyuensis in the Miocene.

Deviation from the Groundplan: a unique new species of freshwater planarian from South Brazil (Platyhelminthes, Tridadida, Paludicola)

The species Girardia biapertura Sluys, sp. nov. is described from the state of Rio Grande do Sul, Brazil. A unique derived character distinguishes this species from its congeners and all other planarians known to date, namely separate openings of the vasa deferentia at the tip of the penial papilla. Present knowledge on the distribution of Girardia species in Rio Grande do Sul is summarized.

Enzyme electrophoresis, 18S rRNA sequences, and levels of phylogenetic resolution among several species of freshwater planarians (Platyhelminthes, Tricladida, Paludicola)

Enzyme polymorphism and 18S rRNA sequences have been used to measure genetic distances between several species of Platyhelminthes belonging to different taxa including freshwater and parasitic forms. We have used these data to address unresolved phylogenetic and taxonomic problems with this group at several different levels ranging from phylum to subgenus. The main conclusions supported by the data seem to be the following: (i) 18S rRNA data strongly suggest that the Platyhelminthes are monophyletic, being a sister-group to the other Eubilateria; a similar conclusion applies to the Paludicola as to the rest of Platyhelminthes studied; (ii) 18S rRNA and enzyme data indicate that the family Dugesiidae of the Paludicola is monophyletic with respect to the other two families, Planariidae and Dendrocoelidae; and (iii) the subgenus Schmidtea of the genus Dugesia is monophyletic with respect to the other two subgenera of Dugesia, Dugesia and Girardia. Other aspects of the relationships of subgenera and families could not be satisfactorily resolved, but point to new problems that should be addressed in future studies, namely the taxonomic status of the family Planariidae and the relationships between the genera and subgenera of the family Dugesiidae.