Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica.

BACKGROUND: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. OBJECTIVE: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. METHODS: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. RESULTS: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. CONCLUSION: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

Insights into the histology of planarian flatworm Phagocata gracilis based on location specific, intact lipid information provided by GCIB-ToF-SIMS imaging.

Planarian flatworms are known as the masters of regeneration, re-growing an entire organism from as little as 1/279th part of their body. While the proteomics of these processes has been studied extensively, the planarian lipodome remains relatively unknown. In this study we investigate the lipid profile of planarian tissue sections with imaging Time-of-Flight - Secondary-Ion-Mass-Spectrometry (ToF-SIMS). ToF-SIMS is a label-free technique capable of gathering intact, location specific lipid information on a cellular scale. Lipid identities are confirmed using LC-MS/MS. Our data shows that different organ structures within planarians have unique lipid profiles. The 22-carbon atom poly unsaturated fatty acids (PUFAs) which occur in unusually high amounts in planarians are found to be mainly located in the testes. Additionally, we observe that planarians contain various odd numbered fatty acid species, that are usually found in bacteria, localized in the reproductive and ectodermal structures of the planarian. An abundance of poorly understood ether fatty acids and ether lipids were found in unique areas in planarians as well as a new, yet unidentified class of potential lipids in planarian intestines. Identifying the location of these lipids in the planarian body provides insights into their bodily functions and, in combination with knowledge about their diet and their genome, enables drawing conclusions about planarian fatty acid processing.

Perfluorooctane sulfonate induced neurotoxicity responses associated with neural genes expression, neurotransmitter levels and acetylcholinesterase activity in planarians Dugesia japonica.

As a persistent and widespread toxic organic pollutant in the environment, perfluorooctane sulfonate (PFOS) has the potential to cause great harm to wildlife. In our study, the effects of PFOS on neurodevelopment gene expression, neurotransmitter content, neuronal morphology, acetylcholinesterase (AChE) activity were examined, and the potential neurotoxicity mechanisms of PFOS were also investigated in planarians, Dugesia japonica. Using quantitative real-time PCR analysis, five neurodevelopmental related genes were measured, among which, DjotxA, DjotxB, DjFoxD, and DjFoxG were found to be down-regulated, while Djnlg was found to be up-regulated, following exposure to PFOS for 10 days compared with control groups. In addition, the neurotransmitters including dopamine, serotonin, and γ-aminobutyricacid as well as the acitivity of AChE were altered by PFOS exposure. Furthermore, PFOS exposure altered brain morphology as well as smaller cephalic ganglia which displayed reduced nerve fiber density decreased brain branches compared to controls. Our results demonstrate that neurotransmission was disturbed after exposure to PFOS and that exposure to this pollutant can cause neurotoxic defects. Results from this study provide valuable information regarding the neuro- and ecological toxicity of PFOS in aquatic animals and aquatic environments.

An adaptable chromosome preparation methodology for use in invertebrate research organisms.

BACKGROUND: The ability to efficiently visualize and manipulate chromosomes is fundamental to understanding the genome architecture of organisms. Conventional chromosome preparation protocols developed for mammalian cells and those relying on species-specific conditions are not suitable for many invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate species, is needed. RESULTS: We optimized a chromosome preparation protocol and applied it to several planarian species (phylum Platyhelminthes), the freshwater apple snail Pomacea canaliculata (phylum Mollusca), and the starlet sea anemone Nematostella vectensis (phylum Cnidaria). We demonstrated that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with deionized water was sufficient for karyotyping; growing cells in culture was not necessary. The obtained karyotypes allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla this protocol could be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization. CONCLUSIONS: Our simple and inexpensive chromosome preparation protocol can be readily adapted to new invertebrate research organisms to accelerate the discovery of novel genomic patterns across the branches of the tree of life.

De novo discovery of neuropeptides in the genomes of parasitic flatworms using a novel comparative approach.

Neuropeptide mediated signalling is an ancient mechanism found in almost all animals and has been proposed as a promising target for the development of novel drugs against helminths. However, identification of neuropeptides from genomic data is challenging, and knowledge of the neuropeptide complement of parasitic flatworms is still fragmentary. In this work, we have developed an evolution-based strategy for the de novo discovery of neuropeptide precursors, based on the detection of localised sequence conservation between possible prohormone convertase cleavage sites. The method detected known neuropeptide precursors with good precision and specificity in the models Drosophila melanogaster and Caenorhabditis elegans. Furthermore, it identified novel putative neuropeptide precursors in nematodes, including the first description of allatotropin homologues in this phylum. Our search for neuropeptide precursors in the genomes of parasitic flatworms resulted in the description of 34 conserved neuropeptide precursor families, including 13 new ones, and of hundreds of new homologues of known neuropeptide precursor families. Most neuropeptide precursor families show a wide phylogenetic distribution among parasitic flatworms and show little similarity to neuropeptide precursors of other bilaterian animals. However, we could also find orthologs of some conserved bilaterian neuropeptides including pyrokinin, crustacean cardioactive peptide, myomodulin, neuropeptide-Y, neuropeptide KY and SIF-amide. Finally, we determined the expression patterns of seven putative neuropeptide precursor genes in the protoscolex of Echinococcus multilocularis. All genes were expressed in the nervous system with different patterns, indicating a hidden complexity of peptidergic signalling in cestodes.

Nanoporous Structures Similar to Those Reported from Squid Sucker Teeth are also Present in Egg Shells of a Terrestrial Flatworm (Platyhelminthes; Rhabditophora; Geoplanidae) from Hachijojima (Izu Islands, Japan).

Shells of the egg cocoon of a terrestrial planarian (Diversibipalium sp.) from Hachijojima were found to be composed of a lattice of parallel nanotubes of ca. 120 nm diameter oriented perpendicular to the shell's surface. The arrangement of the porous proteinaceous tubes closely resembles that has recently been reported from the sucker teeth of squid and to date is the only other example of this kind of structure. Although the array of nanotubes undoubtedly contributes to the stiffness of the shell and helps protecting the embryo, questions such as to how the planary worm produces the array of nanotubes and what exactly their chemical and physical properties are versus those of the squid sucker tooth still remain to be answered.

Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea.

Tissue regeneration is a complex process that involves a mosaic of molecules that vary spatially and temporally. Insights into the chemical signaling underlying this process can be achieved with a multiplex and untargeted chemical imaging method such as mass spectrometry imaging (MSI), which can enablede novostudies of nervous system regeneration. A combination of MSI and multivariate statistics was used to differentiate peptide dynamics in the freshwater planarian flatwormSchmidtea mediterraneaat different time points during cephalic ganglia regeneration. A protocol was developed to makeS. mediterraneatissues amenable for MSI. MS ion images of planarian tissue sections allow changes in peptides and unknown compounds to be followed as a function of cephalic ganglia regeneration. In conjunction with fluorescence imaging, our results suggest that even though the cephalic ganglia structure is visible after 6 days of regeneration, the original chemical composition of these regenerated structures is regained only after 12 days. Differences were observed in many peptides, such as those derived from secreted peptide 4 and EYE53-1. Peptidomic analysis further identified multiple peptides from various known prohormones, histone proteins, and DNA- and RNA-binding proteins as being associated with the regeneration process. Mass spectrometry data also facilitated the identification of a new prohormone, which we have named secreted peptide prohormone 20 (SPP-20), and is up-regulated during regeneration in planarians.

[Own Chemiluminescence of Planarian Neoblasts during Regeneration].

We investigated the kinetics of the luminescence induced by reactive oxygen species in planarians during regeneration process. It was found that regeneration is accompanied with changes in the concentration of reactive oxygen species correlating with energy-intensive processes such as oxidative stress, caused by damage to cell membranes in the dissection of the planarian, phagocytosis of dying cells and mitosis of neoblasts. We showed for the first time that there is an opportunity of registering the physiological state of pluripotent stem cells at the level of the organism in vivo.

Development of an extraction and purification method for the determination of multi-class pharmaceuticals and endocrine disruptors in freshwater invertebrates.

Aquatic organisms from freshwater ecosystems impacted by waste water treatment plant (WWTP) effluents are constantly exposed to constant concentrations of pharmaceuticals, endocrine disruptors and related compounds, among other anthropogenic contaminants. Macroinvertebrates inhabiting freshwater ecosystems might be useful bioindicators of exposure to contaminants, since their lives are long enough to bioaccumulate, but at the same time may integrate short-term changes in the environment. However, studies about potential bioaccumulation of emerging contaminants in these organisms are very scarce. The objectives of this study were to develop an analytical methodology for the analysis of 41 pharmaceuticals and 21 endocrine disruptors in freshwater invertebrates. In addition, bioaccumulation of these contaminants in three macroinvertebrate taxa inhabiting a waste water treatment plant -impacted river was evaluated. The method for the simultaneous extraction of both families of compounds is based on sonication, purification via removal of phospholipids, and analysis by ultra performance liquid chromatography coupled to a mass spectrometer (UPLC-MS/MS) in tandem. Recoveries for pharmaceuticals were 34-125%, and for endocrine disruptors were 48-117%. Method detection limits (MDLs) for EDCs were in the range of 0.080-2.4 ng g(-1), and for pharmaceuticals, 0.060-4.3 ng g(-1). These pollutants were detected in water samples taken downstream the waste water treatment plant effluent at concentrations up to 572 ng L(-1). Two non-esteroidal anti-inflammatory drugs, diclofenac and ibuprofen, and four endocrine disruptors - estrone, bisphenol A, TBEP, and nonylphenol - were detected in at least one macroinvertebrate taxa in concentrations up to 183 ng g(-1) (dry weight). An isobaric interference was identified during the analysis of diclofenac in Hydropsyche samples, which was successfully discriminated via accurate mass determination by TFC-LTQ Orbitrap.

Comprehensive analysis of neurotransmitters from regenerating planarian extract using an ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring method.

RATIONALE: Absolute quantification of neurotransmitters (NTs) from biological systems is imperative to track how changes in concentration of active neurochemicals may affect biological behavior. A sensitive method for the absolute quantification of multiple NTs in a single method is highly needed. METHODS: A stable-isotope dilution ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC/MS/SRM) assay has been developed for a sensitive and quantitative assessment of NTs in planaria. We used this method for the simultaneous quantification of 16 NTs. All analytes showed a linear relationship between concentrations (0.78-50 ng/mL), regression coefficients higher than 0.97, accuracy (91-109%) and low coefficients of variation (CVs). The inter-day CVs for the lowest quality controls (1.56 ng/mL) were in the range between 2-11%. RESULTS: The levels of most of the NTs were similar in both sexual and asexual planarians except for glutamic acid, which was about two-fold higher in asexual compared to sexual planarians. We identified high levels of serotonin and failed to detect tryptamine suggesting that the pathway essential for the conversion of tryptophan into tryptamine is absent in planarians. Interestingly, we also found high levels of dopamine and L-DOPA in regenerating planarians suggesting their possible role in regeneration. CONCLUSIONS: For the first time, we developed novel methodology based on UHPLC/MS/SRM and quantified 16 NTs with high sensitivity and specificity from sexual and asexual strains of planarian Schmidtea mediterranea. This method will also have great application in quantifying various NTs with great precision in different model systems.

Molecular characterization of gap region in 28S rRNA molecules in brine shrimp Artemia parthenogenetica and planarian Dugesia japonica.

In most insects and some other protostomes, a small stretch of nucleotides can be removed from mature 28S rRNA molecules, which could create two 28S rRNA subunits (28Sα and 28Sß). Thus, during electrophoresis, the rRNA profiles of these organisms may differ significantly from the standard benchmark since the two subunits co-migrate with the 18S rRNA. To understand the structure and mechanism of the atypical 28S rRNA molecule, partial fragments of 28Sα and 28Sß in brine shrimp Artemia parthenogenetica and planarian Dugesia japonica were cloned using a modified technology based on terminal transferase. Alignment with the corresponding sequences of 28S rDNAs indicates that there are 41 nucleotides in A. parthenogenetica and 42 nucleotides in D. japonica absent from the mature rRNAs. The AU content of the gap sequences of D. japonica and A. parthenogenetica is high. Both the gaps may form stem-loop structure. In D. japonica a UAAU cleavage signal is identified in the loop, but it is absent in A. parthenogenetica. Thus, it is proposed that the gap processing of 28S rRNA was a late enzyme-dependent cleavage event in the rRNA maturational process based on the AU rich gap sequence and the formation of the stem-loop structure to expose the processing segment, while the deletion of the gap region would not affect the structure and function of the 28S rRNA molecule.

Proteomic profiling of the planarian Schmidtea mediterranea and its mucous reveals similarities with human secretions and those predicted for parasitic flatworms.

The freshwater planarian Schmidtea mediterranea has been used in research for over 100 years, and is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. Exteriorly, planarians are covered in mucous secretions of unknown composition, implicated in locomotion, predation, innate immunity, and substrate adhesion. Although the planarian genome has been sequenced, it remains mostly unannotated, challenging both genomic and proteomic analyses. The goal of the current study was to annotate the proteome of the whole planarian and its mucous fraction. The S. mediterranea proteome was analyzed via mass spectrometry by using multidimensional protein identification technology with whole-worm tryptic digests. By using a proteogenomics approach, MS data were searched against an in silico translated planarian transcript database, and by using the Swiss-Prot BLAST algorithm to identify proteins similar to planarian queries. A total of 1604 proteins were identified. The mucous subproteome was defined through analysis of a mucous trail fraction and an extract obtained by treating whole worms with the mucolytic agent N-acetylcysteine. Gene Ontology analysis confirmed that the mucous fractions were enriched with secreted proteins. The S. mediterranea proteome is highly similar to that predicted for the trematode Schistosoma mansoni associated with intestinal schistosomiasis, with the mucous subproteome particularly highly conserved. Remarkably, orthologs of 119 planarian mucous proteins are present in human mucosal secretions and tear fluid. We suggest planarians have potential to be a model system for the characterization of mucous protein function and relevant to parasitic flatworm infections and diseases underlined by mucous aberrancies, such as cystic fibrosis, asthma, and other lung diseases.

Bioaccumulation and toxicodynamics of cadmium to freshwater planarian and the protective effect of N-acetylcysteine.

Although toxic responses of freshwater planarians after exposure to environmental toxicants can be observed through external toxicological end points, physiological responses inside the bodies of treated planarians have rarely been investigated. The present study was designed, using cadmium (Cd) as a reference toxicant, to determine its bioaccumulation and toxicodynamics in the freshwater planarian, Dugesia japonica, after acute toxicity was obtained. Accumulated Cd concentrations, metallothionein levels, and the oxidative status in planarians were determined after exposure to Cd. Furthermore, we hypothesized that the acute death of Cd-treated planarians was associated with increased oxidative stress. After Cd-treated planarians were coexposed to antioxidant, N-acetylcysteine (NAC), we found that NAC protected planarians from Cd lethality by maintaining the oxidative status and decreasing the bioaccumulation of Cd. The results of the present study support planarians being used as a practical model for toxicological studies of environmental contaminants in the future.

Minimal structural requirements of alkyl γ-lactones capable of antagonizing the cocaine-induced motility decrease in planarians.

We recently reported that the natural cyclic lactone, parthenolide, and related analogs prevent the expression of behavioral effects induced by cocaine in planarians and that parthenolide's γ-lactone ring is required for this effect. In the present work, we tested a series of alkyl γ-lactones with varying chain length (1-8 carbons) to determine their ability to antagonize the planarian motility decrease induced by 200 µM cocaine. Alkyl lactones with up to a 4-carbon alkyl chain did not affect planarian motility or antagonized the cocaine-induced motility decrease; only the compound γ-nonalactone (a γ-lactone with a 5-carbon chain) was able to prevent the cocaine-induced behavioral patterns, while alkyl lactones with longer carbon chains failed to prevent the cocaine-induced effects. Thus, we conclude that the optimal structural features of this family of compounds to antagonize cocaine's effect in this experimental system is a γ-lactone ring with at a 5-carbon long functional group.

Presence of galactosylated core fucose on N-glycans in the planaria Dugesia japonica.

Planarial species are of especial interest to biologists due to the phenomenon of pluripotency and, in comparison to other developmental processes, it can be hypothesised that glycan-lectin interactions may play a role. In order to examine the N-glycans of one of these organisms, Dugesia japonica, peptide:N-glycosidase A was employed and the released glycans were subject to pyridylamination, HPLC and mass spectrometric analysis. A range of oligomannosidic glycans was observed with a trimethylated Man(5) GlcNAc(2) structure being the dominant species. Three glycans were also observed to contain deoxyhexose; in particular, a glycan with the composition Hex(4) HexNAc(2) Fuc(1) Me(2) was revealed by exoglycosidase digestion, in combination with MS/MS, to contain a galactosylated core α1,6-fucose residue, whereas this core modification was found to be capped with a methylhexose residue in the case of a Hex(5) HexNAc(2) Fuc(1) Me(3) structure. This is the first report of these types of structures in a platyhelminth and indicates that the 'GalFuc' modification of N-glycans is not just restricted to molluscs and nematodes.

Structural analysis of N-glycans of the planarian Dugesia japonica.

To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N-glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N-glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2-aminopyridine at their reducing end. The labeled N-glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N-glycans, it was shown that the N-glycans of planarian include high mannose-type and pauci-mannose-type glycans. However, many of the major N-glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3-O-methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non-reducing-end mannose, as the glycans were resistant to α-mannosidase digestion. These results indicate that methylated high-mannose-type glycans are the most abundant structure in planarians.

Glutamate and aspartate measurements in individual planaria by rapid capillary electrophoresis.

INTRODUCTION: Planaria present a unique model organism for studying primitive central nervous systems. The major mammalian excitatory neurotransmitters, glutamate and aspartate, have previously been measured in planaria via high pressure liquid chromatography (HPLC). A faster extraction and analysis procedure using capillary electrophoresis (CE) was developed which confirms the presence of these amino acids in single planaria homogenates. METHOD: Following homogenization and centrifugation of individual planaria in hydrochloric acid/acetonitrile, glutamate and aspartate were derivatized with naphthalene-2, 3-dicarboxaldehyde (NDA). The labeled amino acids were measured using capillary electrophoresis with laser-induced fluorescence (CE-LIF). RESULTS: CE-LIF electropherograms were generated in less than 1 min. The mean ± S.D. amounts of glutamate and aspartate were 1200 ± 500 and 1900 ± 700 pmol/mg-planarian (n=22), respectively. Spiked average recoveries of glutamate and aspartate were 96% and 91%, respectively. DISCUSSION: The high-throughput method provides the ability to quantitate changes in excitatory neurotransmitters under developmental or stimulatory conditions. The capability to monitor multiple neurotransmitter levels offers the opportunity to correlate behavioral responses with biochemical changes in planaria.

Removal of methyl parathion in water, by Dugesia dorotocephala.

The aim of this study was to determine the efficiency of Dugesia dorotocephala on Methyl parathion removal. An initial concentration of 1.25 microg mL(-1) of MeP was used to evaluate the removal capacity of planarian. A first-order removal kinetics was obtained with a disappearance rate constant (k(r)) of 0.49 days(-1) and 69% efficiency on contaminant removal. This is significantly different (p < 0.5) from the degradation occurring in control systems, leading us to conclude that D. dorotocephala effectively removes MeP from contaminated water.

Measurement of glutamate and aspartate in Planaria.

INTRODUCTION: The major excitatory neurotransmitters in the mammalian central nervous system are glutamate and aspartate. We developed a rapid and efficient method for the extraction and measurement of these amino acids in Planaria--a valuable model for mammalian processes because of their simple, centralized nervous system and similar neurotransmitter systems. METHOD: The method utilized buffer extraction (perchloric acid containing 0.025% of L-cystine and Na2EDTA), simple derivatization, high-pressure liquid chromatography (HPLC), and fluorescence detection. RESULTS: The mean+/-S.E.M. amounts of glutamate and aspartate were 322.6+/-43.6 and 188.6+/-27.6 pmol/mg-planarian, respectively. DISCUSSION: The method provides the ability to investigate changes in glutamate and aspartate in response to drug administration or withdrawal.

SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells.

We have identified two genes, smedwi-1 and smedwi-2, expressed in the dividing adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. Both genes encode proteins that belong to the Argonaute/PIWI protein family and that share highest homology with those proteins defined by Drosophila PIWI. RNA interference (RNAi) of smedwi-2 blocks regeneration, even though neoblasts are present, irradiation-sensitive, and capable of proliferating in response to wounding; smedwi-2(RNAi) neoblast progeny migrate to sites of cell turnover but, unlike normal cells, fail at replacing aged tissue. We suggest that SMEDWI-2 functions within dividing neoblasts to support the generation of cells that promote regeneration and homeostasis.