The toxicity of potentially toxic elements (Cu, Fe, Mn, Zn and Ni) to the cnidarian Hydra attenuata at environmentally relevant concentrations.

The domestic, agricultural, industrial, technological and medical applications of potentially toxic elements (PTEs) have led to global pollution in all environments. In this study, the cnidarian Hydra attenuata was exposed individually and to a mixture of 5 metals (copper, iron, manganese, zinc and nickel) at environmentally relevant concentrations (1×) within the Clyde estuary, Scotland and incremental concentrations ranging from 0.0001× to 1000×. Toxicity was investigated using morphology, attachment, hydranth number and feeding behaviour as endpoints. When exposed individually, Cu, Mn and Fe significantly reduced Hydra morphology, feeding and attachment at environmentally relevant concentrations. Hydra mortality was measured, having an LC50 of 0.045× (for the environmentally relevant mixture of metals) and Cu 0.5 mg/l, Fe 3 mg/l, Mn 2 mg/l, Zn 0.1 mg/l, Ni 0.5 mg/l for each element exposed individually. The PTE mixture incurred a significant decrease (p ≤ 0.05) in morphology at 0.0001×, with 100% mortality at 0.1× (containing a concentration of Cu 0.05 mg/l, Fe 0.3 mg/l, Mn 0.2 mg/l, Zn 0.01 mg/l, Ni 0.05 mg/l) and a toxicity threshold (TT) of 0.000005×. Both copper and iron when exposed individually to the concentration of their respective metals found in the environment resulted in 100% mortality for all Hydra exposed. These results indicate that the PTE mixture (including the individual concentrations of copper, iron, manganese and nickel) could potentially prove significantly toxic to the aquatic environment.

Allatoregulatory-like systems and changes in cytosolic Ca2+ modulate feeding behavior in Hydra.

Allatotropin (AT) and allatostatin-C (AST-C) are neuropeptides originally characterized by their ability to modulate the secretion of juvenile hormones in insects. Beyond the allatoregulatory function, these neuropeptides are pleiotropic acting as myoregulators not only in insects, but also in other groups of invertebrates. We have previously proposed the existence of AT and AST-C like systems in Hydra sp., a member of the phylum Cnidaria, which is a basal group of Metazoa, sharing a common ancestor with Bilateria. In the present study we analyze the regulatory effects of both peptides on the activity of the hypostome during feeding in Hydra sp. Furthermore, the importance of changes in the cytosolic Ca2+ levels involved in the response of the hypostome were analyzed. Physiological assays showed that while the presence of food or treatment with AT stimulates the extrusion of the hypostome, AST-C has an inhibitory effect on the behavior induced by both, food and AT. These facts suggest that both systems participate in the regulatory mechanisms associated with feeding and, as in insects, AST-C and AT may exert opposite effects. The use of thapsigargin (TG) and nifedipine, two compounds that modify the levels of cytosolic Ca2+, showed that changes in the levels of this ion are involved in the regulation of the activity of the hypostome. Indeed, these results suggest that the two basic mechanisms operating to increase the cytosolic levels of Ca2+ (i.e. the influx from the extracellular space and the release from endoplasmic reticulum) are relevant for the extrusion of the hypostome. Like in insects, the treatment with TG counteracted the effect of AST-C, suggesting that this peptide acts by reducing cytosolic Ca2+ levels. Furthermore, nifedipine prevented the myostimulatory effect of AT, showing that the effect of this peptide depends on the influx of Ca2+ throughout voltage-gated calcium channels. Altogether, these results suggest that the Allatotropin/Orexin and Allatostatin/Somatostatin regulatory systems could represent an ancestral mechanisms regulating hypostome activity and feeding behavior in Cnidaria.

Dynamics of Mouth Opening in Hydra.

Hydra, a simple freshwater animal famous for its regenerative capabilities, must tear a hole through its epithelial tissue each time it opens its mouth. The feeding response of Hydra has been well-characterized physiologically and is regarded as a classical model system for environmental chemical biology. However, due to a lack of in vivo labeling and imaging tools, the biomechanics of mouth opening have remained completely unexplored. We take advantage of the availability of transgenic Hydra lines to perform the first dynamical analysis, to our knowledge, of Hydra mouth opening and test existing hypotheses regarding the underlying cellular mechanisms. Through cell position and shape tracking, we show that mouth opening is accompanied by changes in cell shape, but not cellular rearrangements as previously suggested. Treatment with a muscle relaxant impairs mouth opening, supporting the hypothesis that mouth opening is an active process driven by radial contractile processes (myonemes) in the ectoderm. Furthermore, we find that all events exhibit the same relative rate of opening. Because one individual can open consecutively to different amounts, this suggests that the degree of mouth opening is controlled through neuronal signaling. Finally, from the opening dynamics and independent measurements of the elastic properties of the tissues, we estimate the forces exerted by the myonemes to be on the order of a few nanoNewtons. Our study provides the first dynamical framework, to our knowledge, for understanding the remarkable plasticity of the Hydra mouth and illustrates that Hydra is a powerful system for quantitative biomechanical studies of cell and tissue behaviors in vivo.

Unraveling the non-senescence phenomenon in Hydra.

Unlike other metazoans, Hydra does not experience the distinctive rise in mortality with age known as senescence, which results from an increasing imbalance between cell damage and cell repair. We propose that the Hydra controls damage accumulation mainly through damage-dependent cell selection and cell sloughing. We examine our hypothesis with a model that combines cellular damage with stem cell renewal, differentiation, and elimination. The Hydra individual can be seen as a large single pool of three types of stem cells with some features of differentiated cells. This large stem cell community prevents "cellular damage drift," which is inevitable in complex conglomerate (differentiated) metazoans with numerous and generally isolated pools of stem cells. The process of cellular damage drift is based on changes in the distribution of damage among cells due to random events, and is thus similar to Muller's ratchet in asexual populations. Events in the model that are sources of randomness include budding, cellular death, and cellular damage and repair. Our results suggest that non-senescence is possible only in simple Hydra-like organisms which have a high proportion and number of stem cells, continuous cell divisions, an effective cell selection mechanism, and stem cells with the ability to undertake some roles of differentiated cells.

Extraocular spectral photosensitivity in the tentacles of Hydra vulgaris.

Previous electrophysiological studies on the cnidarian Hydra vulgaris have shown that hydra have a highly developed and specific photoresponse despite their lack of any structure recognizable as a traditional photoreceptor. In an effort to identify the site of hydra's photoreceptors, we recorded extracellularly from single excised tentacles and from ablated hypostomes lacking tentacles in absolute darkness and during exposure to light of various wavelengths. During recording, after an initial period of absolute darkness, tentacles or hypostomes were exposed to light from 450nm to 600nm, red, and white light. Exposure to light caused a change in the pattern and frequency of impulses in the tentacles that varied with color. The number of large tentacle pulses (TPs) increased at 550 and 600nm relative to darkness, whereas the number of small tentacle pulses (STPs) tended to decrease in 500nm light. Impulse frequency was significantly different among the different wavelengths. In addition to bursts of tentacle contraction pulses, long trains of pulses were observed. A change in lighting caused a switch from bursting to trains or vice versa. In contrast to excised tentacles, no change in electrical activity was seen in ablated hypostomes at any of the wavelengths relative to each other or relative to darkness. These results indicate that isolated tentacles can distinguish among and respond to various colors across the visible spectrum and suggest that electromagnetic information is transmitted from the tentacles to the hypostome where it may be integrated by the hypostomal nervous system, ultimately contributing to hydra's photoreceptive behavior.

Protein SYCP2 is an ancient component of the metazoan synaptonemal complex.

During the first meiotic prophase, chromosome synapsis is mediated by the synaptonemal complex (SC), an evolutionarily conserved meiosis-specific structure. In mammals, 7 SC protein components have been identified so far. Despite some controversy in the past, we have shown that SC proteins are ancient in metazoans and very likely formed an ancestral SC structure in the ancestor of metazoans. Protein components SYCP1, SYCP3, SYCE2, and TEX12 were identified in basal-branching metazoans, while other components (SYCE1 and SYCE3) are more recent elements. However, the evolutionary history of mammalian SYCP2 is not known. Here, we investigated this aspect with the aid of bioinformatic tools as well as with RNA and protein expression analysis. We conclude that SYCP2 belongs to the group of ancient SC proteins that was already present in the common ancestor of metazoans more than 500 million years ago.

A new species of hydra (Cnidaria: Hydrozoa: Hydridae) and molecular phylogenetic analysis of six congeners from China.

A new species of genus Hydra (Cnidaria: Hydrozoa: Hydridae), Hydra shenzhensis sp. nov. from Guangdong Province, China, is described and illustrated. Most polyps have five tentacles. Column length reaches 11 mm when relaxed. Buds do not acquire tentacles synchronously. Stenotele is broad and pyriform in shape, 1.2 times as long as its width. Holotrichous isorhiza is asymmetrical and slender (more than 2.7 times as long as its width), with transverse and slanting coils. Atrichous isorhiza is long, resembling a melon-seed in shape. Desmoneme is asymmetrically pyriform in shape. The new species, belonging to the vulgaris group, is dioecious; sexual reproduction was found to occur mostly during November and December under conditions of dense culture or food shortage. Two to thirteen testes, cone-like shape with papilla, formed beneath the tentacles. One to three ovaries, with an egg cup, milky white in color, formed on body column. Ninety percent of individuals developed only one ovum. On a mother polyp, a fertilized ovum developed an embryonic theca covering its surface. The embryotheca is brown, with a spine-like structure, covering a layer of transparent, membrane-like material. For phylogenetic analysis, the mitochondrial cytochrome oxidase subunit I gene (COI) of six hydra species collected from China was amplified by polymerase chain reaction (PCR) and sequenced. Morphological characters in combination with molecular evidence support the hydra described here as a new species.

Hydra, a model system for environmental studies.

Hydra have been extensively used for studying the teratogenic and toxic potential of numerous toxins throughout the years and are more recently growing in popularity to assess the impacts of environmental pollutants. Hydra are an appropriate bioindicator species for use in environmental assessment owing to their easily measurable physical (morphology), biochemical (xenobiotic biotransformation; oxidative stress), behavioural (feeding) and reproductive (sexual and asexual) endpoints. Hydra also possess an unparalleled ability to regenerate, allowing the assessment of teratogenic compounds and the impact of contaminants on stem cells. Importantly, Hydra are ubiquitous throughout freshwater environments and relatively easy to culture making them appropriate for use in small scale bioassay systems. Hydra have been used to assess the environmental impacts of numerous environmental pollutants including metals, organic toxicants (including pharmaceuticals and endocrine disrupting compounds), nanomaterials and industrial and municipal effluents. They have been found to be among the most sensitive animals tested for metals and certain effluents, comparing favourably with more standardised toxicity tests. Despite their lack of use in formalised monitoring programmes, Hydra have been extensively used and are regarded as a model organism in aquatic toxicology.

Value of the Hydra model system for studying symbiosis.

Green Hydra is used as a classical example for explaining symbiosis in schools as well as an excellent research model. Indeed the cosmopolitan green Hydra (Hydra viridissima) provides a potent experimental framework to investigate the symbiotic relationships between a complex eumetazoan organism and a unicellular photoautotrophic green algae named Chlorella. Chlorella populates a single somatic cell type, the gastrodermal myoepithelial cells (also named digestive cells) and the oocyte at the time of sexual reproduction. This symbiotic relationship is stable, well-determined and provides biological advantages to the algal symbionts, but also to green Hydra over the related non-symbiotic Hydra i.e. brown hydra. These advantages likely result from the bidirectional flow of metabolites between the host and the symbiont. Moreover genetic flow through horizontal gene transfer might also participate in the establishment of these selective advantages. However, these relationships between the host and the symbionts may be more complex. Thus, Jolley and Smith showed that the reproductive rate of the algae increases dramatically outside of Hydra cells, although this endosymbiont isolation is debated. Recently it became possible to keep different species of endosymbionts isolated from green Hydra in stable and permanent cultures and compare them to free-living Chlorella species. Future studies testing metabolic relationships and genetic flow should help elucidate the mechanisms that support the maintenance of symbiosis in a eumetazoan species.

Critical behavior and axis defining symmetry breaking in Hydra embryonic development.

The formation of a hollow cellular sphere is often one of the first steps of multicellular embryonic development. In the case of Hydra, the sphere breaks its initial symmetry to form a foot-head axis. During this process a gene, ks1, is increasingly expressed in localized cell domains whose size distribution becomes scale-free at the axis-locking moment. We show that a physical model based solely on the production and exchange of ks1-promoting factors among neighboring cells robustly reproduces the scaling behavior as well as the experimentally observed spontaneous and temperature-directed symmetry breaking.

Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth) families.

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem ß-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.

Microtubules are involved in regulating body length in hydra.

Little is known about how the size of an adult animal is determined and regulated. To investigate this issue in hydra, we altered the body size by surgically removing a part of the body column and/or by axial grafting, and examined changes of column length with time. When the body column was shortened it elongated and resumed the original length within 24-48 h. This increase in the body column length was not accompanied by an increase in the number of epithelial cells in the body column. Instead, each of the epithelial cells elongated longitudinally, leading to elongation of the body column. When the body column surpassed the original length, the column shortened over time. This was not accompanied by a decrease in cell number but by the shortening and thickening of the epithelial cells. TEM analysis showed that formation of microtubule arrays takes place longitudinally along the body axis in elongated cells and perpendicular to the axis in shortened cells. Treatment with a drug that degrades microtubules completely blocked changes in body length. These observations suggest that microtubules are involved in regulating the length of the hydra body column by altering the shape of the epithelial cells. We propose from these observations that hydra has a mechanism for detecting the metrical distance between the two ends of the body column.

Phylogenomics reveals an anomalous distribution of USP genes in metazoans.

Members of the universal stress protein (USP) family were originally identified in stressed bacteria on the basis of a shared domain, which has since been reported in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants. Although not previously characterized in metazoans, here we report that USP genes are distributed in animal genomes in a unique pattern that reflects frequent independent losses and independent expansions. Multiple USP loci are present in urochordates as well as all Cnidaria and Lophotrochozoa examined, but none were detected in any of the available ecdysozoan or non-urochordate deuterostome genome data. The vast majority of the metazoan USPs are short, single-domain proteins and are phylogenetically distinct from the prokaryotic, plant, protist, and fungal members of the protein family. Whereas most of the metazoan USP genes contain introns, with few exceptions those in the cnidarian Hydra are intronless and cluster together in phylogenetic analyses. Expression patterns were determined for several cnidarian USPs, including two genes belonging to the intronless clade, and these imply diverse functions. The apparent paradox of implied diversity of roles despite high overall levels of sequence (and implied structural) similarity parallels the situation in bacteria. The absence of USP genes in ecdysozoans and most deuterostomes may be a consequence of functional redundancy or specialization in taxon-specific roles.

Nematocyst discharge in Hydra vulgaris: Differential responses of desmonemes and stenoteles to mechanical and chemical stimulation.

The effects of mechanical and chemical stimulation on desmoneme and stenotele discharge in Hydra vulgaris were quantified, in situ, in isolated tentacles and single tentacles with attached hypostomes. Cnidocils of desmonemes and stenoteles were stimulated 24h after tentacle ablation. Single impacts with maximum calculated forces of 3.7x10(-3) to 3.8x10(-2) Newtons were delivered by piezoelectrically-driven glass capillary probes. Video analysis revealed that desmonemes discharged at forces of 3.7x10(-3)N; stenoteles required forces of 1.9x10(-2)N. Desmonemes not directly stimulated discharged if another desmoneme was adequately stimulated; the effect was carried through to at least two adjacent battery cells. Tentacles responded to desmoneme stimulation, by bending at the stimulation site. These findings imply afferent excitatory pathways between nematocytes onto other nematocytes and myonemes. Locomotory nematocysts (atrichous isorhizas) discharged only in hypostome-attached tentacles contacting a substrate; desmonemes and stenoteles did not discharge during substrate attachment, implying differential neuronal inhibition. At low concentrations, bath-applied mucin, a prey-associated glycosylated protein, lowered desmoneme and stenotele firing thresholds, abolishing the force dose-dependency in stenoteles, and allowing them to discharge at previously below threshold forces. At higher concentrations, mucin inhibited discharge, suggesting an involvement in prey-induced feeding inhibition.

Peptidomic approaches to the identification and characterization of functional peptides in Hydra.

Little is known about peptides that control developmental processes such as cell differentiation and pattern formation in metazoans. The cnidarian Hydra is one of the most basal metazoans and is a key model system for studying the peptides involved in these processes. We developed a novel peptidomic approach to the isolation and identification of functional signalling peptides from Hydra (the Hydra peptide project). First, peptides extracted from the tissue of Hydra magnipapillata are purified to homogeneity using high-performance liquid chromatography (HPLC). The isolated peptides are then tested for their ability to alter gene expression in Hydra using differential display-PCR (DD-PCR). If gene expression is altered, the peptide is considered as a putative signalling peptide and is subjected to amino acid sequencing. Following the sequencing, synthetic peptides are produced and compared to their native counterparts by HPLC and/or mass spectrometry (MS). The synthetic peptides, which are available in larger quantities than their native analogues, are then tested in a variety of biological assays in Hydra to determine their functions. Here we present our strategies and a systematic approach to the identification and characterization of novel signalling peptides in Hydra. We also describe our high-throughput reverse-phase nano-flow liquid chromatography matrix-assisted laser desorption ionization time-of-flight mass spectrometry (LC-MALDI-TOF-MS/MS) approach, which was proved to be a powerful tool in the discovery of novel signalling peptides.

A new species of green hydra (Hydrozoa: Hydrida) from China.

A new species of green freshwater hydra (Cnidaria, Hydrozoa: Hydrida), Hydra sinensis, is described from Guangdong Province, China. The chief distinction between H. sinensis sp. nov. and three other green hydras (H. hadleyi, H. viridissima, and H. plagiodesmica) is in the holotrichous isorhizae. Hydra sinensis sp. nov. differs from H. plagiodesmica in the shape of the holotrichous isorhlzae, and from H. viridissima and H. hadleyi in the tubule of the capsule of the holotrichous isorhlzae. The capsule tubule colls two times in 86% and three times in 14% of holotrlchous isorhlzae (n=50) In H. sinensis sp. nov.; we observed no tubules coiling four times. In contrast, the capsule tubule coils three or four times in H. viridissima and H. hadleyi, and no tubules coiling two times have been reported. In addition, holotrichous isorhlzae, which are mainly located around the hypostome, are sparse in the tentacles of H. sinensis sp. nov., whereas the majority of holotrichous isorhlzae is located on the tentacles in most other hydras. A molecular phylogenetic analysis using the nuclear small subunlt (18S) ribosomal RNA gene Indicated a close relationship between H. sinensis and H. viridissima. Hydra viridissima did not group within a clade of four Individuals of H. sinensis, Indicating a possible sister-species relationship between the two species. Morphological characters in combination with the molecular phylogenetic evidence support Hydra sinensis as a new species.

More than just orphans: are taxonomically-restricted genes important in evolution?

Comparative genome analyses indicate that every taxonomic group so far studied contains 10-20% of genes that lack recognizable homologs in other species. Do such 'orphan' or 'taxonomically-restricted' genes comprise spurious, non-functional ORFs, or does their presence reflect important evolutionary processes? Recent studies in basal metazoans such as Nematostella, Acropora and Hydra have shed light on the function of these genes, and now indicate that they are involved in important species-specific adaptive processes. Here we focus on evidence from Hydra suggesting that taxonomically-restricted genes play a role in the creation of phylum-specific novelties such as cnidocytes, in the generation of morphological diversity, and in the innate defence system. We propose that taxon-specific genes drive morphological specification, enabling organisms to adapt to changing conditions.

Apoptotic cells provide an unexpected source of Wnt3 signaling to drive hydra head regeneration.

Decapitated Hydra regenerate their heads via morphallaxis, i.e., without significant contributions made by cell proliferation or interstitial stem cells. Indeed, Hydra depleted of interstitial stem cells regenerate robustly, and Wnt3 from epithelial cells triggers head regeneration. However, we find a different mechanism controlling regeneration after midgastric bisection in animals equipped with both epithelial and interstitial cell lineages. In this context, we see rapid induction of apoptosis and Wnt3 secretion among interstitial cells at the head- (but not foot-) regenerating site. Apoptosis is both necessary and sufficient to induce Wnt3 production and head regeneration, even at ectopic sites. Further, we identify a zone of proliferation beneath the apoptotic zone, reminiscent of proliferative blastemas in regenerating limbs and of compensatory proliferation induced by dying cells in Drosophila imaginal discs. We propose that different types of injuries induce distinct cellular modes of Hydra head regeneration, which nonetheless converge on a central effector, Wnt3.