Starfish Asterias Amurensis Research Articles

Behavioral and physiological responses of nocturnal marine shellfish to predation pressure

Predator-prey interactions are critical in ecological communities. As the wild resources of Pacific abalone are on the verge of exhaustion, efforts to reveal the effects of predation by hostile organisms on the efficiency of its bottom sowing and proliferation are crucial for the success of resource restoration. In this study, abalone and its predator, starfish Asterias amurensis, were placed in a customized monitoring unit. Through continuous infrared video monitoring, predation pressure was found to cause a significant decrease in the distance moved, duration of movement, cumulative feeding duration and orexin levels in hemolymph (P < 0.01). It also caused a significant increase in the average velocity of movement, group cohesion, coordination and cholecystokinin levels in hemolymph (P < 0.01). Surprisingly, when a shelter was set up in the environment, the cumulative feeding duration of abalone was significantly extended in the presence of the predator (P < 0.001), whereas when the predator was removed, the distance moved, duration of movement, and cumulative feeding duration of abalone were found to significantly increase during continuous 24 h monitoring (P < 0.01). These results first identified the anti-predation strategy by nocturnal marine organisms when exposed to predation risk, and supported the pivotal role of shelter in helping abalone make a trade-off between predation pressure and survival needs, which not only provides a new insight to optimizing the strategy for prevention and control of marine ecological disasters and strengthening the protection of marine endangered species, but also results in a productive attempt to restore the marine benthic ecosystem.

Development and Testing of Species-Specific Primers for Detecting the Presence of the Northern Pacific Sea Star (Asterias amurensis) from Environmental DNA.

The starfish Asterias amurensis, a well-known predator of molluscan species in intertidal ecosystems, has caused substantial ecological and economic losses in North China such as offshore Qingdao. Effective monitoring and prevention measures are urged to minimize its negative impacts. Compared with traditional biomonitoring methods, environmental DNA technology has emerged as a powerful and cost-efficient tool for inferring species' presence and abundance. In this study, we developed a pair of species-specific primers (i.e., Ast-F and Ast-R) for the A. amurensis mitochondrial COI gene and tested its utility in amplifying and quantifying the DNA fragments from environmental samples under both laboratory and field conditions. The results of controlled water tank experiments demonstrated that the amount of eDNA released by A. amurensis was positively related to its biomass; after the removal of the starfish, the eDNA degraded significantly in 24h and remained detectable for 8days. The number of eDNA copies enriched tended to increase with smaller pore size of filter membrane and larger volume of filtered water. For field tests, we confirmed the validation of our approach in six locations in Qingdao by filtering 1000ml water per sample with a 0.45-µm pore size filtration. All the amplification products generated a single and bright band via gel electrophoresis, and the quantitative PCR results unveiled significant differences in eDNA copies. This study provided an eDNA-based approach for investigating the distribution and biomass of A. amurensis, which may help to formulate early warning and management strategies in coastal Qingdao and other regions.

Global Analysis of 2-Hydroxy Fatty Acids by Gas Chromatography-Tandem Mass Spectrometry Reveals Species-Specific Enrichment in Echinoderms.

Abnormal levels of 2-hydroxy fatty acids (2-OH FAs) are characterized in multiple diseases, and their quantification in foodstuffs is critical to identify the sources of supplementation for potential treatment. However, due to the structural complexity and limited available standards, the comprehensive profiling of 2-OH FAs remains an ongoing challenge. Herein, an innovative approach based on gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed to determine the full profile of these FA metabolites. MS and MS/MS spectra of the trimethylsilyl (TMS) derivatives of 2-OH fatty acid methyl esters (FAMEs) were collected for peak annotation by their signature fragmentation patterns. The structures were further confirmed by validated structure-dependent retention time (RT) prediction models, taking advantage of the correlation between the RT, carbon chain length, and double bond number from commercial standards and pseudostandards identified in the whole-brain samples from mice. An in-house database containing 50 saturated and monounsaturated 2-OH FAs was established, which is expandible when additional molecular species with different chain lengths and backbone structures are identified in the future. A quantitation method was then developed by scheduled multiple reaction monitoring (MRM) and applied to investigate the profiling of 2-OH FAs in echinoderms. Our results revealed that the levels of total 2-OH FAs in sea cucumber Apostichopus japonicas (8.40 ± 0.28 mg/g dry weight) and starfish Asterias amurensis (7.51 ± 0.18 mg/g dry weight) are much higher than that in sea urchin Mesocentrotus nudus (531 ± 108 μg/g dry weight). Moreover, 2-OH C24:1 is the predominant molecular species accounting for 67.9% of the total 2-OH FA in sea cucumber, while 2-OH C16:0 is the major molecular species in starfish. In conclusion, the current innovative GC-MS approach has successfully characterized distinct molecular species of 2-OH FAs that are highly present in sea cucumbers and starfish. Thus, these findings suggest the possibility of developing future feeding strategies for preventing and treating diseases associated with 2-OH FA deficiency.

Cellular uptake of liposome consisting mainly of glucocerebroside from the starfish Asterias amurensis into Caco-2 cells

Glucocerebroside (GlcCer) is a group of compounds consisting of β-linked glucose and ceramide with various chain lengths, some of which possess anti-tumor activity and improve skin barrier function for atopic patients when administered orally. The amphiphilic GlcCer molecules are generally easy to aggregate in aqueous solution and result in low absorption in the gut, which can be improved by forming a liposome. With a recognition that a relatively large amount of GlcCer is contained in the starfish and is being discarded, we prepared a liposome consisting mainly of GlcCer (over 95%) with 100 nm in diameter. The adsorption efficiency of the liposome into cultured Caco-2 cells was investigated by live-cell imaging using fluorescently labeled liposomes. We found an immediate internalization of GlcCer-liposome on exposure without significant accumulation on the plasma membrane. The membrane fluidity was transiently affected as evidenced by fluorescence recovery after photobleaching (FRAP) experiments without no significant cellular damage, which indicates a liposome with high content of GlcCer might be useful as the carrier of dietary and/or drug molecules.

Morphometry of cellular behavior of coelomocytes from starfish Asterias amurensis.

A comprehensive statistical analysis using a wide range of linear and non-linear morphological parameters enabled identification of the main stages in the in vitro dynamics of cell behavior of immune cells of the marine invertebrate Asterias amurensis (Echinodermata, Asteroidea). Three stages may be distinguished in the cell behavior, which are characterized by the differences in complexity of the cell boundary microsculpture as well as by the size and asymmetry of the cell and convex hull of the cell. The first stage (5 min after placing cells onto a substrate) is characterized by more complex cell morphology and an increase in the process number and spreading area. The second stage (15 min) is characterized by simplification of cell morphology, retraction of some processes, and rounding of cells upon continued cell spreading. At the third stage (60 min), new large processes with rounded contours emerge due to partial retraction of the flattened cell surface. Each stage is characterized by statistically significant differences in several linear and nonlinear parameters of the external morphology for all cell types.

Characterization and comparison of the mitochondrial genomes of the sea star Asterias amurensis (Echinodermata: Asteroidea) in East Asia

The starfish Asterias amurensis is a marine pest native to the northwestern Pacific and has successfully invaded southern Australia. Asterias amurensis have caused substantial environmental and economic impacts in both native and non-native regions. However, little information is available about the genetic features of its native populations, especially for those in North China. Here we sequenced the complete mitochondrial genomes of five individuals from different locations in China and compared their characteristics with three mitogenomes available from Japan. Multiple analyses and comparisons revealed little difference in the gene composition, gene order, codon usage, and nucleotide content among the eight mitogenomes. However, intraspecific phylogenetic reconstruction unveiled two divergent lineages between specimens from North China plus Ushimado (Seto Inland Sea, Japan) and northern Japan (Asamushi and Onagawa). This conclusion was backed by an analysis of pairwise genetic distances, which showed that individuals from different lineages had relatively higher values (all above 2%). Based on knowledge of paleoenvironmental and tectonic activity in the northwestern Pacific, the two lineages might have originated during the Early Pliocene due to the isolation of the East China Sea from the Japan Sea/East Sea during that time, while the present-day distribution of these lineages have likely been influenced by the ocean current system.

A novel G protein-coupled receptor for starfish gonadotropic hormone, relaxin-like gonad-stimulating peptide.

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP.

A novel approach for preparing disulfide-rich peptide-KLH conjugate applicable to the antibody production.

To produce the antiserum against a small peptide, the target peptide-keyhole limpet hemocyanine (KLH) conjugate is generally used as an antigen, although the disulfide-rich peptide-KLH conjugate is still difficult to prepare. In our previous study, we have developed a preparation method of the disulfide-rich peptide-KLH conjugate, and this method was applied to produce the antiserum against a relaxin-like peptide. However, this method is limited to the synthetic peptide antigen, and is not applicable to a native or a recombinant peptide. In this study, to expand the applicability of this method to wide variety of peptides, we newly designed a novel thiol probe enabling the conjugation between various peptides and KLH, and applied it to produce the antiserum against relaxin-like peptide of a starfish Asterias amurensis. The antiserum obtained here showed high antibody-titer and good specificity, strongly suggesting that the method developed in this study is applicable to various peptides.

Isolation of Sphingoid Bases from Starfish Asterias amurensis Glucosylceramides and Their Effects on Sphingolipid Production in Cultured Keratinocytes.

Starfish Asterias amurensis produces sphingoid bases d18:3, 9-methyl-d18:3 (9Me-d18:3), and d22:2, which possess unique structural features. In this study, sphingoid bases prepared from A. amurensis glucosylceramides displayed unexpected elution behaviors from a general octadecyl silyl high-performance liquid chromatography (HPLC) column. For separation and isolation, sphingoid bases were fractionated by octadecyl silyl HPLC after N-acetylation, yielding d18:3, 9Me-d18:3, and two d22:2 isomers. To compare the biological activities of individual sphingoid bases, their effects on sphingolipid production in normal human keratinocytes were evaluated. Treatment with sphingoid bases increased the content of ceramides, glucosylceramides, and sphingomyelins in keratinocytes. Moreover, ceramides, which contain saturated ultra-long-chain fatty acids (C30-34), were significantly increased by treatment with d18:3, but not with other A. amurensis sphingoid bases. The mRNA level of the early differentiation marker keratin 10 was markedly decreased and sphingolipid synthesis-related genes were slightly increased in keratinocytes exposed to A. amurensis-derived d18:3, 9Me-d18:3, and d22:2 isomers. These results suggest that A. amurensis-derived sphingoid bases induce differentiation to varying degrees, sphingolipid production depends on their chemical structures, and d18:3 is the most promising functional sphingoid base.

A unique structural distribution pattern discovered for the cerebrosides from starfish Asterias amurensis

Cerebroside is an important family of the mono-glycosylated ceramides involved in the larger family of glycosphingolipid and sulfatide. Cerebroside is synthesized from ceramide by the transfer of glucose from UDP-glucose, and degraded back to ceramide, which plays an important role at the epidermis protecting interior of the body as a barrier. Because cerebroside is regarded as the source molecule of ceramide and is amphiphilic in nature, cerebroside is considered valuable as the ingredient of cosmetic lotion.Various sources can be considered as raw material of cerebrosides. Starfish is considered as one of such potent source. However, the structure of the ceramide part of cerebroside is not fully investigated. Therefore, the individual structures of cerebroside molecules need to be identified including sphingosine and fatty acyl group composition to assess the potential of the molecule.We investigated and determined the structures of cerebrosides in starfish Asterias amurensis using LC-MS, GC-MS, tandem mass spectrometry (MS/MS), and 1H NMR. We also discovered a characteristic structure distribution that was divided into three major groups: 1) a group composed of a relatively long sphingosine (C22) and a short length of fatty acyl group (less than C16), 2) a group composed of a typical C18 sphingosine and long fatty acyl groups (greater than C23), and 3) a group composed of C18 sphingosine and fatty acyl groups with their length less than C18. The calculated Log P values of cerebrosides ranging from 9 to 11 covered about 80% of the molecules that were in the range of those used in cosmetics, thus showing the potential usefulness of starfish Asterias amurensis as a source of raw material for cerebrosides.

A dynamic energy budget model to describe the reproduction and growth of invasive starfish Asterias amurensis in southeast Australia

The introduction of alien species is a global phenomenon that alters ecosystems structure and functioning. Invasive species are responsible for substantial economic and ecological losses. Invasive species impact resource availability, outcompeting and even causing extinction of native species. The management of invasive species requires knowledge on the ecology, physiology and population dynamics of these species. In a world where environmental conditions are changing fast due to global climate change and other anthropogenic stressors, a more comprehensive knowledge of the life history and physiology of these species is urgently needed. The DEB theory is unique in capturing the metabolic processes of an organism through its entire life cycle, and thus, is a useful tool to model lifetime feeding, growth, reproduction, and responses to changes in biotic and abiotic conditions. In this work, we estimated the parameters of a DEB model for Asterias amurensis. This starfish was introduced in Tasmania and is considered the most serious marine pest in Australia where it has caused local extinctions of several species. Asterias amurensis is a major predator and is a keystone species exerting top-down control of its prey populations by achieving large densities. We determined the influence of biotic and abiotic factors on the performance of A. amurensis. The DEB model presented here includes energy handling rules to describe gonad and pyloric caeca cycles. Model parameters were used to explore population dynamics of populations of A. amurensis in Australia. The DEB model allowed us to characterise the ecophysiology of A. amurensis, providing new insights on the role of food availability and temperature on its life cycle and reproduction strategy. Moreover it is a powerful tool for risk management of already established invasive populations and of regions with a high invasion risk.

Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca2+ levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+ . Yet, the contribution of signaling molecules downstream of cAMP and Ca2+ in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.

English

The starfish Asterias amurensis is one of the common species in Japanese coastal area. The species is considered an extremely rich source of biological active compounds, including saponin. Saponins are secondary metabolites and exhibit a wide spectrum of biological activities. The species of starfish caught in Kobe, Hyogo prefecture, Japan, was analyzed for species identification and micronutrient content. The starfish extract was evaluated for different biological properties and its fractions obtained using thin layer chromatography (TLC). Nucleotide sequence analysis of the 16S rRNA gene fragment of mitochondrial DNA indicated that partial sequences of PCR products of the species was identical with that of A. amurensis. The micronutrient contents results showed that nitrogen (N) content of the starfish was 1.50% of dry weight and the copper content was 2.10 µg/g. The crude extract of A. amurensis exhibited predominant growth inhibitory activity against six human fungal pathogens and also showed hemolytic activity against 2% sheep erythrocyte. The present findings suggest the possible pharmacological applications of A. amurensis that can be used as food ingredients and antifungal component. Key words: Asterias amurensis, crude extract, biological property, saponin, antifungal component.

The small cells of coelomic fluid and coelomic epithelium isolated from starfish Asterias rubens and Asterias amurensis (Echinodermata: Asteroidea): Comparative analysis of cell morphology and proliferative activity in vivo and in vitro

Earlier we revealed the probable candidates for the role of Asteroidea stem cells in the starfish Asterias rubens L., small coelomic epithelial cells (SECs-1) with a high nuclear–cytoplasmic ratio that were able to proliferate in vivo and in vitro. To check the existence of a similar cell type in other members of Asteroidea, the small cells in suspensions of coelomic fluid (CF) and coelomic epithelium (CE) of A. amurensis were analyzed with respect to their morphology and proportion in the total cell pool. The morphology of proliferating cells and the proliferative activity of CF and CE cells in vivo and in vitro were studied. The small cells with parameters identical to those of A. rubens SECs-1, were found both in CF and CE of related species. The subpopulation of weakly attached CE cells, highly enriched with SECs-1, was detected. These cells were able to migrate from CE and to proliferate in vivo and in vitro. Additionally, large proliferating cells were described in both starfish. The dynamics of proliferative activity in primary cell cultures of these starfish had some distinctions. Moreover, for the first time, the formation of “crystals”, the potential centres of spiculogenesis, was observed in primary culture of A. amurensis CE cells. The data prove that SECs may fulfil the common functions in two members of Asteroidea.

Amino acid sequence analysis and characterization of a ribonuclease from starfishAsterias amurensis

The aim of this study was to phylogenetically characterize the location of the RNase T2 enzyme in the starfish (Asterias amurensis). We isolated an RNase T2 ribonuclease (RNase Aa) from the ovaries of starfish and determined its amino acid sequence by protein chemistry and cloning cDNA encoding RNase Aa. The isolated protein had 231 amino acid residues, a predicted molecular mass of 25,906 Da, and an optimal pH of 5.0. RNase Aa preferentially released guanylic acid from the RNA. The catalytic sites of the RNase T2 family are conserved in RNase Aa; furthermore, the distribution of the cysteine residues in RNase Aa is similar to that in other animal and plant T2 RNases. RNase Aa is cleaved at two points: 21 residues from the N-terminus and 29 residues from the C-terminus; however, both fragments may remain attached to the protein via disulfide bridges, leading to the maintenance of its conformation, as suggested by circular dichroism spectrum analysis. The phylogenetic analysis revealed that starfish RNase Aa is evolutionarily an intermediate between protozoan and oyster RNases.

Antitumour activity of EPA-enriched phospholipids liposomes against S180 ascitic tumour-bearing mice

The health benefits of eicosapentaenoic acid (EPA)-enriched phospholipids (PL) have witnessed a recent upsurge. In the present study, PL from starfish Asterias amurensis (SFP) and sea cucumber Cucumaria frondosa (SCP) were extracted and the liposomes were prepared. Both SFP and SCP liposomes showed antitumour effects in vitro and exhibited high transport and uptake effects in small intestinal epithelial cell models. The results also demonstrated that dietary SFP and SCP liposomes prolonged the life span and ameliorated oxidative stress status of S180 ascitic tumour-bearing mice. Results also showed that dietary SFP and SCP liposomes up-regulated the expression of Bax and down-regulated Bcl-2 level in tumour cells, then induced release of cytochrome c from mitochondria to cytosol, thus the activation of caspase 9 and caspase 3. Taken together, these findings suggest that SFP and SCP liposomes exert antitumour effects mainly via activation of mitochondrial apoptotic pathways in tumour-bearing mice.

EPA-enriched phospholipids ameliorate cancer-associated cachexia mainly via inhibiting lipolysis.

Excessive loss of fat mass is considered as a key feature of body weight loss in cancer-associated cachexia (CAC). It affects the efficacy and tolerability of cancer therapy and reduces the quality and length of cancer patients' lives. The aim of the present study was to evaluate the effects of EPA-enriched phospholipids (EPA-PL) derived from starfish Asterias amurensis on cachectic weight loss in mice bearing S180 ascitic tumor, and TNF-α-stimulated lipolysis in 3T3-L1 adipocytes and to elucidate the possible mechanisms involved. Our findings revealed that oral administration of EPA-PL at 100 mg per kg body weight (BW) per day for 14 days prevented body weight loss in CAC mice by preserving the white adipose tissue (WAT) mass. We found that serum levels of nonesterified fatty acid (NEFA) and pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin (IL)-6 increased in CAC mice but decreased significantly after oral treatment of EPA-PL. In addition, EPA-PL treatment also suppressed the overexpression of several key lipolytic factors and raised the mRNA levels of some adipogenic factors in the WAT of CAC mice. Moreover, treatment of EPA-PL (200 and 400 μM) markedly inhibited TNF-α-stimulated lipolysis in adipocytes. Furthermore, the antilipolytic effects of EPA-PL were stimulated by the extracellular signal-regulated kinase 1/2 (ERK 1/2) inhibitor PD 98059 and blocked via the AMP-activated protein kinase (AMPK) inhibitor compound C and the phosphoinositide-3-kinase (PI3K) inhibitor LY 294002. Taken together, these data suggest that the dietary EPA-PL ameliorates CAC mainly via inhibiting lipolysis and at least in part for recovering the function of adipogenesis.

Complete NMR assignments of undegraded asterosaponins from Asterias amurensis.

Four asterosaponins, thornasteroside A (1), versicoside A (2), anasteroside B (3), and asteronylpentaglycoside sulfate (4), were isolated from the predatory starfish Asterias amurensis Lütken. Unlike previous studies focusing on structure elucidation by degradation of the complex saponin molecules, complete nuclear magnetic resonance (NMR) assignment for the intact molecules was accomplished using 600MHz high magnetic field NMR. The complete set of NMR assignments can help in the structure elucidation of asterosaponins isolated in low yields without resorting to chemical degradation. Furthermore, this approach can be extended to other complex steroidal saponins, which may accelerate the discovery of bioactive secondary metabolites from this invasive starfish species.

아무르 불가사리(Asterias amurensis)의 관족으로부터 신경성 펩타이드의 분리 및 정제

Two neuropeptides were purified from the acidified tube feet extract of the starfish Asterias amurensis by C18 reversed phase and size-exclusion high-performance liquid chromatography (HPLC). The tube feet extract and the purified peptides (AST-I and AST-II) showed potent contractile activity on dorsal retractor muscle (DRM) of the starfish Asterina pectinifera and intestine (smooth muscle) of the panther puffer Takifugu pardalis. Treatment of the purified peptides with dithiothreitol (DTT) for 60 min at <TEX>$37^{\circ}C$</TEX> significantly altered their retention times, suggesting that these compounds contained disulfide bonds. The molecular weights of AST-I and AST-II were determined to be 2064.2 Da and 6137.2 Da, respectively, by MALDI-TOF mass spectrometry.

Erratum to: Purification and characterization of a lectin from the starfish Asterias amurensis

Erratum to: Purification and characterization of a lectin from the starfish Asterias amurensis