Evaluation of different types of adjuvants in a malaria transmission-blocking vaccine.

Adjuvants are critical components for vaccines, which enhance the strength and longevity of the antibody response and influence the types of immune response. Limited research has been conducted on the immunogenicity and protective efficacy of various adjuvants in malaria transmission-blocking vaccines (TBVs). In this study, we formulated a promising TBV candidate antigen, the P. berghei ookinete surface antigen PSOP25, with different types of adjuvants, including the TLR4 agonist monophosphoryl lipid A (MPLA), the TLR9 agonist cytosine phosphoguanosine oligodeoxynucleotides (CpG ODN 1826) (CpG), a saponin adjuvant QS-21, aluminum hydroxide (Alum), and two combination adjuvants MPLA + QS-21 and QS-21 + CpG. We demonstrated that adjuvanted vaccines results in elevated elicited antibody levels, increased proliferation of plasma cells, and efficient formation of germinal centers (GCs), leading to enhanced long-term protective immune responses. Furthermore, CpG group exhibited the most potent inhibition of ookinete formation and transmission-blocking activity. We found that the rPSOP25 with CpG adjuvant was more effective than MPLA, QS-21, MPLA + QS-21, QS-21 + CpG adjuvants in dendritic cells (DCs) activation and differentiation. Additionally, the CpG adjuvant elicited more rubust immune memory response than Alum adjuvant. CpG and QS-21 adjuvants could activate the Th1 response and promote the secretion of IFN-γ and TNF-α. PSOP25 induced a higher number of Tfh cells in splenocytes when combined with MPLA, CpG, and QS-21 + CpG; and there was no increase in these cell populations when PSOP25 was administered with Alum. In conclusion, CpG may confer enhanced efficacy for the rPSOP25 vaccine, as evidenced by the ability of the elicited antisera to induce protective immune responses and improved transmission-blocking activity.

Endocrine regulation of reproductive biology in echinoderms: An evolutionary perspective from closest marine invertebrate relatives to chordates.

Echinoderms are a phylum of invertebrate deuterostomes, which contain echinoids, asteroids, holothuroids, crinoids, and ophiuroids. Echinoderms have special evolutionary position and unique characteristics, including pentamerous radial body structure, elaborate calcareous endoskeletons, and versatile water vascular system. Echinoderms exhibit extraordinarily diverse reproductive modes: asexual reproduction, sexual reproduction, sexual reversal, etc. Endocrine regulation plays important well-known roles in sex differentiation, gonadal development and maturation, gametogenesis, and reproductive behavior in vertebrates. However, the entire picture of reproductive endocrinology in echinoderms as an evolutionary model of the closest marine invertebrate relatives to chordates has not been revealed. Here, we reviewed previous and recent research progress on reproductive endocrinology in echinoderms, mainly including two sections: Sex steroids in echinoderms and neuropeptide regulation in echinoderm reproduction. This review introduces a variety of endocrine regulatory mechanisms in reproductive biology of echinoderms. It discusses the vertebrate-like sex steroids, putative steroidogenic pathway and metabolism, and reproduction-related neuropeptides. The review will provide a deeper understanding about endocrine regulatory mechanisms of gonadal development in lower deuterostomes and the application of endocrine control in economic echinoderm species in aquaculture.

A dual-antigen malaria vaccine targeting Pb22 and Pbg37 was able to induce robust transmission-blocking activity.

BACKGROUND: Despite years of effort to develop an effective vaccine against malaria infection, a vaccine that provides individuals with sufficient protection against malaria illness and death in endemic areas is not yet available. The development of transmission-blocking vaccines (TBVs) is a promising strategy for malaria control. A dual-antigen malaria vaccine targeting both pre- and post-fertilization antigens could effectively improve the transmission-blocking activity of vaccines against the sexual stages of the parasite. METHODS: A chimeric recombinant protein Pb22-Pbg37 (Plasmodium berghei 22-P. berghei G37) composed of 19-218 amino acids (aa) of Pb22 and the N-terminal 26-88 aa of Pbg37 was designed and expressed in the Escherichia coli expression system. The antibody titers of the fusion (Pb22-Pbg37) and mixed (Pb22+Pbg37) antigens, as well as those of Pb22 and Pbg37 single antigens were evaluated by enzyme-linked immunosorbent assay. Immunofluorescence and western blot assays were performed to test the reactivity of the antisera with the native proteins in the parasite. The induction of transmission-blocking activity (TBA) by Pb22-Pbg37 and Pb22+Pbg37 were evaluated by in vitro gametocyte activation, gamete and exflagellation center formation, ookinete conversion, and in the direct mosquito feeding assay. RESULTS: The Pb22-Pbg37 fusion protein was successfully expressed in vitro. Co-administration of Pb22 and Pbg37 as a fusion or mixed protein elicited comparable antibody responses in mice and resulted in responses to both antigens. Most importantly, both the mixed and fusion antigens induced antibodies with significantly higher levels of TBA than did each of the individual antigens when administered alone. In addition, the efficacy of vaccination with the Pb22-Pbg37 fusion protein was equivalent to that of vaccination with the mixed single antigens. CONCLUSIONS: Dual-antigen vaccines, which expand/lengthen the period during which the transmission-blocking antibodies can act during sexual-stage development, can provide a promising higher transmission-reducing activity compared to single antigens.

Chromosome-level genome assembly of the northern Pacific seastar Asterias amurensis.

Asterias amurensis has attracted widespread concern because of its population outbreaks, which has impacted fisheries and aquaculture, as well as disrupting local ecosystems. A high-quality reference genome is necessary to better investigate mechanisms of outbreak and adaptive changes. Combining PacBio HiFi and Hi-C sequencing data, we generated a chromosome-level A. amurensis genome with a size of 491.53 Mb. The contig N50 and scaffold N50 were 8.05 and 23.75 Mb, respectively. The result of BUSCO analysis revealed a completeness score of 98.85%. A total of 16,531 protein-coding genes were predicted in the genome, of which 94.63% were functionally annotated. The high-quality genome assembly resulting from this study will provide a valuable genetic resource for future research on the mechanism of population outbreaks and invasion ecology.

Morphology and Chemical Messenger Regulation of Echinoderm Muscles.

The muscular systems of echinoderms play important roles in various physiological and behavioral processes, including feeding, reproduction, movement, respiration, and excretion. Like vertebrates, echinoderm muscle systems can be subdivided into two major divisions, somatic and visceral musculature. The former usually has a myoepithelial organization, while the latter contains muscle bundles formed by the aggregation of myocytes. Neurons and their processes are also detected between these myoepithelial cells and myocytes, which are capable of releasing a variety of neurotransmitters and neuropeptides to regulate muscle activity. Although many studies have reported the pharmacological effects of these chemical messengers on various muscles of echinoderms, there has been limited research on their receptors and their signaling pathways. The muscle physiology of echinoderms is similar to that of chordates, both of which have the deuterostome mode of development. Studies of muscle regulation in echinoderms can provide new insights into the evolution of myoregulatory systems in deuterostomes.

Enhanced external counterpulsation treatment regulates blood flow and wall shear stress metrics in femoral artery: An in vivo study in healthy subjects.

As a non-invasive assisted circulation therapy, enhanced external counterpulsation (EECP) has demonstrated potential in treatment of lower-extremity arterial disease (LEAD). However, the underlying hemodynamic mechanism remains unclear. This study aimed to conduct the first prospective investigation of the EECP-induced responses of blood flow behavior and wall shear stress (WSS) metrics in the femoral artery. Twelve healthy male volunteers were enrolled. A Doppler ultrasound-basedapproach was introduced for the in vivo determination of blood flow in the common femoral artery (CFA) and superficial femoral artery (SFA) during EECP intervention, with incremental treatment pressures ranging from 10 to 40 kPa. Three-dimensional subject-specific numerical models were developed in 6 subjects to quantitatively assess variations in WSS-derived hemodynamic metrics in the femoral bifurcation. A mesh-independence analysis was performed. Our results indicated that, compared to the pre-EECP condition, both the antegrade and retrograde blood flow volumes in the CFA and SFA were significantly augmented during EECP intervention, while the heart rate remained constant. The time average shear stress (TAWSS) over the entire femoral bifurcation increased by 32.41%, 121.30%, 178.24%, and 214.81% during EECP with treatment pressures of 10 kPa, 20 kPa, 30 kPa, and 40 kPa, respectively. The mean relative resident time (RRT) decreased by 24.53%, 61.01%, 69.81%, and 77.99%, respectively. The percentage of area with low TAWSS in the femoral artery dropped to nearly zero during EECP with a treatment pressure greater than or equal to 30 kPa. We suggest that EECP is an effective and non-invasive approach for regulating blood flow and WSS in lower extremity arteries.

A Putative Role of Vasopressin/Oxytocin-Type Neuropeptide in Osmoregulation and Feeding Inhibition of Apostichopus japonicus.

Vasopressin/oxytocin (VP/OT)-type neuropeptide is an ancient neurophysin-associated neuropeptide and has been intensively studied to be involved in multiple physiological processes in protostomian and deuterostome vertebrates. However, little is known about the functions of VP/OT-type neuropeptide in deuterostome invertebrates especially in echinoderms. Here, we firstly report VP/OT-type neuropeptide signaling in an important economic species, Apostichopus japonicus, which is widely cultured in Asia, with high nutritional and medicinal values. Molecular characterization analysis of holotocin and its precursor revealed the highly conserved features of VP/OT family. The candidate receptor for holotocin (AjHOR) was confirmed to be able to activate the signaling via cAMP-PKA and possible Ca2+-PKC pathway, and further activated the downstream ERK1/2 cascade. Holotocin precursor expression profile showed that they were mainly concentrated in circumoral nerve ring. Furthermore, in vitro pharmacological experiments demonstrated that holotocin caused contractile responses in preparations from A. japonicus. And in vivo functional studies indicated that short-term injection of holotocin resulted in body bloat and long-term injection resulted in reduced body mass, suggesting potential roles of holotocin in osmoregulation and feeding co-inhibition with holotocin-CCK. Our findings provided a comprehensive description of AjHOR-holotocin signaling, revealed ancient roles of holotocin in osmoregulation and feeding inhibition by controlling muscle contractions.

Identification of sex-specific splicing via comparative transcriptome analysis of gonads from sea cucumber Apostichopus japonicus.

Alternative splicing (AS) is an essential post-transcriptional regulation mechanism for sex differentiation and gonadal development, which has rarely been reported in marine invertebrates. Sea cucumber (Apostichopus japonicus) is an economically important marine benthic echinoderm with a potential XX/XY sex determination mechanism, whose molecular mechanism in the gonadal differentiation has not been clearly understood. In this study, we analyzed available RNA-seq datasets of male and female gonads to explore if AS mechanism exerts an essential function in sex differentiation and gonadal development of A. japonicus. In our results, a total of 20,338 AS events from 7219 alternatively spliced genes, and 189 sexually differential alternative splicing (DAS) events from 156 genes were identified in gonadal transcriptome of sea cucumber. Gene Ontology analysis indicated that these DAS genes were significantly enriched in spermatogenesis-related GO terms. Maximal Clique Centrality (MCC) was then applied for protein-protein interaction (PPI) analysis to search for protein interactions and hub DAS gene. Among all DAS genes, we identified 10 DAS genes closely related to spermatogenesis and (or) sperm motility and a hub gene dnah1. Thus, this study revealed that alternative isoforms were generated from certain genes in female and male gonads through alternative splicing, which may provide direct evidence that alternative splicing mechanisms participate in female and male gonads. These results suggested a novel perspective for explaining the molecular mechanisms underlying gonadal differentiation between male and female sea cucumbers.

Nervous System Development and Neuropeptides Characterization in Embryo and Larva: Insights from a Non-Chordate Deuterostome, the Sea Cucumber Apostichopus japonicus.

Here, we described the complex nervous system at five early developmental stages (blastula, gastrula, auricularia, doliolaria and pentactula) of a holothurian species with highly economic value, Apostichopus japonicus. The results revealed that the nervous system of embryos and larvae is mainly distributed in the anterior apical region, ciliary bands or rings, and the feeding and attachment organs, and that serotonergic immunoreactivity was not observed until the embryo developed into the late gastrula; these are evolutionarily conserved features of echinoderm, hemichordate and protostome larvae. Furthermore, based on available transcriptome data, we reported the neuropeptide precursors profile at different embryonic and larval developmental stages. This analysis showed that 40 neuropeptide precursors present in adult sea cucumbers were also identified at different developmental stages of embryos and larvae, and only four neuropeptide precursors (SWYG precursor 2, GYWKDLDNYVKAHKT precursor, Neuropeptide precursor 14-like precursor, GLRFAmprecursor-like precursor) predicted in adults were absent in embryos and larvae. Combining the quantitative expression of ten specific neuropeptide precursor genes (NPs) by qRT-PCR, we revealed the potential important roles of neuropeptides in embryo development, feeding and attachment in A. japonicus larvae. In conclusion, this work provides novel perspectives on the diverse physiological functions of neuropeptides and contributes to understanding the evolution of neuropeptidergic systems in echinoderm embryos and larvae.

Molecular and functional characterization of the luqin-type neuropeptide signaling system in the sea cucumber Apostichopus japonicus.

The functional characteristics of neuropeptides in marine invertebrates have attracted significant attention recently although functional studies of luqin-type neuropeptides are still very limited, especially in deuterostomes. The sea cucumber, Apostichopus japonicus, is a representative species of deuterostomian Holothurian invertebrates. The species has high nutritional and medicinal value in China. In this study, we report the first comprehensive histological, biochemical and pharmacological characterization of luqin-type neuropeptide signaling in the sea cucumber A. japonicus. The A. japonicus luqin-like neuropeptide precursor (AjLQP) contains a single typical deuterostomian luqin-like neuropeptide AjLQ with an xFxRWamide motif. AjLQ was identified as the ligand for a luqin-type neuropeptide receptor AjLQR, that was previously predicted to be a tachykinin-type receptor, and triggers a rapid intracellular mobilization of Ca2+, followed by receptor internalization and a transient increase in ERK1/2 phosphorylation. In situ hybridization, immunohistochemistry and qRT-PCR analysis revealed extensive expression of AjLQP and AjLQ in A. japonicus tissues, especially in locomotion-related organs. In vitro pharmacological tests revealed that AjLQ caused 12.69% ± 1.99% (p < 0.01) relaxation of longitudinal muscle preparations at 10-7 M concentration. Furthermore, we observed significantly increased expression of AjLQP (about 17.63 fold, p < 0.01) in intestine of deeply aestivating sea cucumbers, which suggests that AjLQ might be involved in feeding inhibition during aestivation. The present study provides a first insight into the experimental characterization of luqin-type neuropeptide signaling in a sea cucumber. The results will broaden our understanding of the potential function of neuropeptides during important biological processes in marine invertebrates and provide theoretical support for optimizing sea cucumber aquaculture technology.

Identification of sex determination locus in sea cucumber Apostichopus japonicus using genome-wide association study.

BACKGROUND: Sex determination mechanisms are complicated and diverse across taxonomic categories. Sea cucumber Apostichopus japonicus is a benthic echinoderm, which is the closest group of invertebrates to chordate, and important economic and ecologically aquaculture species in China. A. japonicus is dioecious, and no phenotypic differences between males and females can be detected before sexual maturation. Identification of sex determination locus will broaden knowledge about sex-determination mechanism in echinoderms, which allows for the identification of sex-linked markers and increases the efficiency of sea cucumber breeding industry. RESULTS: Here, we integrated assembly of a novel chromosome-level genome and resequencing of female and male populations to investigate the sex determination mechanisms of A. japonicus. We built a chromosome-level genome assembly AJH1.0 using Hi-C technology. The assembly AJH1.0 consists of 23 chromosomes ranging from 22.4 to 60.4 Mb. To identify the sex-determination locus of A. japonicus, we conducted genome-wide association study (GWAS) and analyses of distribution characteristics of sex-specific SNPs and fixation index FST. The GWAS analysis showed that multiple sex-associated loci were located on several chromosomes, including chromosome 4 (24.8%), followed by chromosome 9 (10.7%), chromosome 17 (10.4%), and chromosome 18 (14.1%). Furthermore, analyzing the homozygous and heterozygous genotypes of plenty of sex-specific SNPs in females and males confirmed that A. japonicus might have a XX/XY sex determination system. As a physical region of 10 Mb on chromosome 4 included the highest number of sex-specific SNPs and higher FST values, this region was considered as the candidate sex determination region (SDR) in A. japonicus. CONCLUSIONS: In the present study, we integrated genome-wide association study and analyses of sex-specific variations to investigate sex determination mechanisms. This will bring novel insights into gene regulation during primitive gonadogenesis and differentiation and identification of master sex determination gene in sea cucumber. In the sea cucumber industry, investigation of molecular mechanisms of sex determination will be helpful for artificial fertilization and precise breeding.

Genome-Wide DNA Methylation Signatures of Sea Cucumber Apostichopus japonicus during Environmental Induced Aestivation.

Organisms respond to severe environmental changes by entering into hypometabolic states, minimizing their metabolic rates, suspending development and reproduction, and surviving critical ecological changes. They come back to an active lifestyle once the environmental conditions are conducive. Marine invertebrates live in the aquatic environment and adapt to environmental changes in their whole life. Sea cucumbers and sponges are only two recently known types of marine organisms that aestivate in response to temperature change. Sea cucumber has become an excellent model organism for studies of environmentally-induced aestivation by marine invertebrates. DNA methylation, the most widely considered epigenetic marks, has been reported to contribute to phenotypic plasticity in response to environmental stress in aquatic organisms. Most of methylation-related enzymes, including DNA methyltransferases, Methyl-CpG binding domain proteins, and DNA demethylases, were up-regulated during aestivation. We conducted high-resolution whole-genome bisulfite sequencing of the intestine from sea cucumber at non-aestivation and deep-aestivation stages. Further DNA methylation profile analysis was also conducted across the distinct genomic features and entire transcriptional units. A different elevation in methylation level at internal exons was observed with clear demarcation of intron/exon boundaries during transcriptional unit scanning. The lowest methylation level occurs in the first exons, followed by the last exons and the internal exons. A significant increase in non-CpG methylation (CHG and CHH) was observed within the intron and mRNA regions in aestivation groups. A total of 1393 genes were annotated within hypermethylated DMRs (differentially methylated regions), and 749 genes were annotated within hypomethylated DMRs. Differentially methylated genes were enriched in the mRNA surveillance pathway, metabolic pathway, and RNA transport. Then, 24 hypermethylated genes and 15 hypomethylated genes were Retrovirus-related Pol polyprotein from transposon (RPPT) genes. This study provides further understanding of epigenetic control on environmental induced hypometabolism in aquatic organisms.

De novo transcriptome analysis of stressed blood clam (Anadara broughtonii) and identification of genes associated with hemoglobin.

BACKGROUND: Blood clam (Anadara broughtonii) is a commercially important marine bivalve characterised by the red blood. Recently, the clams have been subjected to severe resource recession. Multiple environmental stressors are indispensable for the recession. OBJECTIVE: We aimed to investigate the transcriptome information of blood clam under environmental stressors. METHODS: Paired-end Illumina HiSeq™ 2500 sequencing technology was employed for cDNA library construction and Illumina sequencing. Several public databases were introduced for gene annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathways analyses. The Open Reading Frame of annotated hemoglobin (Hb) was predicted and validated by DNAMAN 6.0 and NCBI BLASTx, respectively. RESULTS: A total of 242,919 transcripts were generated, 116,264 unigenes were subsequently assembled with an average length of 747 base pairs, and 33,776 unigenes were successfully annotated. Gene Ontology (GO) categories indicated that the terms of cellular processes, metabolic processes, cell, cell part, binding, and catalytic activity were dominant. KEGG pathway analyses suggested ribosome, oxytocin, focal adhesion, Ras, and PI3K-Akt were the largest signaling pathway groups, and many presented pathways (Ras, Rap1, and MAPK, etc.) were related to apoptosis, immune and stress response. In addition, a total of 19,306 potential simple sequence repeats (SSRs) were detected in 15,852 sequences. Six hemoglobin-related genes with complete conserved domain sequences were identified and 3 of them were predicted as HbI, HbIIα, and HbIIß. CONCLUSION: This study provides transcriptome responses to multiple environmental stressors in blood clams and would provide interesting hints for further studies.

MiR-200-3p Is Potentially Involved in Cell Cycle Arrest by Regulating Cyclin A during Aestivation in Apostichopus japonicus.

The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally induced aestivation in marine invertebrates. We hypothesized that mechanisms that arrest energy-expensive cell cycle activity would contribute significantly to establishing the hypometabolic state during aestivation. Cyclin A is a core and particularly interesting cell cycle regulator that functions in both the S phase and in mitosis. In the present study, negative relationships between miR-200-3p and AjCA expressions were detected at both the transcriptional and the translational levels during aestivation in A. japonicus. Dual-luciferase reporter assays confirmed the targeted location of the miR-200-3p binding site within the AjCA gene transcript. Furthermore, gain- and loss-of-function experiments were conducted in vivo with sea cucumbers to verify the interaction between miR-200-3p and AjCA in intestine tissue by qRT-PCR and Western blotting. The results show that the overexpression of miR-200-3p mimics suppressed AjCA transcript levels and translated protein production, whereas transfection with a miR-200-3p inhibitor enhanced both AjCA mRNA and AjCA protein in A. japonicus intestine. Our findings suggested a potential mechanism that reversibly arrests cell cycle progression during aestivation, which may center on miR-200-3p inhibitory control over the translation of cyclin A mRNA transcripts.

Comparative physiology and aquaculture: Toward Omics-enabled improvement of aquatic animal health and sustainable production.

Omics-technologies have revolutionized biomedical research over the past two decades, and are now poised to play a transformative role in aquaculture. This article serves as an introduction to a Virtual Special Issue of Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics (CBPD), with the objective to showcase the state-of-the-science for Omics in aquaculture. In this editorial, we describe the role that Omics can play in aquaculture, and provide a synopsis for each of the Special Issue articles that use these technologies to improve aquaculture practices. Current genomic resources available for some aquaculture species are also described. The number of datasets is impressive for species such as Atlantic salmon and rainbow trout, totaling in the thousands (NCBI Gene Expression Omnibus and Sequence Read Archive). We present a conceptual framework that describes how Omics can be leveraged to understand complex responses of aquatic animals in culture for relevant physiological outcomes, such as fecundity, growth, and immunity. Lastly, knowledge gaps and new directions are identified to address current obstacles in aquaculture. Articles in this Special Issue on aquaculture in CBPD highlight the diversity and scope of Omics in aquaculture. As the technology becomes more cost-effective, it is anticipated that genomics, transcriptomics, proteomics, metabolomics and lipidomics will play increasingly important roles in stock diagnostics (e.g. genetics, health, performance). The timing is right, as global concerns are reaching critical levels over food availability/security and water restrictions for humankind.

Neuropeptide precursors and neuropeptides in the sea cucumber Apostichopus japonicus: a genomic, transcriptomic and proteomic analysis.

The sea cucumber Apostichopus japonicus is a foodstuff with very high economic value in China, Japan and other countries in south-east Asia. It is at the heart of a multibillion-dollar industry and to meet demand for this product, aquaculture methods and facilities have been established. However, there are challenges associated with optimization of reproduction, feeding and growth in non-natural environments. Therefore, we need to learn more about the biology of A. japonicus, including processes such as aestivation, evisceration, regeneration and albinism. One of the major classes of molecules that regulate physiology and behaviour in animals are neuropeptides, and a few bioactive peptides have already been identified in A. japonicus. To facilitate more comprehensive investigations of neuropeptide function in A. japonicus, here we have analysed genomic and transcriptomic sequence data and proteomic data to identify neuropeptide precursors and neuropeptides in this species. We identified 44 transcripts encoding neuropeptide precursors or putative neuropeptide precursors, and in some instances neuropeptides derived from these precursors were confirmed by mass spectrometry. Furthermore, analysis of genomic sequence data enabled identification of the location of neuropeptide precursor genes on genomic scaffolds and linkage groups (chromosomes) and determination of gene structure. Many of the precursors identified contain homologs of neuropeptides that have been identified in other bilaterian animals. Precursors of neuropeptides that have thus far only been identified in echinoderms were identified, including L- and F-type SALMFamides, AN peptides and others. Precursors of several peptides that act as modulators of neuromuscular activity in A. japonicus were also identified. The discovery of a large repertoire of neuropeptide precursors and neuropeptides provides a basis for experimental studies that investigate the physiological roles of neuropeptide signaling systems in A. japonicus. Looking ahead, some of these neuropeptides may have effects that could be harnessed to enable improvements in the aquaculture of this economically important species.

The transcriptomic responses of the ark shell, Anadara broughtonii, to sulfide and hypoxia exposure.

Sulfide and hypoxia threaten marine organisms in various ways. Anadara broughtonii, a commercial marine bivalve in China which has great potential exposure to sulfide and hypoxia, was selected to test the responses to these stresses. Digital gene expression profile (DGE) analysis was performed on the juveniles' gills after exposed to normal condition (CG group), hypoxia (LO group), and low/high concentration of sulfide (LS/HS group, administered in hypoxia), respectively, using RNA-seq technology. A total of over 30 million clean reads were filtered from each DGE library and over 90% of them were annotated successfully. In total, 774 significant differentially expressed genes (DEGs) were detected and assigned to Gene ontology (GO) classification and KEGG Pathway enrichment analysis. The results show that many of the upregulated DEGs are related to hemoglobin, immunology, and stress responding. In the stressed A. broughtonii, cytochrome P450 and phosphoenolpyruvate carboxykinase may stimulate the glycolysis process to reduce oxygen consumption; Aminoacyl-tRNA synthetases, heat shock protein and protein disulfide isomerase probably help to maintain the genome integrity; Baculoviral IAP repeat-containing protein 2/3, mitogen-activated protein kinase and tumor necrosis factor pathways were probably responsible for protein repair, proteolysis, apoptosis and immune responses to high concentration of sulfide. Combined challenges also induced alternative oxidase and sushi repeat-containing protein, which have indistinct but probably indispensable function in invertebrates. For the first time, comprehensive transcriptome information on A. broughtonii in response to sulfide and hypoxia were provided. Our research offers new insights into the molecular mechanism behind the resistance of shellfish to sulfide and hypoxia.

Metabolic response of longitudinal muscles to acute hypoxia in sea cucumber Apostichopus japonicus (Selenka): A metabolome integrated analysis.

Hypoxia is a severe problem in aquatic environments worldwide and has caused mass mortality of sea cucumbers (& other species) for decades, seriously affecting the sustainable development of aquaculture. Investigations of the metabolic disruptions and biochemical responses associated with acute hypoxia stress in sea cucumbers can provide a theoretical basis and guidance for improving aquaculture. A metabolomics approach to characterize changes in the profiles of endogenous small molecules in response to acute hypoxia can help to identify the main underlying causes of metabolic damage and potentially suggest solutions to alleviate to improve viability. The current study uses liquid chromatography-mass spectrometry (LC-MS) and multivariate analysis methods to evaluate the metabolic profile of longitudinal muscles from A. japonicus exposed to acute hypoxia stress (by bubbling the aquaria water with nitrogen aeration to decrease dissolved oxygen to 2 mg/L in 2 min) for 6 or 24 h (experimental groups EG6 or EG24) and control group (CG, n = 10, respectively). The results showed that 29 and 62 metabolites were influenced significantly in EG6 and EG24, respectively, mainly including lipids, glycosides and their derivatives. Levels of most lipids (fatty acids, glycerolipids, glycerophospholipids, sphingolipids and sterols) were elevated in both experimental groups, and increased with elongation of hypoxia, implying that the homeostasis of synthesis and degradation of lipids and their derivatives was strongly affected by hypoxia stress. Pathway enrichment analysis was performed to further assess the importance of differential metabolite expression to the development of the A. japonicus response to hypoxia, showing that 4 (fatty acid biosynthesis, d-glutamine and d-glutamate metabolism, glycolysis/gluconeogenesis, glyoxylate and dicarboxylate metabolism) and 2 (steroid biosynthesis, longevity regulating pathway) pathways were markedly enriched in EG6 and EG24, respectively. These results suggested that fatty acid synthesis was strengthened significantly in both treatment groups, and the degree was higher in EG24 than in EG6, providing valuable information towards understanding the special adaptive mechanism of A. japonicus to hypoxia stress.

The potential contribution of miRNA-200-3p to the fatty acid metabolism by regulating AjEHHADH during aestivation in sea cucumber.

The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally-induced aestivation by a marine invertebrate more recently. In the present study, we hypothesized that miRNA-200-3p may contribute to establish rapid biological control to regulate fatty acid metabolism during a estivation. The peroxisomal bi-functional enzyme (EHHADH) is a crucial participant of the classical peroxisomal fatty acid ß-oxidation pathway, the relative mRNA transcripts and protein expressions of EHHADH were analyzed in intestine from sea cucumbers experienced long-term aestivation. Both mRNA transcripts and protein expressions of EHHADH in intestine decreased significantly during deep-aestivation as compared with non-aestivation controls. Analysis of the 3' UTR of AjEHHADH showed the presence of a conserved binding site for miR-200-3p. Level of miR-200-3p showed an inverse correlation with EHHADH mRNA transcripts and protein levels in intestine, implicating miR-200-3p may directly targeted AjEHHADH by inducing the degradation of AjEHHADH mRNA in the aestivating sea cucumber, further dual-luciferase reporter assay validated the predicted role of miRNA-200-3p in regulating AjEHHADH. In order to further understand their regulatory mechanism, we conducted the functional experiment in vivo. The overexpression of miR-200-3p in sea cucumber significantly decreased mRNA and protein expression levels of AjEHHADH. Taken together, these findings suggested the potential contribution of miRNA-200-3p to the fatty acid metabolism by regulating AjEHHADH during aestivation in sea cucumber.

A potential antiapoptotic regulation: The interaction of heat shock protein 70 and apoptosis-inducing factor mitochondrial 1 during heat stress and aestivation in sea cucumber.

The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally induced aestivation in marine invertebrates. A characteristic feature of aestivation in this species is the degeneration of the intestine. In the current study, we hypothesized that energy conservation and cytoprotective strategies need to be coordinated in the intestine to ensure long-term survival during aestivation, and there was potential relationship between heat shock protein 70 (HSP70) and apoptosis-inducing factor mitochondrial 1 (AIFM1) during extreme environmental stress. AIFM1 is a bifunctional flavoprotein that is involved in the caspase-independent activation of apoptosis. The gene and protein expression profiles of AjAIFM1 and AjHSP70 in intestinal tissue during aestivation were analyzed and results showed an inverse correlation between them, AjAIFM1 being suppressed during aestivation whereas AjHSP70 was strongly upregulated. Comparable responses were also seen when intestinal cells were isolated and analyzed in vitro for responses to heat stress at 25°C (a water temperature typical during aestivation), compared with 15°C control cells. Combined with co-immunoprecipitation studies in vivo and in vitro, our results suggested that AjHSP70 protein may have potential interaction with AjAIFM1. To determine the influence of heat stress on apoptotic rate of intestinal cells, we also assessed the DNA fragmentation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and results also supported a potential antiapoptotic response in sea cucumber during heat stress. This type of cytoprotective mechanism could be used to preserve the existing cellular components during long-term aestivation in sea cucumber.