The Holothuria leucospilota genome elucidates sacrificial organ expulsion and bioadhesive trap enriched with amyloid-patterned proteins.

Some tropical sea cucumbers of the family Holothuriidae can efficiently repel or even fatally ensnare predators by sacrificially ejecting a bioadhesive matrix termed the Cuvierian organ (CO), so named by the French zoologist Georges Cuvier who first described it in 1831. Still, the precise mechanisms for how adhesiveness genetically arose in CO and how sea cucumbers perceive and transduce danger signals for CO expulsion during defense have remained unclear. Here, we report the first high-quality, chromosome-level genome assembly of Holothuria leucospilota, an ecologically significant sea cucumber with prototypical CO. The H. leucospilota genome reveals characteristic long-repeat signatures in CO-specific outer-layer proteins, analogous to fibrous proteins of disparate species origins, including spider spidroin and silkworm fibroin. Intriguingly, several CO-specific proteins occur with amyloid-like patterns featuring extensive intramolecular cross-ß structures readily stainable by amyloid indicator dyes. Distinct proteins within the CO connective tissue and outer surface cooperate to give the expelled matrix its apparent tenacity and adhesiveness, respectively. Genomic evidence offers further hints that H. leucospilota directly transduces predator-induced mechanical pressure onto the CO surface through mediation by transient receptor potential channels, which culminates in acetylcholine-triggered CO expulsion in part or in entirety. Evolutionarily, innovative events in two distinct regions of the H. leucospilota genome have apparently spurred CO's differentiation from the respiratory tree to a lethal defensive organ against predators.

Transcriptomic analysis of sea cucumber (Holothuria leucospilota) coelomocytes revealed the echinoderm cytokine response during immune challenge.

BACKGROUND: The sea cucumber Holothuria leucospilota belongs to echinoderm, which is evolutionally the most primitive group of deuterostomes. Sea cucumber has a cavity between its digestive tract and the body wall that is filled with fluid and suspended coelomic cells similar to blood cells. The humoral immune response of the sea cucumber is based on the secretion of various immune factors from coelomocytes into the coelomic cavity. The aim of this study is to lay out a foundation for the immune mechanisms in echinoderms and their origins in chordates by using RNA-seq. RESULTS: Sea cucumber primary coelomocytes were isolated from healthy H. leucospilota and incubated with lipopolysaccharide (LPS, 10 µg/ml), polyinosinic-polycytidylic acid [Poly (I:C), 10 µg/ml] and heat-inactived Vibrio harveyi (107 cell/ml) for 24 h, respectively. After high-throughput mRNA sequencing on an Illumina HiSeq2500, a de novo transcriptome was assembled and the Unigenes were annotated. Thirteen differentially expressed genes (DEGs) were selected randomly from our data and subsequently verified by using RT-qPCR. The results of RT-qPCR were consistent with those of the RNA-seq (R2 = 0.61). The top 10 significantly enriched signaling pathways and immune-related pathways of the common and unique DEGs were screened from the transcriptome data. Twenty-one cytokine candidate DEGs were identified, which belong to 4 cytokine families, namely, BCL/CLL, EPRF1, IL-17 and TSP/TPO. Gene expression in response to LPS dose-increased treatment (0, 10, 20 and 50 µg/ml) showed that IL-17 family cytokines were significantly upregulated after 10 µg/ml LPS challenge for 24 h. CONCLUSION: A de novo transcriptome was sequenced and assembled to generate the gene expression profiling across the sea cucumber coelomocytes treated with LPS, Poly (I:C) and V. harveyi. The cytokine genes identified in DEGs could be classified into 4 cytokine families, in which the expression of IL-17 family cytokines was most significantly induced after 10 µg/ml LPS challenge for 24 h. Our findings have laid the foundation not only for the research of molecular mechanisms related to the immune response in echinoderms but also for their origins in chordates, particularly in higher vertebrates.

A fibrinogen-related protein, LvFREP2, from Litopenaeus vannamei facilitates the clearance of Vibrio harveyi.

Fibrinogen-related proteins (FREPs) play a crucial role in invertebrate immune response. In this study, we acquired a novel fibrinogen-related protein gene in Litopenaeus vannamei coding for one kind of fibrinogen-related protein, designated as LvFREP2. The complete cDNA sequence of LvFREP2 was 1903 bp long, containing an open reading frame of 1479 bp coding for LvFREP2. The LvFREP2 protein contained a putative signal peptide and a fibrinogen-related protein domain. qRT-PCRs indicated that LvFREP2 mRNA ubiquitously distributed in all examined tissues, and it was up-regulated in gills after V. harveyi and LPS challenges. The recombinant LvFREP2 agglutinated Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Vibrio alginolyticus, V. cholerae, V. vulnificus, V. parahaemolyticus, V. harveyi, Pseudomonas aeruginosa, P. fluorescens) in a calcium-dependent manner. LvFREP2 also facilitated the clearance of Vibrio harveyi in vivo. Therefore, our results suggested that lvFREP2 may have important roles in the anti-bacterial immunity of L. vannamei.

Applications of Environmental DNA (eDNA) in Monitoring the Endangered Status and Evaluating the Stock Enhancement Effect of Tropical Sea Cucumber Holothuria Scabra.

The tropical sea cucumber Holothuria scabra is naturally found in the Indo-West Pacific. However, due to their commercial value, natural H. scabra populations have declined significantly in recent years, resulting in its status as an endangered species. Surveys of H. scabra resource pose a challenge due to its specific characteristics, such as sand-burrowing behavior. To overcome this problem, our study established a convenient and feasible method for assessing H. scabra resources using environmental DNA (eDNA) monitoring technology. First, H. scabra-specific TaqMan primers and probe were designed based on its cox1 gene, followed by the development of an eDNA monitoring method for H. scabra in two separate sea areas (Xuwen and Daya Bay). The method was subsequently employed to investigate the distribution of H. scabra and assess the effects of aquaculture stock enhancement through juvenile releasing in the Weizhou Island sea area. The H. scabra eDNA monitoring approach was found to be more appropriate and credible than traditional methods, and a positive impact of stocking on H. scabra populations was observed. In summary, this is the first report to quantify eDNA concentration in a Holothuroidea species, and it provides a convenient and accurate method for surveying H. scabra resources. This study introduces novel concepts for eDNA-based detection of endangered marine benthic animals and monitoring their population distribution and abundance.

Characterization and phylogenetic analysis of the complete mitochondrial genome of a tropical sea cucumber, Holothuria fuscocinerea.

In this study, the mitochondrial genome (mitogenome) of Holothuria fuscocinerea was unraveled to be 15,890 bp in length, containing 13 protein-coding genes (PCGs), 22 tRNA genes, and 2 rRNA genes. The PCGs were initiated by four initiation codons (ATG, TAC, ATC, and ATA). Only one PCG (nad6) and five tRNA genes (tRNASer(UCN) , tRNAGln , tRNAAla , tRNAVal, and tRNAAsp ) were encoded on the light chain, and the other genes were encoded on the heavy chain. A phylogenetic tree constructed with 16S rRNA sequences showed that H. fuscocinerea is most closely related to H. leucospilota.

Fine Mapping of the High-pH Tolerance and Growth Trait-Related Quantitative Trait Loci (QTLs) and Identification of the Candidate Genes in Pacific White Shrimp (Litopenaeus vannamei).

High-pH tolerance and growth are important traits for the shrimp culture industry in areas with saline-alkali water. In the present study, an F1 full-sib family of Pacific white shrimp (Litopenaeus vannamei) was generated with a new "semidirectional cross" method, and double-digest restriction site-associated DNA sequencing (ddRAD-Seq) technology was applied to genotype the 2 parents and 148 progenies. A total of 3567 high-quality markers were constructed for the genetic linkage map, and the total map length was 4161.555 centimorgans (cM), showing 48 linkage groups (LGs) with an average interlocus length of 1.167 cM. With a constrained logarithm of odds (LOD) score ≥ 2.50, 12 high-pH tolerance and 2 growth (body weight) QTLs were located. L. vannamei genomic scaffolds were used to assist with the detection of 21 stress- and 5 growth-related scaffold genes. According to the high-pH transcriptome data of our previous study, 6 candidate high-pH response genes were discovered, and 5 of these 6 genes were consistently expressed with the high-pH transcriptome data, validating the locations of the high-pH tolerance trait-related QTLs in this study. This paper is the first report of fine-mapping high-pH tolerance and growth (body weight) trait QTLs in one L. vannamei genetic map. Our results will further benefit marker-assisted selection work and might be useful for promoting genomic research on the shrimp L. vannamei.

Na+/H+ exchanger (NHE) in Pacific white shrimp (Litopenaeus vannamei): Molecular cloning, transcriptional response to acidity stress, and physiological roles in pH homeostasis.

Na+/H+ exchangers are the most common membrane proteins involved in the regulation of intracellular pH that concurrently transport Na+ into the cells and H+ out of the cells. In this study, the full-length cDNA of the Na+/H+ exchanger (NHE) from the Pacific white shrimp (Litopenaeus vannamei) was cloned. The LvNHE cDNA is 3167 bp long, contains a 5'-untranslated region (UTR) of 74 bp and a 3'-UTR of 456 bp and an open reading frame (ORF) of 2637 bp, coding for a protein of 878 amino acids with 11 putative transmembrane domains and a long cytoplasmic tail. LvNHE shows high sequence homology with mud crab NHE at the amino acid level. LvNHE mRNA was detected in the hepatopancreas, gill, eyestalk, skin, heart, intestine, muscle, brain and stomach, with the highest abundance in the intestine. In the shrimp intestinal fragment cultures exposed to gradually declining pH medium (from pH 8.0 to pH 6.4), the LvNHE mRNA expression was significantly stimulated, with the highest response when incubated in pH 7.0 medium for 6 h. To investigate the functional roles of LvNHE in pH regulation at the physiological and cellular levels, the LvNHE mRNA expression was silenced by siRNA knockdown. Upon low-pH challenge, the hemolymph pH was significantly reduced in the LvNHE mRNA knockdown shrimp. In addition, knockdown of LvNHE mRNA reduced the recovery capacity of intracellular pH in intestinal fragment cultures after acidification. Altogether, this study demonstrates the role of NHE in shrimp response to low pH stress and provides new insights into the acid/base homeostasis mechanisms of crustaceans.

The complete mitochondrial genome of a tropical sea cucumber, Holothuria leucospilota.

The mitochondrial genome of Holothuria leucospilota was 15,906 bp in length, containing 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. There were four initiation codons (ATG, ATT, ATC, and ATA) for the PCGs, and the termination codon of most PCGs was TAA, except for nad4 (TAG) and nad6 (TAG). Only one PCG (nad6) and five tRNA genes (tRNASer(UCN) , tRNAGln , tRNAAla , RNAVal , and tRNAAsp ) were encoded on the light chain; the other genes were encoded on the heavy chain. H. leucospilota was most closely related to Holothuria scabra in a phylogenetic tree.

Analysis of the transcriptome data in Litopenaeus vannamei reveals the immune basis and predicts the hub regulation-genes in response to high-pH stress.

Soil salinization erodes the farmlands and poses a serious threat to human life, reuse of the saline-alkali lands as cultivated resources becomes increasingly prominent. Pacific white shrimp (Litopenaeus vannamei) is an important farmed aquatic species for the development and utilization of the saline-alkali areas. However, little is known about the adaptation mechanism of this species in terms of high-pH stress. In the present study, a transcriptome analysis on the gill tissues of L. vannamei in response to high-pH stress (pH 9.3 ± 0.1) was conducted. After analyzing, the cyclic nucleotide gated channel-Ca2+ (CNGC-Ca2+) and patched 1 (Ptc1) were detected as the majority annotated components in the cAMP signaling pathway (KO04024), indicating that the CNGC-Ca2+ and Ptc1 might be the candidate components for transducing and maintaining the high-pH stress signals, respectively. The immunoglobulin superfamily (IgSF), heat shock protein (HSP), glutathione s-transferase (GST), prophenoloxidase/phenoloxidase (proPO/PO), superoxide dismutase (SOD), anti-lipopolysaccharide factor (ALF) and lipoprotein were discovered as the major transcribed immune factors in response to high-pH stress. To further detect hub regulation-genes, protein-protein interaction (PPI) networks were constructed; the genes/proteins "Polymerase (RNA) II (DNA directed) polypeptide A" (POLR2A), "Histone acetyltransferase p300" (EP300) and "Heat shock 70kDa protein 8" (HSPA8) were suggested as the top three hub regulation-genes in response to acute high-pH stress; the genes/proteins "Heat shock 70kDa protein 4" (HSPA4), "FBJ murine osteosarcoma viral oncogene homolog" (FOS) and "Nucleoporin 54kDa" (NUP54) were proposed as the top three hub regulation-genes involved in adapting endurance high-pH stress; the protein-interactions of "EP300-HSPA8" and "HSPA4-NUP54" were detected as the most important biological interactions in response to the high-pH stress; and the HSP70 family genes might play essential roles in the adaptation of the high-pH stress environment in L. vannamei. These findings provide the first insight into the molecular and immune basis of L. vannamei in terms of high-pH environments, and the construction of a PPI network might improve our understanding in revealing the hub regulation-genes in response to abiotic stress in shrimp species and might be beneficial for further studies.