An ancient whole-genome duplication in barnacles contributes to their diversification and intertidal sessile life adaptation.

INTRODUCTION: Whole-genome duplication (WGD) is one of the most sudden and dramatic events rarely reported in invertebrates, but its occurrence can lead to physiological, morphological, and behavioral diversification. WGD has also never been reported in barnacles, which is one of the most unique groups of crustaceans with extremely speciallized morphology (calcareous shells) and habits (intertidal sessile lifestyle). OBJECTIVES: To investigate whether WGD has occurred in barnacles and examine its potential role in driving the adaptive evolution and diversification of barnacles. METHODS: Based on a newly sequenced and assembled chromosome-level barnacle genome, a novel WGD event has been identified in barnacles through a comprehensive analysis of interchromosomal synteny, the Hox gene cluster, and synonymous substitution distribution. RESULTS: We provide ample evidences for WGD in the barnacle genomes. Comparative genomic analysis indicates that this WGD event predates the divergence of Thoracicalcarea, occurring more than 247 million years ago. The retained ohnologs from the WGD are primarily enriched in various pathways related to environmental information processing, shedding light on the adaptive evolution and diversification of intertidal sessile lifestyle. In addition, transcriptomic analyses show that most of these ohnologs were differentially expressed following the ebb of tide. And the cytochrome P450 ohnologs with differential expression patterns are subject to subfunctionalization and/or neofunctionalization for intertidal adaptation. Besides WGD, parallel evolution underlying intertidal adaptation has also occurred in barnacles. CONCLUSION: This study revealed an ancient WGD event in the barnacle genomes, which is potentially associated with the origin and diversification of thoracican barnacles, and may have contributed to the adaptive evolution of their intertidal sessile lifestyle.

LvCD14L Acts as a Novel Pattern Recognition Receptor and a Regulator of the Toll Signaling Pathway in Shrimp.

Leucine-rich repeat (LRR) is a structural motif has important recognition function in immune receptors, such as Tolls and NOD-like receptors (NLRs). The immune-related LRR proteins can be divided into two categories, LRR-containing proteins and LRR-only proteins. The latter contain LRR motifs while they are without other functional domains. However, the functional mechanisms of the LRR-only proteins were still unclear in invertebrates. Here, we identified a gene encoding a secretory LRR-only protein, which possessed similarity with vertebrate CD14 and was designated as LvCD14L, from the Pacific whiteleg shrimp Litopenaeus vannamei. Its transcripts in shrimp hemocytes were apparently responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvCD14L with dsRNA resulted in significant increase of the viable bacteria in the hepatopancreas of shrimp upon V. parahaemolyticus infection. Further functional studies revealed that LvCD14L could bind to microorganisms' PAMPs, showed interaction with LvToll1 and LvToll2, and regulated the expression of LvDorsal and LvALF2 in hemocytes. These results suggest that LvCD14L functions as a pattern recognition receptor and activates the NF-κB pathway through interaction with LvTolls. The present study reveals a shrimp LvCD14L-Tolls-NF-κB signaling pathway like the CD14/TLR4/NF-κB signaling pathway in mammalians, which enriches the functional mechanism of secretory LRR-only immune receptors during pathogens infection in invertebrates.

IAG Regulates the Expression of Cytoskeletal Protein-Encoding Genes in Shrimp Testis.

Insulin-like androgenic gland hormone (IAG) is the master regulator of sexual differentiation and testis development in male crustaceans. However, the molecular mechanism on how IAG functions during testis development is still largely unknown. Here, the transcriptional changes were analyzed in the testes of shrimp after LvIAG knockdown in Litopenaeus vannamei. Differential expression analysis identified 111 differentially expressed genes (DEGs), including 48 upregulated DEGs and 63 downregulated DEGs, in testes of shrimp after LvIAG knockdown. Gene ontology (GO) analysis showed that these DEGs were apparently enriched in cytoskeleton-related GO items. Gene function analysis showed that genes enriched in these GO items mainly encoded actin, myosin, and heat shock protein. Interestingly, these genes were all downregulated in testis after LvIAG knockdown, which was confirmed by qRT-PCR detection. Furthermore, injection of LvIAG protein that was recombinantly expressed in insect cells upregulated the expression levels of these genes. The present study revealed that shrimp IAG might function in testis development through regulating the expression of cytoskeletal protein-encoding genes, which would provide new insights into understanding the functional mechanisms of IAG on male sexual development of crustaceans.

Genome of a giant isopod, Bathynomus jamesi, provides insights into body size evolution and adaptation to deep-sea environment.

BACKGROUND: The deep-sea may be regarded as a hostile living environment, due to low temperature, high hydrostatic pressure, and limited food and light. Isopods, a species-rich group of crustaceans, are widely distributed across different environments including the deep sea and as such are a useful model for studying adaptation, migration, and speciation. Similar to other deep-sea organisms, giant isopods have larger body size than their shallow water relatives and have large stomachs and fat bodies presumably to store organic reserves. In order to shed light on the genetic basis of these large crustaceans adapting to the oligotrophic environment of deep-sea, the high-quality genome of a deep-sea giant isopod Bathynomus jamesi was sequenced and assembled. RESULTS: B. jamesi has a large genome of 5.89 Gb, representing the largest sequenced crustacean genome to date. Its large genome size is mainly attributable to the remarkable proliferation of transposable elements (84%), which may enable high genome plasticity for adaptive evolution. Unlike its relatives with small body size, B. jamesi has expanded gene families related to pathways of thyroid and insulin hormone signaling that potentially contribute to its large body size. Transcriptomic analysis showed that some expanded gene families related to glycolysis and vesicular transport were specifically expressed in its digestive organs. In addition, comparative genomics and gene expression analyses in six tissues suggested that B. jamesi has inefficient lipid degradation, low basal metabolic rate, and bulk food storage, suggesting giant isopods adopt a more efficient mechanism of nutrient absorption, storage, and utilization to provide sustained energy supply for their large body size. CONCLUSIONS: Taken together, the giant isopod genome may provide a valuable resource for understanding body size evolution and adaptation mechanisms of macrobenthic organisms to deep-sea environments.

A Novel TRIM9 Protein Promotes NF-κB Activation Through Interacting With LvIMD in Shrimp During WSSV Infection.

The TRIpartite Motif (TRIM) proteins play key roles in cell differentiation, apoptosis, development, autophagy, and innate immunity in vertebrates. In the present study, a novel TRIM9 homolog (designated as LvTRIM9-1) specifically expressed in the lymphoid organ of shrimp was identified from the Pacific whiteleg shrimp Litopenaeus vannamei. Its deduced amino acid sequence possesses the typical features of TRIM proteins, including a RING domain, two B-boxes, a coiled-coil domain, a FN3 domain, and a SPRY domain. The transcripts of LvTRIM9-1 were mainly located in the lymphoid tubules of the lymphoid organ. Knockdown of LvTRIM9-1 could apparently inhibit the transcriptions of some genes from white spot syndrome virus (WSSV) and reduce the viral propagation in the lymphoid organ. Overexpression of LvTRIM9-1 in mammalian cells could activate the promoter activity of NF-κB, and an in vivo experiment in shrimp showed that knockdown of LvTRIM9-1 reduced the expression of LvRelish in the lymphoid organ. Yeast two-hybridization and co-immunoprecipitation (Co-IP) assays confirmed that LvTRIM9-1 could directly interact with LvIMD, a key component of the IMD pathway, through its SPRY domain. These data suggest that LvTRIM9-1 could activate the IMD pathway in shrimp via interaction with LvIMD. This is the first evidence to show the regulation of a TRIM9 protein on the IMD pathway through its direct interaction with IMD, which will enrich our knowledge on the role of TRIM proteins in innate immunity of invertebrates.

tRNA copy number and codon usage in the sea cucumber genome provide insights into adaptive translation for saponin biosynthesis.

Genomic tRNA copy numbers determine cytoplasmic tRNA abundances, which in turn influence translation efficiency, but the underlying mechanism is not well understood. Using the sea cucumber Apostichopus japonicus as a model, we combined genomic sequence, transcriptome expression and ecological food resource data to study its codon usage adaptation. The results showed that, unlike intragenic non-coding RNAs, transfer RNAs (tRNAs) tended to be transcribed independently. This may be attributed to their specific Pol III promoters that lack transcriptional regulation, which may underlie the correlation between genomic copy number and cytoplasmic abundance of tRNAs. Moreover, codon usage optimization was mostly restrained by a gene's amino acid sequence, which might be a compromise between functionality and translation efficiency for stress responses were highly optimized for most echinoderms, while enzymes for saponin biosynthesis (LAS, CYPs and UGTs) were especially optimized in sea cucumbers, which might promote saponin synthesis as a defence strategy. The genomic tRNA content of A. japonicus was positively correlated with amino acid content in its natural food particles, which should promote its efficiency in protein synthesis. We propose that coevolution between genomic tRNA content and codon usage of sea cucumbers facilitates their saponin synthesis and survival using food resources with low nutrient content.

Clustering genomic organization of sea cucumber miRNAs impacts their evolution and expression.

Echinoderms are marine deuterostomes with fascinating adaptation features such as aestivation and organ regeneration. However, post-transcriptional gene regulation by microRNAs (miRNAs) underlying these features are largely unexplored. Here, using homology-based and de novo approaches supported by expression data, we provided a comprehensive annotation of miRNA genes in the sea cucumber Apostichopus japonicus. By linkage and phylogenic analyses, we characterized miRNA genomic organization, evolutionary history and expression regulation. The results showed that sea cucumbers evolved a large number of new miRNAs, which tended to form polycistronic clusters via tandem duplication that had been especially active in the echinoderms. Most new miRNAs were weakly expressed, but miRNA clustering increased the expression level of clustered new miRNAs. The most abundantly expressed new miRNAs were organized in a single tandem cluster (cluster n2), which was activated during aestivation and intestine regeneration. Overall, our analyses suggest that clustering of miRNAs is important for their evolutionary origin, expression control, and functional cooperation.

Isoflurane produces antidepressant effects inducing BDNF-TrkB signaling in CUMS mice.

RATIONALE: The volatile anesthetic isoflurane is suggested to produce a rapid and robust antidepressive effect in preliminary clinical trials. Recently, isoflurane was found to activate the tropomyosin receptor kinase B (TrkB) signaling which is the underlying mechanism of the rapid antidepressant ketamine. OBJECTIVE: Our study investigated the effect of isoflurane anesthesia on chronic unpredictable mild stressed (CUMS) model in mice and verified the role of brain-derived neurotrophic factor (BDNF)/TrkB/ the mammalian target of rapamycin (mTOR) signaling in the antidepressant effect of isoflurane. METHODS: We employed the CUMS model of depression to assess the rapid antidepressant effect of isoflurane by the forced swimming test (FST), the sucrose preference test (SPT), and the novelty suppressed feeding test (NSFT). The protein expression of BDNF and TrkB/protein kinase B (PKB or Akt)/mTOR was determined through Western blot. The dendritic spine density in the hippocampus and medial prefrontal cortex (PFC) was measured by the Golgi staining. RESULTS: A brief burst-suppressing isoflurane anesthesia rapidly reversed the behavioral deficits caused by CUMS procedure, normalized the expression of BDNF and further activated the TrkB signaling pathway in CUMS-induced stressed mice in both prefrontal cortex (PFC) and hippocampus (HC). All of those behavioral and proteomic effects were blocked by K252a, a selective receptor inhibitor of TrkB. Isoflurane significantly promoted the formation of dendritic spines in both medial prefrontal cortex (mPFC), CA1, CA3, and DG of the hippocampus. CONCLUSION: Our study indicates that isoflurane exerts a rapid antidepressant-like effect in CUMS depression animal model, and the activation of BDNF/TrkB signaling pathway plays an indispensable role in the biological and behavioral antidepressant effects of isoflurane. A single exposure to isoflurane could repair synaptic damage caused by chronic stimulation.

Wnt Signaling Pathway Linked to Intestinal Regeneration via Evolutionary Patterns and Gene Expression in the Sea Cucumber Apostichopus japonicus.

Many echinoderms are regenerative species that exhibit exceptional regenerative capacity, and sea cucumber is a representative organism that could regenerate the whole intestine after evisceration. There are many signaling pathways participate in the regeneration process, but it is not clear which is essential for the intestinal regeneration. In this study, we performed genome-wide comprehensive analyses on these regeneration-related signaling pathways, and found the Wnt signaling pathway was one of the most conservative pathways among regenerative species. Additionally, among these signaling pathways, we found that the Wnt signaling pathway was the only one under positive selection in regenerative echinoderms, and the only one enriched by differentially expressed genes during the intestinal regeneration. Thus, it suggests both coding sequence and gene expression of the Wnt signaling pathway have been shaped by natural selection to provide the genetic architecture for intestinal regeneration. Wnt7, Fz7, and Dvl are the three positively selected genes and also happen to be three upstream genes in the Wnt signaling pathway. They are all significantly upregulated at the early stages of regeneration, which may contribute significantly to the early activation of Wnt signaling and the initiation of intestinal regeneration. Expression knockdown of Wnt7 and Dvl by RNA interference significantly inhibit intestinal extension, implying that they are essential for intestinal regeneration. As an important regeneration-related gene, the downstream gene c-Myc is also conserved and highly expressed during the whole regeneration stages, which may make the Wnt/c-Myc signaling to be an important way to promote intestinal regeneration. Therefore, it is reasonable to conclude that the Wnt signaling pathway is the chosen one to play an important role in intestinal regeneration of sea cucumbers, or even in the regeneration of other echinoderms.

Penaeid shrimp genome provides insights into benthic adaptation and frequent molting.

Crustacea, the subphylum of Arthropoda which dominates the aquatic environment, is of major importance in ecology and fisheries. Here we report the genome sequence of the Pacific white shrimp Litopenaeus vannamei, covering ~1.66 Gb (scaffold N50 605.56 Kb) with 25,596 protein-coding genes and a high proportion of simple sequence repeats (>23.93%). The expansion of genes related to vision and locomotion is probably central to its benthic adaptation. Frequent molting of the shrimp may be explained by an intensified ecdysone signal pathway through gene expansion and positive selection. As an important aquaculture organism, L. vannamei has been subjected to high selection pressure during the past 30 years of breeding, and this has had a considerable impact on its genome. Decoding the L. vannamei genome not only provides an insight into the genetic underpinnings of specific biological processes, but also provides valuable information for enhancing crustacean aquaculture.

Development of a primary culture system for haematopoietic tissue cells from Cherax quadricarinatus and an exploration of transfection methods.

Various known and unknown viral diseases can threaten crustacean aquaculture. To develop prophylactic and therapeutic strategies against viruses, crustacean cell lines are urgently needed for immunology and virology studies. However, there are currently no permanent crustacean cell lines available. In this study, we developed a new method for preparing crayfish plasma (CP) and found that CP enhanced the proliferative capacity of haematopoietic tissue (hpt) cells from Cherax quadricarinatus by an EdU (5-ethynyl-2'-deoxyuridine) assay. The optimal CP concentration for hpt cell culture and the optimal subculture method are discussed. To achieve efficient expression of a foreign gene in hpt cells cultured in vitro, different transfection methods and vectors were analysed. We found that Lipofectamine 2000 could be used to efficiently transfect a foreign vector into hpt cells and exhibited a lower level of cytotoxicity than the other methods tested, and transfection of pEGFP-N1/w249 and pDHsp70-EGFP-FLAG resulted in high EGFP expression. By transmission electron microscopy (TEM) and virus copy number analysis, we found that white spot syndrome virus (WSSV) could infect hpt cells and multiply efficiently. Our results implied that the crayfish hpt cell culture system we improved could be used as a replacement for immortal crustacean cell lines in viral infection studies. Our findings provide a solid foundation for future immortalization and gene function studies in crustacean cells.

The sea cucumber genome provides insights into morphological evolution and visceral regeneration.

Apart from sharing common ancestry with chordates, sea cucumbers exhibit a unique morphology and exceptional regenerative capacity. Here we present the complete genome sequence of an economically important sea cucumber, A. japonicus, generated using Illumina and PacBio platforms, to achieve an assembly of approximately 805 Mb (contig N50 of 190 Kb and scaffold N50 of 486 Kb), with 30,350 protein-coding genes and high continuity. We used this resource to explore key genetic mechanisms behind the unique biological characters of sea cucumbers. Phylogenetic and comparative genomic analyses revealed the presence of marker genes associated with notochord and gill slits, suggesting that these chordate features were present in ancestral echinoderms. The unique shape and weak mineralization of the sea cucumber adult body were also preliminarily explained by the contraction of biomineralization genes. Genome, transcriptome, and proteome analyses of organ regrowth after induced evisceration provided insight into the molecular underpinnings of visceral regeneration, including a specific tandem-duplicated prostatic secretory protein of 94 amino acids (PSP94)-like gene family and a significantly expanded fibrinogen-related protein (FREP) gene family. This high-quality genome resource will provide a useful framework for future research into biological processes and evolution in deuterostomes, including remarkable regenerative abilities that could have medical applications. Moreover, the multiomics data will be of prime value for commercial sea cucumber breeding programs.

Establishment and characterization of a skin epidermal cell line from mud loach, Misgurnus anguillicaudatus, (MASE) and its interaction with three bacterial pathogens.

A continuous skin epidermal cell line from mud Loach (Misgurnus anguillicaudatus) (MASE cell line) was established with its application in bacteria infection demonstrated in this study. Primary MASE cell culture was initiated at 26 °C in Dulbecco's modified Eagle medium/F12 medium (1:1; pH7.2) supplemented with 20% fetal bovine serum (FBS). The primary MASE cells in spindle morphology proliferated into a confluent monolayer within 2 weeks, and were continuously subcultured even in 10% FBS- DMEM/F12 after 10 passages. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 26 °C. The MASE cells have been subcultured steadily over Passage 90 with a population doubling time of 53.3 h at Passage 60. Chromosome analysis revealed that 60.5% of MASE cells at Passage 60 maintained the normal diploid chromosome number (50) with a normal karyotype of 10m+4sm + 36t. Bacteria from the three species (Aeromonas veronii, Vibrio parahaemolyticus and Escherichia coli) were used to investigate the interactions between bacteria and cellular hosts. The three strains could be attached to the MASE cells and replicate at different levels. A. veronii could induce apoptosis in the MASE cells, with highest adherence rate among the three strains, whereas V. parahaemolyticus could cause highest cell death rate through a non-apoptotic cell death pathway, with high level of replication. The results revealed that different bacteria could interact with the MASE cells in different manners, and divergent pathways might lie in mediating cell death when cellular hosts confronted with pathogen infection. Therefore, the MASE cell line may serve as a useful tool for studying the interaction between skin bacteria and fish cells.

Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytose beads, E. coli and WSSV, but in different ways.

The hemocytes of the red claw crayfish Cherax quadricarinatus are classified by morphologic observation into the following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Density gradient centrifugation with Percoll was developed to separate these three subpopulations of hemocytes. Beads, Escherichia coli, and FITC labeling WSSV were used to investigate the characteristics of granulocytes by using scanning electron microscope, transmission electron microscope, and laser scan confocal microscope. Results showed that granulocytes could phagocytose beads and E. coli by endocytic pathways. WSSV could rely on caveolae-mediated endocytosis to mainly enter into granulocytes. These results could elucidate the mechanism of the innate immunity function of granulocytes, and it also showed the mechanism by which WSSV invaded granulocytes in the red claw crayfish.

A novel tumor necrosis factor ligand superfamily member (CsTL) from Ciona savignyi: molecular identification and expression analysis.

A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi. The CsTL cDNA consisted of 995 nucleotides and encoded 281 amino acids. A conserved TNF family signature and several motifs of TNF ligand superfamily were identified in deduced amino acid sequence of CsTL. Phylogenetic analysis grouped CsTL, CiTNF (predicted TNF ligand superfamily homolog in Ciona intestinalis) and urchin TL1A with their own cluster apart from mammalian TNFalpha, LTA, TNFSF15 and fish TNFalpha proteins. Expression studies demonstrated that CsTL mRNA is present in all tested tissues from unchallenged ascidians and its expression was significantly upregulated in hemocytes following LPS injection. The recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells. Present results indicated that TNF ligand superfamily molecules are present in marine invertebrates.