Spatio-temporal patterns of ovarian development and VgR gene silencing reduced fecundity in parthenogenetic Artemia.

The halophilic zooplankton brine shrimp Artemia has been used as an experimental animal in multidisciplinary studies. However, the reproductive patterns and its regulatory mechanisms in Artemia remain unclear. In this study, the ovarian development process of parthenogenetic Artemia (A. parthenogenetica) was divided into five stages, and oogenesis or egg formation was identified in six phases. The oogenesis mode was assumed to be polytrophic. We also traced the dynamic translocation of candidate germline stem cells (cGSCs) using EdU labelling and elucidated several key cytological events in oogenesis through haematoxylin and eosin staining and fluorescence imaging. Distinguished from the ovary structure of insects and crustaceans, Artemia germarium originated from ovariole buds and are located at the base of the ovarioles. RNA-seq based on five stages of ovarian development identified 2657 upregulated genes related to reproduction by pair-to-pair comparison. Gbb, Dpp, piwi, vasa, nanos, VgA and VgR genes associated with cGSCs recognition and reproductive development were screened and verified using qPCR. Silencing of the VgR gene in A. parthenogenetica (Ap-VgR) at ovarian development Stage II led to a low level of gene expression (less than 10%) within 5 days, which resulted in variations in oogenesis-related gene expression and significantly inhibited vitellogenesis, impeded oocyte maturation, and eventually decreased the number of offspring. In conclusion, we have illustrated the patterns of ovarian development, outlined the key spatio-temporal features of oogenesis and identified the negative impacts of VgR gene knockdown on oogenesis using A. parthenogenetica as an experimental animal. The findings of this study also lay a foundation for the further study of reproductive biology of invertebrates.

Penaeid Shrimp Chromosome Studies Entering the Post-Genomic Era.

Chromosome studies provide the foundation for comprehending inheritance, variation, systematics, and evolution. Penaeid shrimps are a group of crustaceans with great economic importance. Basic cytogenetic information obtained from these shrimps can be used to study their genome structure, chromosome relationships, chromosome variation, polyploidy manipulation, and breeding. The study of shrimp chromosomes experienced significant growth in the 1990s and has been closely linked to the progress of genome research since the application of next-generation sequencing technology. To date, the genome sequences of five penaeid shrimp species have been published. The availability of these genomes has ushered the study of shrimp chromosomes into the post-genomic era. Currently, research on shrimp cytogenetics not only involves chromosome counting and karyotyping, but also extends to investigating submicroscopic changes; exploring genome structure and regulation during various cell divisions; and contributing to the understanding of mechanisms related to growth, sexual control, stress resistance, and genome evolution. In this article, we provide an overview of the progress made in chromosome research on penaeid shrimp. We emphasize the mutual promotion between studies on chromosome structure and genome research and highlight the impact of chromosome-level assembly on studies of genome structure and function. Additionally, we summarize the emerging trends in post-genomic-era shrimp chromosome research.

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.

Convergent evolution of barnacles and molluscs sheds lights in origin and diversification of calcareous shell and sessile lifestyle.

The calcareous shell and sessile lifestyle are the representative phenotypes of many molluscs, which happen to be present in barnacles, a group of unique crustaceans. The origin of these phenotypes is unclear, but it may be embodied in the convergent genetics of such distant groups (interphylum). Herein, we perform comprehensive comparative genomics analysis in barnacles and molluscs, and reveal a genome-wide strong convergent molecular evolution between them, including coexpansion of biomineralization and organic matrix genes for shell formation, and origination of lineage-specific orphan genes for settlement. Notably, the expanded biomineralization gene encoding alkaline phosphatase evolves a novel, highly conserved motif that may trigger the origin of barnacle shell formation. Unlike molluscs, barnacles adopt novel organic matrices and cement proteins for shell formation and settlement, respectively, and their calcareous shells have potentially originated from the cuticle system of crustaceans. Therefore, our study corroborates the idea that selection pressures driving convergent evolution may strongly act in organisms inhabiting similar environments regardless of phylogenetic distance. The convergence signatures shed light on the origin of the shell and sessile lifestyle of barnacles and molluscs. In addition, notable non-convergence signatures are also present and may contribute to morphological and functional specificities.

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 newly identified NLR-like gene participates in bacteria and virus infection possibly through regulating hemocytes apoptosis in shrimp.

The nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play important roles in innate immunity. Previously, we identified an NLR-like gene, LvNLRPL1, and found that it participated in Vibrio infection and regulated hemocytes apoptosis in the Pacific whiteleg shrimp Litopenaeus vannamei. However, it is still unclear whether other NLR-like genes exist in shrimp and how they function during virus infection. In the present study, a novel NLR-like gene (LvNLRPL2) was identified and functionally characterized in L. vannamei. LvNLRPL2 was highly expressed in hemocytes and responsive to both Vibrio parahaemolyticus and white spot syndrome virus (WSSV) infection. Knockdown of LvNLRPL2 could markedly increase the proliferation of Vibrio and the mortality of shrimp infected with V. parahaemolyticus, whereas inhibit in vivo WSSV propagation in shrimp, indicating its distinct roles during Vibrio and WSSV infection. After LvNLRPL2 knockdown, the apoptotic rate of hemocytes increased, and the expression levels of LvCaspase 2, 3 and 5 were significantly up-regulated. In addition, LvNLRPL2 could form a hetero-dimer with LvNLRPL1 through their NACHT domains. These results suggest that LvNLRPL2 might resist bacterial infection while promote WSSV propagation by forming hetero-dimer with LvNLRPL1 and then inhibiting apoptosis of hemocytes. These data will be helpful for understanding the functions of NLR-like genes and their regulation mechanisms in crustaceans.

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.

The immune function of a NLR like gene, LvNLRPL1, in the Pacific whiteleg shrimp Litopenaeus vannamei.

NOD-like receptors (NLRs) are a kind of pattern recognition receptors, which are vital for detection of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and then trigger downstream immune responses in vertebrates. Although many NLR like genes have been identified in invertebrates in recent years, knowledge about their immune functions is still very limited. In the present study, a NLR like gene, designated as LvNLRPL1, was identified in Litopenaeus vannamei. It was widely expressed in multiple tissues and responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvNLRPL1 could accelerate the proliferation of Vibrio in hepatopancreas and increase the mortality rate of shrimp after Vibrio infection. Meanwhile, knockdown of LvNLRPL1 also up-regulated the expression of Caspase 2, 3 and 5 in hemocytes, which caused apoptosis of more hemocytes. These results indicated that LvNLRPL1 played important immune functions in shrimp during Vibrio infection through regulating the apoptosis of hemocytes in shrimp. To our knowledge, this is the first time to reveal the immune function of a NLR like gene in crustaceans.

Comparison of Gene Expression Between Resistant and Susceptible Families Against VPAHPND and Identification of Biomarkers Used for Resistance Evaluation in Litopenaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) has caused a heavy loss to shrimp aquaculture since its outbreak. Vibrio parahaemolyticus (VPAHPND) is regarded as one of the main pathogens that caused AHPND in the Pacific white shrimp Litopenaeus vannamei. In order to learn more about the mechanism of resistance to AHPND, the resistant and susceptible shrimp families were obtained through genetic breeding, and comparative transcriptome approach was used to analyze the gene expression patterns between resistant and susceptible families. A total of 95 families were subjected to VPAHPND challenge test, and significant variations in the resistance of these families were observed. Three pairs of resistant and susceptible families were selected for transcriptome sequencing. A total of 489 differentially expressed genes (DEGs) that presented in at least two pairwise comparisons were screened, including 196 DEGs highly expressed in the susceptible families and 293 DEGs in the resistant families. Among these DEGs, 16 genes demonstrated significant difference in all three pairwise comparisons. Gene set enrichment analysis (GSEA) of all 27,331 expressed genes indicated that some energy metabolism processes were enriched in the resistant families, while signal transduction and immune system were enriched in the susceptible families. A total of 32 DEGs were further confirmed in the offspring of the detected families, among which 19 genes were successfully verified. The identified genes in this study will be useful for clarifying the genetic mechanism of shrimp resistance against Vibrio and will further provide molecular markers for evaluating the disease resistance of shrimp in the breeding program.

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.

Corrigendum: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei.

[This corrects the article DOI: 10.3389/fphys.2019.01525.].

Genome Sequencing and Assembly Strategies and a Comparative Analysis of the Genomic Characteristics in Penaeid Shrimp Species.

Penaeid shrimp (family Penaeidae) represents one of the most economically and ecologically important groups of crustaceans. However, their genome sequencing and assembly have encountered extreme difficulties during the last 20 years. In this study, based on our previous genomic data, we investigated the genomic characteristics of four penaeid shrimp species and identified potential factors that result in their poor genome assembly, including heterozygosity, polyploidization, and repeats. Genome sequencing and comparison of somatic cells (diploid) of the four shrimp species and a single sperm cell (haploid) of Litopenaeus vannamei identified a common bimodal distribution of K-mer depths, suggesting either high heterozygosity or abundant homo-duplicated sequences present in their genomes. However, penaeids have not undergone whole-genome duplication as indicated by a series of approaches. Besides, the remarkable expansion of simple sequence repeats was another outstanding character of penaeid genomes, which also made the genome assembly highly fragmented. Due to this situation, we tried to assemble the genome of penaeid shrimp using various genome sequencing and assembly strategies and compared the quality. Therefore, this study provides new insights about the genomic characteristics of penaeid shrimps while improving their genome assemblies.

Identification of Growth-Associated Genes by Genome-Wide Association Study and Their Potential Application in the Breeding of Pacific White Shrimp (Litopenaeus vannamei).

The Pacific white shrimp (Litopenaeus vannamei) is the most widely cultured shrimp in the world. A great attention has been paid to improve its body weight (BW) at harvest through genetic selection for decades. Genome-wide association study (GWAS) is a tool to dissect the genetic basis of the traits. In this study, a GWAS approach was conducted to find genes related to BW through genotyping 94,113 single nucleotide polymorphisms (SNPs) in 200 individuals from a breeding population. Four BW-related SNPs located in LG19 and LG39 were identified. Through further candidate gene association analysis, the SNPs in two candidate genes, deoxycytidylate deaminase and non-receptor protein tyrosine kinase, were found to be related with the body weight of the shrimp. Marker-assisted best linear unbiased prediction (MA-BLUP) based on the SNPs in these two genes was used to estimate the breeding values, and the result showed that the highest prediction accuracy of MA-BLUP was increased by 9.4% than traditional BLUP. These results will provide useful information for the marker-assisted breeding in L. vannamei.

The Chinese mitten crab genome provides insights into adaptive plasticity and developmental regulation.

The infraorder Brachyura (true or short-tailed crabs) represents a successful group of marine invertebrates yet with limited genomic resources. Here we report a chromosome-anchored reference genome and transcriptomes of the Chinese mitten crab Eriocheir sinensis, a catadromous crab and invasive species with wide environmental tolerance, strong osmoregulatory capacity and high fertility. We show the expansion of specific gene families in the crab, including F-ATPase, which enhances our knowledge on the adaptive plasticity of this successful invasive species. Our analysis of spatio-temporal transcriptomes and the genome of E. sinensis and other decapods shows that brachyurization development is associated with down-regulation of Hox genes at the megalopa stage when tail shortening occurs. A better understanding of the molecular mechanism regulating sexual development is achieved by integrated analysis of multiple omics. These genomic resources significantly expand the gene repertoire of Brachyura, and provide insights into the biology of this group, and Crustacea in general.

Chitin Synthesis and Degradation in Crustaceans: A Genomic View and Application.

Chitin is among the most important components of the crustacean cuticular exoskeleton and intestinal peritrophic matrix. With the progress of genomics and sequencing technology, a large number of gene sequences related to chitin metabolism have been deposited in the GenBank database in recent years. Here, we summarized the genes and pathways associated with the biosynthesis and degradation of chitins in crustaceans based on genomic analyses. We found that chitin biosynthesis genes typically occur in single or two copies, whereas chitin degradation genes are all multiple copies. Moreover, the chitinase genes are significantly expanded in most crustacean genomes. The gene structure and expression pattern of these genes are similar to those of insects, albeit with some specific characteristics. Additionally, the potential applications of the chitin metabolism genes in molting regulation and immune defense, as well as industrial chitin degradation and production, are also summarized in this review.

A Lymphoid Organ Specific Anti-Lipopolysaccharide Factor from Litopenaeus vannamei Exhibits Strong Antimicrobial Activities.

Different shrimp species are known to possess apparent distinct resistance to different pathogens in aquaculture. However, the molecular mechanism underlying this finding still remains unknown. One kind of important antimicrobial peptides, anti-lipopolysaccharide factors (ALF), exhibit broad-spectrum antimicrobial activities. Here, we reported a newly identified ALF from the shrimp Litopenaeus vannamei and compared the immune function with its counterpart in the shrimp Fenneropenaeus chinensis. The ALF, designated as LvALF8, was specifically expressed in the lymphoid organ of L. vannamei. The expression level of LvALF8 was apparently changed after white spot syndrome virus (WSSV) or Vibrio parahaemolyticus challenges. The synthetic LBD peptide of LvALF8 (LvALF8-LBD) showed strong antibacterial activities against most tested Gram-negative and Gram-positive bacteria. LvALF8-LBD could also inhibit the in vivo propagation of WSSV similar as FcALF8-LBD, the LBD of LvALF8 counterpart in F. chinensis. However, LvALF8-LBD and FcALF8-LBD exhibited apparently different antibacterial activity against V. parahaemolyticus, the main pathogen causing acute hepatopancreatic necrosis disease (AHPND) of affected shrimp. A structural analysis showed that the positive net charge and amphipathicity characteristics of LvALF8-LBD peptide were speculated as two important components for its enhanced antimicrobial activity compared to those of FcALF8-LBD. These new findings may not only provide some evidence to explain the distinct disease resistance among different shrimp species, but also lay out new research ground for the testing and development of LBD-originated antimicrobial peptides to control of shrimp diseases.

Simple sequence repeats drive genome plasticity and promote adaptive evolution in penaeid shrimp.

Simple sequence repeats (SSRs) are rare (approximately 1%) in most genomes and are generally considered to have no function. However, penaeid shrimp genomes have a high proportion of SSRs (>23%), raising the question of whether these SSRs play important functional and evolutionary roles in these SSR-rich species. Here, we show that SSRs drive genome plasticity and adaptive evolution in two penaeid shrimp species, Fenneropenaeus chinensis and Litopenaeus vannamei. Assembly and comparison of genomes of these two shrimp species at the chromosome-level revealed that transposable elements serve as carriers for SSR expansion, which is still occurring. The remarkable genome plasticity identified herein might have been shaped by significant SSR expansions. SSRs were also found to regulate gene expression by multi-omics analyses, and be responsible for driving adaptive evolution, such as the variable osmoregulatory capacities of these shrimp under low-salinity stress. These data provide strong evidence that SSRs are an important driver of the adaptive evolution in penaeid shrimp.

Cadmium-induced oxidative stress, metabolic dysfunction and metal bioaccumulation in adult palaemonid shrimp Palaemon macrodactylus (Rathbun, 1902).

The Palaemonid shrimp Palaemon macrodactylus is widely distributed in coastal areas and estuaries which are easily contaminated by various pollutants. However, the responses of this species to environmental toxicants are not well described. In the present study, adult individuals of P. macrodactylus were exposed to gradient concentrations of Cadmium (Cd) to evaluate its acute toxic effects, including bioaccumulation, induced oxidative stress and changed energy metabolism in this species. The medium lethal concentration (LC50) of Cd at 24 h, 48 h, 72 h, and 96 h were 2.60, 0.88, 0.49 and 0.37 mg/L, respectively. Cd bioaccumulations in tissues of shrimp increased in a concentration-dependent manner, and higher concentration (50% 96 h-LC50, 0.185 mg/L) of Cd exposure led to a maximum increase of Cd concentration by 14.8, 145.5 and 15.8 folds in gill, hepatopancreas and abdominal muscle. Cd exposure caused a significant inhibition on the activity of catalase (CAT), and total superoxide dismutase (T-SOD), decrease in the total antioxidant capacity (T-AOC), and an increase of malonadehyde (MDA) content, which indicated a damage to the antioxidant system of shrimp. Meanwhile, Cd exposure also led to a significant up-regulation in the expression level of metallothionein gene (MT), and down-regulations at the mRNA level of heat shock protein 70 (HSP70) and CAT. Moreover, Cd exposure significantly inhibited the oxygen consumption rate (22%), and increased the ammonia excretion rate (43%), hence lead to a significant decrease of the O:N ratio (45%) in shrimp. The results indicated that Cd exposure could induce obvious oxidative stress, energy metabolic dysfunction and bioaccumulation of Cd in P. macrodactylus. The data obtained from the present study would provide useful information for further understanding on the toxicological mechanism of Cd to crustaceans in coastal areas and estuaries.