CRISPR/Cas9-mediated gene mutation of EcIAG leads to sex reversal in the male ridgetail white prawn Exopalaemon carinicauda.

In the culture of crustaceans, most species show sexual dimorphism. Monosex culture is an effective approach to achieve high yield and economic value, especially for decapods of high value. Previous studies have developed some sex control strategies such as manual segregation, manipulation of male androgenic gland and knockdown of the male sexual differentiation switch gene encoding insulin-like androgenic gland hormone (IAG) in decapods. However, these methods could not generate hereditable changes. Genetic manipulation to achieve sex reversal individuals is absent up to now. In the present study, the gene encoding IAG (EcIAG) was identified in the ridgetail white prawn Exopalaemon carinicauda. Sequence analysis showed that EcIAG encoded conserved amino acid structure like IAGs in other decapod species. CRISPR/Cas9-mediated genome editing technology was used to knock out EcIAG. Two sgRNAs targeting the second exon of EcIAG were designed and microinjected into the prawn zygotes or the embryos at the first cleavage with commercial Cas9 protein. EcIAG in three genetic males was knocked out in both chromosome sets, which successfully generated sex reversal and phenotypic female characters. The results suggest that CRISPR/Cas9-mediated genome editing technology is an effective way to develop sex manipulation technology and contribute to monosex aquaculture in crustaceans.

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.

CRISPR/Cas9-mediated mutation on an insulin-like peptide encoding gene affects the growth of the ridgetail white prawn Exopalaemon carinicauda.

Insulin-like peptides (ILPs) play key roles in animal growth, metabolism and reproduction in vertebrates. In crustaceans, one type of ILPs, insulin-like androgenic gland hormone (IAG) had been reported to be related to the sex differentiations. However, the function of other types of ILPs is rarely reported. Here, we identified another type of ILPs in the ridgetail white prawn Exopalaemon carinicauda (EcILP), which is an ortholog of Drosophila melanogaster ILP7. Sequence characterization and expression analyses showed that EcILP is similar to vertebrate insulin/IGFs and insect ILPs in its heterodimeric structure and expression profile. Using CRISPR/Cas9 genome editing technology, we generated EcILP knockout (KO) prawns. EcILP-KO individuals have a significant higher growth-inhibitory trait and mortality than those in the normal group. In addition, knockdown of EcILP by RNA interference (RNAi) resulted in slower growth rate and higher mortality. These results indicated that EcILP was an important growth regulator in E. carinicauda.

Transcriptome Analysis of the Accumulation of Astaxanthin in Haematococcus pluvialis Treated with White and Blue Lights as well as Salicylic Acid.

Haematococcus pluvialis is the most commercially valuable microalga for the production of natural astaxanthin, showing enhanced production of astaxanthin with the treatments of high-intensity light and hormones. The molecular mechanisms regulating the biosynthesis of astaxanthin in H. pluvialis treated with white light, blue light, and blue light with salicylic acid (SA) were investigated based on the transcriptome analysis. Results showed that the combined treatment with both blue light and SA generated the highest production of astaxanthin. A total of 109,443 unigenes were identified to show that the genes involved in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), and the astaxanthin biosynthesis were significantly upregulated to increase the production of the substrates for the synthesis of astaxanthin, i.e., pyruvate and glyceraldehyde-3-phosphate generated in the TCA cycle and PPP, respectively. Results of transcriptome analysis were further verified by the quantitative real-time PCR (qRT-PCR) analysis, showing that the highest content of astaxanthin was obtained with the expression of the bkt gene significantly increased. Our study provided the novel insights into the molecular mechanisms regulating the synthesis of astaxanthin and an innovative strategy combining the exogenous hormone and physical stress to increase the commercial production of astaxanthin by H. pluvialis.

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.

Transcriptome Analysis Provides Insights into the Mechanism of Astaxanthin Enrichment in a Mutant of the Ridgetail White Prawn Exopalaemon carinicauda.

A mutant of the ridgetail white prawn, which exhibited rare orange-red body color with a higher level of free astaxanthin (ASTX) concentration than that in the wild-type prawn, was obtained in our lab. In order to understand the underlying mechanism for the existence of a high level of free astaxanthin, transcriptome analysis was performed to identify the differentially expressed genes (DEGs) between the mutant and wild-type prawns. A total of 78,224 unigenes were obtained, and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant in comparison with the wild-type prawns. Based on Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis, as well as further investigation of annotated DEGs, we found that the biological processes related to astaxanthin binding, transport, and metabolism presented significant differences between the mutant and the wild-type prawns. Some genes related to these processes, including crustacyanin, apolipoprotein D (ApoD), cathepsin, and cuticle proteins, were identified as DEGs between the two types of prawns. These data may provide important information for us to understand the molecular mechanism of the existence of a high level of free astaxanthin in the prawn.

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.

Transcriptomic and Proteomic Characterizations of the Molecular Response to Blue Light and Salicylic Acid in Haematococcus pluvialis.

Haematococcus pluvialis accumulates a large amount of astaxanthin under various stresses, e.g., blue light and salicylic acid (SA). However, the metabolic response of H. pluvialis to blue light and SA is still unclear. We investigate the effects of blue light and SA on the metabolic response in H. pluvialis using both transcriptomic and proteomic sequencing analyses. The largest numbers of differentially expressed proteins (DEPs; 324) and differentially expressed genes (DEGs; 13,555) were identified on day 2 and day 7 of the treatment with blue light irradiation (150 µmol photons m-2s-1), respectively. With the addition of SA (2.5 mg/L), a total of 63 DEPs and 11,638 DEGs were revealed on day 2 and day 7, respectively. We further analyzed the molecular response in five metabolic pathways related to astaxanthin synthesis, including the astaxanthin synthesis pathway, the fatty acid synthesis pathway, the heme synthesis pathway, the reactive oxygen species (ROS) clearance pathway, and the cell wall biosynthesis pathway. Results show that blue light causes a significant down-regulation of the expression of key genes involved in astaxanthin synthesis and significantly increases the expression of heme oxygenase, which shows decreased expression by the treatment with SA. Our study provides novel insights into the production of astaxanthin by H. pluvialis treated with blue light and SA.

Characterization and Function Analysis of the Beta-Carotene Oxygenase-like Genes in Carotenoids Metabolism of the Ridgetail White Prawn Exopalaemon carinicauda.

Carotenoids are almost universally distributed in living organisms. The oxidative metabolism by carotene oxygenase contributes to the metabolic processes of carotenoids. 15,15'-beta-carotene oxygenase (BCO1) and 9',10'-beta-carotene oxygenase (BCO2) are two important carotenoid oxygenases. In order to understand the function of carotenoid oxygenases in crustaceans, seven genes encoding carotenoid oxygenases (named EcBCO-like) were isolated from the transcriptome database of Exopalaemon carinicauda. After phylogenetic analysis with carotenoid oxygenases reported in other species, EcBCO-like1, EcBCO-like3, and EcBCO-like6 were chosen for further functional study. The prawns after EcBCO-like1 knockdown suffered continuous death, which suggested its important role for the survival of the animals. For the prawns after EcBCO-like3 knockdown, no phenotype change was observed. The prawns after EcBCO-like6 knockdown showed color changes in their hepatopancreas when they were fed with carotenoids-containing diet, and the content of carotenoid in their hepatopancreas was much higher than that in the control prawns. The present study will pave the way for further understanding the carotenoids metabolism in the prawns.

Enhancing Mechanical Properties of the Spark Plasma Sintered Inconel 718 Alloy by Controlling the Nano-Scale Precipitations.

The present study aimed to optimize the phase constituents and mechanical properties of the spark plasma sintered (SPS) Inconel 718 (IN718) alloy. A series of heat treatment routes were designed based on the phase relations in IN718 and performed for the optimization. The microstructure and phase compositions of the SPS IN718 alloys were examined by using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy disperse spectroscopy (EDS), and transmission electron microscopy (TEM). The mechanical properties of the samples were characterized at room temperature and at 650 °C. The results showed that large amounts of γ" (Ni3Nb) and γ' (Ni3(Al, Ti)) strengthening phases precipitated in the IN718 alloy after direct aging (DA) of the as-fabricated sample. Moreover, the mechanical properties of the DA sample were comparable to that of the best one of the solution-treated and aged counterparts. The analysis showed that the rapid sintering and solid solution treatment of the IN718 alloy were achieved simultaneously by SPS. In the case of the SPS IN718 material, the direct aging regime had the same heat treatment effect as the conventional solid solution and aging treatment. This contributes toward improving the production efficiency and reduces manufacturing costs in the actual production process.

Identification of Single Nucleotide Polymorphisms Related to the Resistance Against Acute Hepatopancreatic Necrosis Disease in the Pacific White Shrimp Litopenaeus vannamei by Target Sequencing Approach.

Acute hepatopancreatic necrosis disease (AHPND) is a major bacterial disease in Pacific white shrimp Litopenaeus vannamei farming, which is caused by Vibrio parahaemolyticus. AHPND has led to a significant reduction of shrimp output since its outbreak. Selective breeding of disease-resistant broodstock is regarded as a key strategy in solving the disease problem. Understanding the relationship between genetic variance and AHPND resistance is the basis for marker-assisted selection in shrimp. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) associated with the resistance against AHPND in L. vannamei. In this work, two independent populations were used for V. parahaemolyticus challenge and the resistant or susceptible shrimp were evaluated according to the survival time after Vibrio infection. The above two populations were genotyped separately by a SNP panel designed based on the target sequencing platform using a pooling strategy. The SNP panel contained 508 amplicons from DNA fragments distributed evenly along the genome and some immune-related genes of L. vannamei. By analyzing the allele frequency in the resistant and susceptible groups, 30 SNPs were found to be significantly associated with the resistance of the shrimp against V. parahaemolyticus infection (false discovery rate corrected at P < 0.05). Three SNPs were further validated by individual genotyping in all samples of population 1. Our study illustrated that target sequencing and pooling sequencing were effective in identifying the markers associated with economic traits, and the SNPs identified in this study could be used as molecular markers for breeding disease-resistant shrimp.

Highly microporous carbon with nitrogen-doping derived from natural biowaste for high-performance flexible solid-state supercapacitor.

Highly microporous carbon material with nitrogen doping has been synthesized via a facile one-step approach by employing natural biowaste miscellaneous wood fibers derived hydrochar as precursor and melamine as nitrogen source respectively. The added melamine not only results in the incorporation of some nitrogen into the carbon framework but also increases the specific surface area of carbon material. Such resultant N-doped microporous carbon possesses the functionalized nitrogen doping (1.75 at. %), a large specific surface area (∼1807 m2 g-1) and abundant highly interconnected micropores. Benefiting from the synergistic effect of high specific surface area, well-developed pore size distribution and functionalized groups, this carbon material delivers a high specific capacitance of 345 F g-1 at 0.5 A g-1, an excellent capacitance retention with 270 F g-1 at up to 30 A g-1, and a remarkable cycle ability with 91.3% retention after 10,000 cycles at 5.0 A g-1. Based on it, the as-developed flexible symmetric solid-state supercapacitor delivers a high energy density of 7.92 W h kg-1 at the power density of 250 W kg-1. Evidently, this work provides a facile and cost-effective route for functionalized natural biowaste-based carbon materials and further opens up a way for highly value-added recycling of biowaste-like materials.

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.

Isolation and identification of the main carotenoid pigment from a new variety of the ridgetail white prawn Exopalaemon carinicauda.

The ridgetail white prawn (Exopalaemon carinicauda) is an important economic species in China. In order to know the molecular basis and evaluate the potential nutrition value of a new variety of this species with orange-red color, the main carotenoid pigments were extracted and identified through high performance liquid chromatography. It showed that higher concentration of free astaxanthin existed in the new variety which might cause the color variation. The concentration of total astaxanthin in muscle and waste of the new variety was 3.3 and 1.9 fold higher than that in natural prawn under the same culture condition (P < 0.05). The predominant geometric and optical isomers of astaxanthin in muscle of the new variety were identified as all-trans and 3S,3'S astaxanthin. This is the first report of astaxanthin-enriched E. carinicauda, which also provides an alternative source of natural astaxanthin and the possibility for improving the nutrition value in this species.

Multiple Isoforms of Anti-Lipopolysaccharide Factors and Their Antimicrobial Functions in the Ridgetail Prawn Exopalaemon carinicauda.

As a kind of antimicrobial peptides (AMP) in crustacean, anti-lipopolysaccharide factors (ALFs) have broad spectrum antimicrobial activities. In the present study, we identified four ALF genes, EcALF2-5, from the ridgetail prawn Exopalaemon carinicauda. Tissue distribution analysis showed that EcALF2 and EcALF4 transcripts were mainly located in gill, epidermis, and stomach, while EcALF3 and EcALF5 were mainly in hemocytes. Peptides corresponding to the LPS binding domain (LBD) of EcALFs were synthesized for analyzing their antimicrobial activities. Minimal inhibitory concentration (MIC) analysis showed that the synthetic LBD peptides of EcALF3 and EcALF4 could inhibit the growth of Gram-positive and Gram-negative bacteria, while the synthetic LBD peptides of EcALF2 and EcALF5 showed antibacterial activity against Vibrio. Incubation of white spot syndrome virus (WSSV) with the synthetic LBD peptides of EcALF3, EcALF4, and EcALF5 could reduce the in vivo viral copy number in WSSV-infected prawns. After silencing of EcALFs, Vibrio exhibited a rapid proliferation in the hepatopancreas of the prawn. The present data showed the important function of different EcALFs in modulating the in vivo bacterial and viral propagation in E. carinicauda. This study will provide new clues into the disease control in aquaculture.

Acute toxic effects of zinc and mercury on survival, standard metabolism, and metal accumulation in juvenile ridgetail white prawn, Exopalaemon carinicauda.

Ridgetail white prawn (Exopalaemon carinicauda) is widely distributed in Chinese coastal zones, especially in the Yellow Sea and Bohai Sea. It is not only considered as an important economic species in China, but also taken as a potential indicator species for the environmental pollution in the estuaries. At present, the responses of this species to environmental toxicants, including trace metal are not well understood. In this study, the acute toxic effects of zinc (Zn) and mercury (Hg) on the survival, oxygen consumption, ammonia-N excretion, and metal accumulation were investigated in the juveniles of E. carinicauda. The median lethal concentrations (LC50) of Zn were 76.4, 44.0, 30.2, and 17.2mg/L, respectively, after the juveniles were exposed in for 24, 48, 72, and 96h, and the LC50 of Hg was 0.212, 0.096, 0.084, and 0.065mg/L under the same exposure duration. The juveniles decreased the oxygen consumption by 51.4%, and increased ammonia-N excretion by 129% when they were exposed in Zn at the concentration of 76.4mg/L compared with their controls without Zn exposure, therefore the O:N ratio decreased by 82.9% compared with the control. Hg exposure with the concentration of 0.212mg/L caused the inhibition of oxygen consumption by 48.1% and increasement of ammonia-N excretion by 161%, and the atomic ratio of consumed oxygen to excreted ammonia-nitrogen (O:N ratio) decreased by 80.6% in the juveniles in comparison with the control. A concentration-dependent accumulation of heavy metals was observed in the gills, hepatopancreas and muscles of the experimental animals, with a maximum accumulation of 16.3 folds for Zn and 72.8 fold for Hg in the gills of the juveniles after 24h exposure. The data obtained from the present study would provide useful information for help further understanding on the toxicological responses of this species to trace metals.

Gene set based association analyses for the WSSV resistance of Pacific white shrimp Litopenaeus vannamei.

White Spot Syndrome Virus (WSSV) is regarded as a virus with the strongest pathogenicity to shrimp. For the threshold trait such as disease resistance, marker assisted selection (MAS) was considered to be a more effective approach. In the present study, association analyses of single nucleotide polymorphisms (SNPs) located in a set of immune related genes were conducted to identify markers associated with WSSV resistance. SNPs were detected by bioinformatics analysis on RNA sequencing data generated by Illimina sequencing platform and Roche 454 sequencing technology. A total of 681 SNPs located in the exons of immune related genes were selected as candidate SNPs. Among these SNPs, 77 loci were genotyped in WSSV susceptible group and resistant group. Association analysis was performed based on logistic regression method under an additive and dominance model in GenABEL package. As a result, five SNPs showed associations with WSSV resistance at a significant level of 0.05. Besides, SNP-SNP interaction analysis was conducted. The combination of SNP loci in TRAF6, Cu/Zn SOD and nLvALF2 exhibited a significant effect on the WSSV resistance of shrimp. Gene expression analysis revealed that these SNPs might influence the expression of these immune-related genes. This study provides a useful method for performing MAS in shrimp.

Acute effects of cadmium and copper on survival, oxygen consumption, ammonia-N excretion, and metal accumulation in juvenile Exopalaemon carinicauda.

Ridgetail white prawn (Exopalaemon carinicauda), a commercially important species in China, is a potential candidate for evaluating impairments caused by environmental pollutants in coastal and estuarine areas. The main purpose of the present study was to investigate the acute effects of cadmium (Cd) and copper (Cu) on survival, oxygen consumption, ammonia-N excretion, and metal accumulation in E. carinicauda. The feasibility of using this species for pollution monitoring was also evaluated. Results showed that the median lethal concentrations (LC50) for 24h, 48h, 72h, and 96h were 0.66mg/L, 0.379mg/L, 0.343mg/L, and 0.258mg/L for Cd, and 0.932mg/L, 0.748mg/L, 0.725mg/L, and 0.712mg/L for Cu. Cd exposure (0.66mg/L) caused an inhibition in oxygen consumption of 21.1 percent and an increase in ammonia-N excretion of 47.1 percent, thereby decreasing the atomic ratio of oxygen consumed to nitrogen consumed (O:N ratio) of 46.32 percent relative to the control. Cu exposure (0.932mg/L) also resulted in an inhibition in oxygen consumption of 34.8 percent and a decrease in the O:N ratio of 23.9 percent in relation to the control, but the ammonia-N excretion was not influenced by the Cu exposure. Concentration-depended accumulation was observed in the experimental animals, which a maximum of 244.8 folds and 1.1 folds increase of mental concentration was measured upon exposure to 24h LC50 of Cd and Cu for 24h, respectively. The change in O:N ratio indicated an alteration in energy utilization. Based on its sensitivity to heavy metals and its availability all year round, E. carinicauda can be used as a test organism to monitor for metal pollution.

Transcriptome analysis of the initial stage of acute WSSV infection caused by temperature change.

White spot syndrome virus (WSSV) is the most devastating virosis threatening the shrimp culture industry worldwide. Variations of environmental factors in shrimp culture ponds usually lead to the outbreak of white spot syndrome (WSS). In order to know the molecular mechanisms of WSS outbreak induced by temperature variation and the biological changes of the host at the initial stage of WSSV acute infection, RNA-Seq technology was used to analyze the differentially expressed genes (DEGs) in shrimp with a certain amount of WSSV cultured at 18°C and shrimp whose culture temperature were raised to 25°C. To analyze whether the expression changes of the DEGs were due to temperature rising or WSSV proliferation, the expression of selected DEGs was analyzed by real-time PCR with another shrimp group, namely Group T, as control. Group T didn't suffer WSSV infection but was subjected to temperature rising in parallel. At the initial stage of WSSV acute infection, DEGs related to energy production were up-regulated, whereas most DEGs related to cell cycle and positive regulation of cell death and were down-regulated. Triose phosphate isomerase, enolase and alcohol dehydrogenase involved in glycosis were up-regulated, while pyruvate dehydrogenase, citrate synthase and isocitrate dehydrogenase with NAD as the coenzyme involved in TCA pathway were down-regulated. Also genes involved in host DNA replication, including DNA primase, DNA topoisomerase and DNA polymerase showed down-regulated expression. Several interesting genes including crustin genes, acting binding or inhibiting protein genes, a disintegrin and metalloproteinase domain-containing protein 9 (ADAM9) gene and a GRP 78 gene were also analyzed. Understanding the interactions between hosts and WSSV at the initial stage of acute infection will not only help to get a deep insight into the pathogenesis of WSSV but also provide clues for therapies.