Ridgetail White Prawn Exopalaemon Carinicauda Research Articles

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.

The Responses of the Ovary and Eyestalk in Exopalaemon carinicauda under Low Salinity Stress

As a euryhaline shrimp, the ridgetail white prawn Exopalaemon carinicauda is strongly adaptable to salinity. Exploring the effect of long-term low salinity stress on ovarian development in E. carinicauda is essential to promote its culture in a non-marine environment. In this study, we performed biochemical assays and ovary histology analysis, finding that the E. carinicauda can adapt to low salinity stress through osmotic adjustment, and there was no substantial damage to the ovary of E. carinicauda under low salinity stress. Then, the ovarian development of E. carinicauda under low salt stress was further explored by RNA sequencing of eyestalk and ovarian tissues. A total of 389 differentially expressed genes (DEGs) in ovary tissue were identified under low salinity stress, and the 16 important DEGs were associated with ovarian development. The majority of the DEGs were enriched in ECM-receptor interaction, folate biosynthesis, arginine biosynthesis, insect hormone biosynthesis and lysosome which were involved in the ovarian development of E. carinicauda. A total of 1223 DEGs were identified in eyestalk tissue under low salinity stress, and the 18 important DEGs were associated with ovarian development. KEGG enrichment analysis found that ECM-receptor interaction, folate biosynthesis, lysosome, arginine biosynthesis and retinol metabolism may be involved in the ovarian development under low salinity stress. Our results provided new insights and revealed new genes and pathways involved in ovarian development of E. carinicauda under long-term low salinity stress.

Identification and functional characterization of WNT5 in the ovarian maturation of Exopalaemon carinicauda (Holthuis, 1950) (Caridea, Palaemonidae)

Abstract The Wnt family, the ligand of the Wnt signal pathway, is highly conserved and plays an important role in various cellular processes including reproduction and development. In this study, wnt5 was cloned from the ridgetail white prawn Exopalaemon carinicauda (Ec-wnt5) and its function in ovarian maturation was investigated. The results showed that the Ec-wnt5 cDNA was 2422 bp, including an ORF with 1116 bp encoding a polypeptide with 371 amino acids. Ec-WNT5 had the highest homology with WNT5 from Penaeus vannamei (93.1%). Ec-wnt5 was expressed in 9 tissues with the most expression in ovary and increased significantly () along with ovarian maturation, while decreased significantly () with vitellogenin and cathepsin L in E. carinicauda ovary post Ec-wnt5 RNA interference. All gonadosmatic indices of the RNAi group were significantly lower than those of the control (). The results suggest that Ec-WNT5 might be the key member of the Wnt family involved in ovarian maturation.

Effects of Long-Term High Carbonate Alkalinity Stress on the Ovarian Development in Exopalaemon carinicauda

Saline–alkaline water limits the growth and survival of aquatic animals due to its high carbonate alkalinity, high pH, and various ion imbalances. The ridgetail white prawn Exopalaemon carinicauda is strongly adaptable to the saline–alkaline water, making it an excellent candidate species for large-scale aquaculture in saline–alkaline areas. To explore the effect of long-term high carbonate alkalinity stress on ovarian development in E. carinicauda for assisting the development of saline–alkaline aquaculture, we performed ovary histology analysis and RNA–sequencing of the eyestalk and ovary in order to compare the transcriptomic responses of individuals in high carbonate alkalinity (8 mmol/L) with a control group (2 mmol/L) for 60 days. It was found that high carbonate alkalinity stress resulted in a loose arrangement of oogonia and a small number of surrounding follicular cells. A total of 1102 differentially expressed genes (DEGs) in ovary tissue were identified under high carbonate alkalinity stress, and the 18 important DEGs were associated with ovarian development. The majority of the DEGs were enriched in ECM–receptor interaction, Folate biosynthesis, the FoxO signaling pathway, insect hormone biosynthesis, and lysosome, which were involved in the ovarian development of E. carinicauda. A total of 468 DEGs were identified in eyestalk tissue under high carbonate alkalinity stress, and the 13 important DEGs were associated with ovarian development. KEGG enrichment analysis found that ECM–receptor interaction, folate biosynthesis, lysosome, metabolic pathways, and retinol metabolism may be involved in the ovarian development under high carbonate alkalinity stress. Our results provide new insights and reveal the genes and pathways involved in the ovarian development of E. carinicauda under long-term high carbonate alkalinity stress.

Analysis of genetic diversity and population structure of the ridgetail white prawn Exopalaemon carinicauda in China

The ridgetail white prawn Exopalaemon carinicauda, an important commercial shrimp species, is naturally distributed along the coasts of China. In order to assess the level of genetic diversity, 268 individuals from seven different geographical regions of China were analyzed using microsatellite markers. Based on the allelic diversity, E. carinicauda populations in Lianyungang and Ninghai showed high level of genetic diversity, and the other five populations (Dandong, Binzhou, Haiyang, Nantong, Guangzhou) showed moderate level of genetic diversity. Pairwise comparison of Fst indicated that low level of genetic differentiation existed in these seven populations, among which the highest genetic variation existed between Guangzhou and Binzhou. Phylogenetic analysis and STRUCTURE analysis showed that these seven populations could be separated into three clusters, which is consistent with their geographical location. This study provided information for genetic improvement, sustainable exploitation and it was also helpful to develop appropriate strategies for the conservation of E. carinicauda.

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.

Comparative Transcriptomic and Proteomic Analysis of Exopalaemon carinicauda in Response to Alkalinity Stress

Saline-alkaline waters are stressful environments where most aquatic animals can’t survive normally, and alkalinity is one of the key limited environmental factors. Due to strong adaptability to environment, the ridgetail white prawn Exopalaemon carinicauda is a potential good species suitable for large-scale culture in saline-alkaline waters. Exploring its alkaline adaptability mechanism will help to guide more marine crustaceans to saline-alkaline culture. In this study, an integrative analysis of the gill-specific transcriptome and proteome at 0, 12, and 36 h after alkalinity stress was performed to identify important regulators and pathways involved in alkalinity adaption of E. carinicauda. A total of 3,157 differentially expressed genes (DEGs) and 443 differentially expressed proteins (DEPs) were identified at 12 and 36 h compared with 0 h. Base on the transcriptome analysis, the Gene Ontology (GO) enriched terms were mainly related to ion transport, including “calcium-transporting ATPase activity,” “ATPase coupled ion transmembrane transporter activity,” “divalent inorganic cation transmembrane transporter activity,” etc., and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways mainly refer to the processes of endocrine system at both 12, and 36 h. Based on the proteomic analysis, KEGG pathways related to lipolysis and amino acids metabolism were significantly enriched at 12 h, and carbohydrate metabolism and immune response were significantly enriched at 36 h. There were significantly up-regulated expressions of ion transport related genes including aquaporin, carbonic anhydrase, ammonium transporter Rh type A-like, Na+/H+-exchanger, etc., as well as ion transport proteins including V-type proton ATPase 116 kDa subunit a-like isoform X1, sodium-potassium ATPase beta, vesicle associated membrane protein, etc. after alkalinity exposure, which indicating their important roles in response to alkalinity stress. The results of integrated analysis between proteome and transcriptome showed that up-regulated DEG/DEP (aldehyde dehydrogenase) was significantly enriched at 12 h and the up-regulated DEG/DEP (peptidylglycine alpha) was significantly enriched at 36 h, suggesting the two molecules may be critical in response to alkalinity change. This study reveals the first time-course, gill-specific, combined transcriptomic and proteomic profiling associated with alkalinity adaption of E. carinicauda and provides new insights into the mechanisms underlying the molecular response to alkalinity stress in shrimp.

Characterization and functional analysis of tandem threonine containing C-type lectin (Thr-Lec) from the ridgetail white prawn Exopalaemon carinicauda

As an important pattern-recognition receptor (PRR), C-type lectins (CTLs) play significant roles in recognizing microbes and battle against pathogenic microorganism in innate immunity. In this study, two tandem threonine containing CTLs (designated as EcThr-LecA and EcThr-LecB) were identified from Exopalaemon carinicauda. The full-length cDNA of EcThr-LecA and EcThr-LecB consisted of 1521 and 1518 bp with 1251 and 1242 bp open reading frame encoding a protein with 412 and 413 amino acids, respectively. The genome structure of EcThr-LecA included 10 exons and 9 introns, and the sequences of intron6 and intron7 were variable. The nucleotide sequence of intron2 in EcThr-LecB was specific and different with that of EcThr-LecA. EcThr-LecA and EcThr-LecB proteins were predicted to have a signal peptide, two conserved carbohydrate recognition domain (CRD), and tandem threonine region. The expression levels of EcThr-LecA and EcThr-LecB in the intestine were significantly up-regulated after Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenge. RNA interference (RNAi) was used to explore the effects of EcThr-LecB silencing on the mRNA expression of anti-lipopolysaccharide factor (ALF), crustin (CRU), and lysozyme (LYSO). Knock down of EcThr-LecB could evidently down-regulate the expression of eight different antibacterial peptides (AMPs), including EcALF2, EcCRU1, EcCRU3, EcCRU4, EcLYSO1, EcLYSO2, EcLYSO3, and EcLYSO4, whereas make no effect on the transcription of EcALF1, EcALF3, EcCRU2, and EcLYSO5. The recombinant two CRD domains and tandem threonine region (RLecB) of EcThr-LecB could bind diverse bacteria, lipopolysaccharide, and peptidoglycans in vitro. In addition, RLecB could accelerate the clearance of V. parahaemolyticus in vivo. The present data indicated that new-found tandem threonine containing CTLs in E. carinicauda may act as PRR to participate in the innate immune defense against pathogens by the recognition of non-self, regulation of AMPs, and clearance of invaders.

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.

Full-length transcriptome sequences of ridgetail white prawn Exopalaemon carinicauda provide insight into gene expression dynamics during thermal stress

Marine heat waves and extreme high temperature become more frequent and intense in these years, which affected the survival of aquaculture animals. The ridgetail white prawn Exopalaemon carinicauda is an important economic species in eastern China, which has remarkable thermal tolerance. However, there has been little study of its thermal-adaptation mechanisms due to the complex genetic structure and unknown genome. To better understand the molecular mechanisms of E. carinicauda to adapt to the changing temperature, a combination of Illumina-based short reads RNA-seq and single molecule real-time-based full-length transcriptome sequencing was used in this study. In total, 17,212 unigenes from high-quality transcripts of E. carinicauda were generated and 14,663 complete ORFs were detected with an average length of 1980 bp. In addition, the transcriptome profiles of E. carinicauda treated with 34 °C heat stress for 6 and 24 h were analyzed. These differentially expressed genes were primarily enriched in oxidation-reduction process (Gene Ontology enrichment, GO) and the pathways of starch and sucrose metabolism (Kyoto Encyclopedia of Genes and Genomes enrichment, KEGG) after 6 h thermal stress, which indicated that E. carinicauda was suffering the attack by reactive oxygen species. After 24 h thermal stress, these differentially expressed genes were enriched in the pathway of lysosome, glycine, serine and threonine metabolism, fatty acid metabolism (KEGG), which indicated the oxidative stress was decreased. Interestingly, 40 genes for hemocyanin were found to be downregulated after 6 h heat stress, which indicated that the immunocompetence of E. carinicauda decreased after short term thermal stress (6 h). After 24 h thermal stress, E. carinicauda showed transcriptional adaptation to high temperature by upregulating of 11 genes encoding molecular chaperones, including HSP40 and HSP90 which were firstly reported to be related to thermal stress in E. carinicauda. These results promote a better understanding of the thermal-adaptation mechanism of E. carinicauda.

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. 15,15ʹ-beta-carotene oxygenase (BCO1) and 9ʹ,10ʹ-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.

Ovarian development pattern and vitellogenesis of ridgetail white prawn, Exopalaemon carinicauda.

The ridgetail white prawn Exopalaemon carinicauda has the potential to be used as a model organism in crustacean research because it has a transparent body, available draft genome, and short life cycle. However, their ovarian development pattern remains unclear under laboratory culture conditions. This study investigated the changes of ovarian external feature, ovarian histology, gonadosomatic index (GSI), and hepatosomatic index (HSI), as well as the expression and localization of vitellogenin in the ovary and the hepatopancreas during the first ovarian development cycle of E. carinicauda under laboratory-reared condition. The results demonstrated that (1) the first ovarian development cycle of E. carinicauda could be divided into 5 different stages in which the ovary changes its color from white to yellow during the vitellogenesis process in parallel with increasing GSI. (2) After pubertal molt, most females reached ovarian stage II while the females reached stage V after premating molt. (3) During the ovarian development, GSI increased smoothly and HSI relatively stable during the period of stages I to IV, while GSI increased but HSI decreased significantly from stages IV to V. (4) In situ hybridization (ISH) revealed that EcVg was slightly expressed in the oocyte cytoplasm of previtellogenic oocytes. The positive signal was mainly detected in hepatopancreatic fibrillar cells, and a strong signal was found in the hepatopancreas at stage IV. Moreover, the expression level of EcVg-mRNA in the hepatopancreas is stage-specific, and the hepatopancreas contributes majority of vitellin precursor protein to support the ovarian development of E. carinicauda.

Effects of acute salinity stress on the survival and prophenoloxidase system of Exopalaemon carinicauda

The ridgetail white prawn Exopalaemon carinicauda is a euryhaline shrimp species in the estuarine and coastal areas of China. In this study, survival rates, transcription levels of two prophenoloxidase system-related genes (EcLGBP and EcproPO) and PO activity were determined quantitatively in juvenile and adult E. carinicauda under different salinity levels. The results showed that E. carinicauda juveniles could survive in a wider range of salinity conditions than adults. For juvenile E. carinicauda, the expression levels of EcLGBP and EcProPO were up-regulated in low salinities and showed no significant difference at 20–40, while PO activities in low salinities were higher compared to those in high salinities. For adult E. carinicauda, the expression profiles of EcLGBP and EcproPO had a different trend of up-regulation in salinity stress treatments and no obvious difference was observed in the gene expression levels and PO activity between 30 and 40. The salinity tolerance range of immunity for juvenile and adult E. carinicauda is 20–40 and 30–40, respectively.

Effects of inbreeding on growth and survival rates, and immune responses of ridgetail white prawn Exopalaemon carinicauda against the infection of Vibrio parahaemolyticus

Inbreeding could lead to a decrease in genetic variation within a population and subsequent depression of performance traits. The purpose of this study was to investigate the effects of inbreeding on the growth-related traits, immune responses, antioxidant status and the acute hepatopancreatic necrosis disease (AHPND) resistant capacity of ridgetail white prawn (Exopalaemon carinicauda) using a new inbred line named EC5. The results demonstrated that the genetic polymorphisms of EST-SSR markers decreased with an increase in artificial inbreeding, and there was less growth traits and a lower survival rate among inbred populations than the control population. Inbreeding effects on the immune responses and antioxidant status were studied in experimental full-sibling inbred populations of E. carinicauda at five levels of inbreeding coefficient (F = 0.785, F = 0.816, F = 0.859, F = 0.886, F = 0.908) under controlled laboratory conditions. Inbreeding affected the total haemocyte count, antibacterial activity and phenoloxidase (PO) activity, and the haemocyanin (HEM) concentration decreased after the ninth generation. Antioxidant status showed a similar pattern: superoxide dismutase (SOD) and catalase (CAT) activity in the cell-free haemolymph decreased. The effects of inbreeding on survival after infection with Vibrio parahaemolyticus were significant, as the cumulative mortality of shrimp in the control population (CP) was significantly lower than that in the F9 generation of EC5 inbred family during 24–72 h after infection with V. parahaemolyticus (P < .05). Additionally, the levels of antibacterial activity, immune enzymes including lysozyme (LZM) activity, alkaline phosphatase (AKP) activity and PO activity in cell-free haemolymph were significantly higher in the control population than those in the F9 generation 6 h after infection with V. parahaemolyticus (P < .05). Furthermore, the expression levels of anti-lipopolysaccharide factor (ALF), LZM, Tollip, evolutionarily conserved signaling intermediate in Toll pathway (ECSIT), Toll and signal transducer and activator of transcription (STAT) in the hepatopancreas, and Crustin, LZM and Tollip in haemocytes were significantly higher in the control population than those in the F9 generation after infection with V. parahaemolyticus (P < .05). These results suggested that the F9 generation has a weaker disease-resistant capability than the control population when infected with V. parahaemolyticus. In conclusion, this study demonstrates that a high level of inbreeding has a negative effect on growth-related traits and physiological health of E. carinicauda, thus highlighting the need to maintain genetic diversity in selective breeding programs.

Genome survey and high-resolution backcross genetic linkage map construction of the ridgetail white prawn Exopalaemon carinicauda applications to QTL mapping of growth traits

BackgroundHigh-resolution genetic linkage map is critical for QTL mapping, genome sequence assembly and marker-assisted selection in aquaculture species. The ridgetail white prawn Exopalaemon carinicauda is one of the most economic shrimp species naturally distributed in the coasts of eastern China and western Korea. However, quite limited genomics and genetics information have been exploited for genetic improvement of economic traits in this species.ResultsIn the present study, we conducted genome survey and constructed high-resolution genetic linkage maps of the ridgetail white prawn with reciprocal-cross mapping family genotyped using next-generation sequencing approaches. The estimated genome size was 9.33 Gb with a heterozygosity of 0.26% and a repeat sequence ratio of 76.62%. 65,772 protein-coding genes were identified by genome annotation. A total of 10,384 SNPs were used to high-throughput genotyping and assigned to 45 linkage groups (LGs) from reciprocal backcross families of E. carinicauda, and the average marker distances were 0.73 cM and 0.55 cM, respectively. Based on the high-resolution linkage map, twenty-three QTLs related to five growth traits were detected. All QTLs could explain 8.8–15.7% of the total growth-traits variation.ConclusionsThe genome size of E. carinicauda was estimated more accurately by genome survey analysis, which revealed basic genomic architecture. The first high-resolution backcross genetic linkage map and QTLs related to growth traits will provide important information for QTL fine mapping, genome assembly and genetic improvement of E. carinicauda and other palaemon shrimps.

Characterization, functional analysis, and expression levels of three carbonic anhydrases in response to pH and saline–alkaline stresses in the ridgetail white prawn Exopalaemon carinicauda

Carbonate alkalinity, salinity, and pH are three important stress factors for aquatic animals in saline-alkaline water. Carbonic anhydrases (CAs) catalyze the reversible reaction of CO2 reported to play an important role in the acid-base regulation in vertebrates. To explore the molecular mechanism of CAs efficacy in shrimp after their transfer into saline-alkaline water, the cDNAs of three CAs (EcCAc, EcCAg, and EcCAb) were cloned from Exopalaemon carinicauda. Sequence analysis showed that EcCAc and EcCAg both possessed a conserved α-CA domain and a proton acceptor site, and EcCAb contained a Pro-CA domain. Tissue expression analysis demonstrated that EcCAc and EcCAg were most abundantly in gills, and EcCAb was highly expressed in muscle. The cumulative mortalities remained below 25% under exposure to pH (pH6 and pH9), low salinity (5ppt), or high carbonate alkalinity (5 and 10mmol/L) after 72h of exposure. However, mortalities increased up to 70% under extreme saline-alkaline stress (salinity 5ppt, carbonate alkalinity 10mmol/L, and pH9) after 14days of exposure. The EcCAc and EcCAg expressions in gills were significantly upregulated during the early period of pH and saline-alkaline stresses, while the EcCAb expressions showed no regular or large changes. The two-way ANOVA found significant interactions between salinity and carbonate alkalinity observed in EcCAc, EcCAg, and EcCAb expressions (p < 0.05). Furthermore, an RNA interference experiment resulted in increased mortality of EcCAc- and EcCAg-silenced prawns under saline-alkaline stress. EcCAc knockdown reduced expressions of Na+/H+ exchanger (EcNHE) and sodium bicarbonate cotransporter (EcNBC), and EcCAg knockdown reduced EcCAc, EcNHE, EcNBC, and V-type H+-ATPase (EcVTP) expressions. These results suggest EcCAc and EcCAg as important modulators in response to pH and saline-alkaline stresses in E. carinicauda.

Characterization and expression patterns of wnt4 in Exopalaemon carinicauda (Holthuis, 1950) (Caridea, Palaemonidae) during embryonic and larval development

Abstract Wnt4 (Wingless-type MMTV integration site family member 4) has been demonstrated to play critical roles in a wide variety of biological processes, including embryonic development, cell proliferation, and differentiation in vertebrates, but its function in crustaceans is still not clear. In the present study, the full-length wnt4 cDNA sequence was cloned and characterized for the ridgetail white prawn Exopalaemon carinicauda. The expression patterns of the wnt4 mRNA in embryos and larvae at different stages were investigated. The tissue distribution showed that wnt4 was obviously expressed in eyestalk and hepatopancreas. During embryonic development, the wnt4 was highly expressed in all developmental stages except the zygote, two-cell stage, and late zoaea stage. The wnt4 mRNA was expressed in Z1-Z5 and post-larval stages. Taken together, the present study indicates that the wnt4 gene may be involved in the regulation of embryonic and larval development in the ridgetail white prawn.

Isolation and characterization of three skeletal troponin genes and association with growth-related traits in Exopalaemon carinicauda.

Growth is among the most important traits for animal breeding. Muscle growth is controlled by different cellular and molecular pathways and environments, and it also relies heavily on high-quality muscle contractions. The troponin complex, composed of troponin T (TnT), troponin C (TnC) and troponin I (TnI), plays a vital role in the regulation of muscle contraction. In this study, the cDNA of EcTnT, EcTnC and EcTnI of the ridgetail white prawn Exopalaemon carinicauda were cloned and characterized. The full length cDNA of EcTnT, EcTnC and EcTnI were 1 373bp, 692bp, and 1 475bp, encoding a protein of 385, 150 and 193 amino acid residues, respectively. The expression of all genes was predominantly detected in abdominal muscle, while extremely lesser expressed in gill and hepatopancreas. Higher expression level of EcTnI was observed in heavier shrimp of the same age during different developmental stages, excepted for 120 days. Eleven single nucleotide polymorphisms (SNPs) were revealed in the three skeletal troponin genes, and only c.TnI66 A>G from EcTnI was significantly associated with both body weight and body length (P < 0.05). In summary, the result of this study suggested that EcTnI is growth-related gene of the troponin complex gene and the presence of SNP suggests that it could be a candidate gene for shrimp genetic improvement research.

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.

CRISPR/Cas9-mediated deletion of EcMIH shortens metamorphosis time from mysis larva to postlarva of Exopalaemon carinicauda

The recently emerged CRISPR/Cas9 technology is the most flexible means to produce targeted mutations at the genomic loci in a variety of organisms. In Crustaceans, molt-inhibiting hormone (MIH) is an important negative-regulatory factor and plays a key role in suppressing the molting process. However, whether precise disruption of MIH in crustacean can be achieved and successfully used to improve the development and growth has not been proved. In this research, the complementary DNA (cDNA) and genomic DNA, including flanking regions of the MIH gene (EcMIH) of ridgetail white prawn Exopalaemon carinicauda, were cloned and sequenced. Sequence analysis revealed that EcMIH was composed of three exons and two introns. Analysis by RT-PCR showed that EcMIH mainly expressed in eyestalks. During different development periods, EcMIH was highest in juvenile stage and extremely low in others but adult prawns eyestalks. In addition, we applied CRISPR/Cas9 technology to generate EcMIH knock-out (KO) prawns and then analyzed the changes in their phenotypes. We efficiently generated 12 EcMIH-KO prawns out of 250 injected one-cell stage embryos and the mutant rate reached 4.8% after embryo injection with one sgRNA targeting the second exon of EcMIH. The EcMIH-KO prawns exhibited increased the body length and shortened the metamorphosis time of larvae from mysis larva to postlarva. Meanwhile, EcMIH-KO did not cause the health problems such as early stage death or deformity. In conclusion, we successfully obtained EcMIH gene and generated EcMIH-KO prawns using CRISPR/Cas9 technology. This study will certainly lead to a wide application prospect of MIH gene in prawns breeding.