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pH has a great impact on the distribution, growth, behavior, and physiology in many aquatic animals. The comparison of proteomics between normal and high pH stress samples was successfully achieved using iTRAQ proteomic analysis to screen key response proteins and pathways. After high pH stress, 124 upregulated and 41 downregulated proteins were identified. The higher expression levels of proteins like citrate synthase, glutathione S-transferase, glutathione peroxidase, and cytochrome c oxidase are associated with oxidative stress and mitochondrial dysfunction. The upregulation of glucose-regulated protein 78 indicated that the endoplasmic reticulum stress is induced by high pH stress. There were significant upregulation expressions of V-type H+-ATPase, Na+, K+-ATPase, 14-3-3 protein, as well as ATP-binding cassette transmembrane transporters after high pH exposure, which indicating their important roles in response to high pH stress. The abundance of proteins involved in protein glycosylation, oxidative pentose phosphate pathway, protein export, and glutathione metabolism were found enriched in high pH group than in control group. Quantitative proteomic profiling and integrated analysis with transcriptomic data provide new insights into the mechanisms underlying the molecular response to high pH stress in Fenneropenaeus chinensis.

pH has a great impact on the distribution, growth, behaviour and physiology in many aquatic animals. MicroRNAs (miRNAs), a class of small non‐coding RNAs play essential roles in animal responses to environmental stress. In this study, we constructed two small RNA (sRNA) libraries from control (pH 7.4) and high pH (9.4) treated shrimp to identify high pH stress‐responsive miRNAs. Using deep sequencing, we identified 72 (3 conserved and 69 novel) up‐ and 4 (2 conserved and 2 novel) down‐regulated miRNAs from high pH challenged shrimp hepatopancreas. Gene Ontology annotations and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways. Moreover, we validated the expression patterns of selected miRNAs and their target genes by qRT‐PCR. The present work adds new insights into the large‐scale identification and characterization of Fenneropenaeus chinensis miRNAs and their potential targets and represents a foundation for underlying mechanisms of animal responses to high pH stress at post‐transcriptional level.

pH has a great impact on the distribution, growth, behavior, and physiology in many aquatic animals. Here, we analyzed miRNA expression profiles of Chinese shrimp (Fenneropenaeus chinensis) from control pH (8.2) and low pH (6.5)-treated shrimp. Expression analysis identified 6 known miRNAs and 23 novel miRNAs with significantly different expression between control pH 8.2 and low pH 6.5; the predicted target genes of differentially expressed miRNAs were significantly enriched in organic acid metabolic process, oxidoreductase activity, coenzyme binding, cofactor binding, and collagen trimer. Moreover, target genes were significantly enriched in several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including citrate cycle, pyruvate metabolism, cytokine-cytokine receptor interaction, tight junction, carbon metabolism, etc. Our survey expanded the number of known shrimp miRNAs and provided comprehensive information about miRNA response to low pH stress. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-019-00989-x) contains supplementary material, which is available to authorized users.

Fenneropenaeus chinensis is a commercially cultured shrimp in China. F. chinensis adults show significant sexual dimorphism, with larger females than males. However, sex determination (SD) of F. chinensis has not yet been elucidated. Clarification of the sex-determining system of F. chinensis could enrich our knowledge of the sex differentiation mechanism in crustaceans and facilitate the study of sex-controlling technologies. Here, we studied the sex-determining system of F. chinensis using the fixation index (FST) between the sexes to detect the genetic differentiation in resequencing data of multiple males and females. We located the candidate sex chromosome in the genome of F. chinensis and concluded the female heterogametic (ZW) SD system. We also assembled female-specific sequences, which could be used as molecular markers to identify the sex of F. chinensis. However, the differentiation of the F. chinensis Z and W chromosome is limited. RNA-seq data detected many genes with male-biased expression in the Z-specific region, which possibly could further intensify the divergency between the Z and W chromosomes.

A high-quality reference genome is necessary to determine the molecular mechanisms underlying important biological phenomena; therefore, in the present study, a chromosome-level genome assembly of the Chinese shrimp Fenneropenaeus chinensis was performed. Muscle of a male shrimp was sequenced using PacBio platform, and assembled by Hi-C technology. The assembled F. chinensis genome was 1.47 Gb with contig N50 of 472.84 Kb, including 57.73% repetitive sequences, and was anchored to 43 pseudochromosomes, with scaffold N50 of 36.87 Mb. In total, 25,026 protein-coding genes were predicted. The genome size of F. chinensis showed significant contraction in comparison with that of other penaeid species, which is likely related to migration observed in this species. However, the F. chinensis genome included several expanded gene families related to cellular processes and metabolic processes, and the contracted gene families were associated with virus infection process. The findings signify the adaptation of F. chinensis to the selection pressure of migration and cold environment. Furthermore, the selection signature analysis identified genes associated with metabolism, phototransduction, and nervous system in cultured shrimps when compared with wild population, indicating targeted, artificial selection of growth, vision, and behavior during domestication. The construction of the genome of F. chinensis provided valuable information for the further genetic mechanism analysis of important biological processes, and will facilitate the research of genetic changes during evolution.

A high-quality reference genome is necessary to determine the molecular mechanisms underlying important biological phenomena; therefore, in the present study, a chromosome-level genome assembly of the Chinese shrimp Fenneropenaeus chinensis was performed. Muscle of a male shrimp was sequenced using PacBio platform, and assembled by Hi-C technology. The assembled F. chinensis genome was 1,465.32 Mb with contig N50 of 472.84 Kb, including 57.73% repetitive sequences, and was anchored to 43 pseudochromosomes, with scaffold N50 of 36.87 Mb. In total, 25,026 protein-coding genes were predicted. The genome size of F. chinensis showed significant contraction in comparison with that of other penaeid species, which is likely related to migration observed in this species. However, the F. chinensis genome included several expanded gene families related to cellular processes and metabolic processes, and the contracted gene families were associated with virus infection process. The findings signify the adaptation of F. chinensis to the selection pressure of migration and cold environment. Furthermore, the selection signature analysis identified genes associated with metabolism, phototransduction, and nervous system in cultured shrimps when compared with wild population, indicating targeted, artificial selection of growth, vision, and behavior during domestication. The construction of the genome of F. chinensis provided valuable information for the further genetic mechanism analysis of important biological processes, and will facilitate the research of genetic changes during evolution.

Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular response to inflammatory cytokines, environmental stress and pathogenic infection, However, compared with mammals and insects, the potential function of p38 MAPK from Chinese shrimp (Fenneropenaeus chinensis) in response to viruses and bacterial infection is limited. In the study, the immune responses of four genes MKK3, MKK4, p38 and ATF-2 from F. chinensis were investigated in defending against white spot syndrome virus (WSSV), Vibrio anguillarum (V. anguillarum) and Staphylococcus aureus (S. aureus) infection. Results demonstrated that the expression levels of these four genes were apparently modulated in hemocytes and gills when shrimp were stimulated by WSSV or bacteria, particularly at 3-24h after infection. MKK3, p38 and ATF-2 were most sensitive to V. anguillarum (Gram-negative bacteria), followed by WSSV and S. aureus (Gram-positive bacteria), while MKK4 gene was most sensitive to S. aureus, followed by WSSV and V. anguillarum. Knockdown of Fcp38 by RNA interference (RNAi) resulted in a reduction in the expression of FcMKK3 and FcATF-2. The results indicate that p38MAPK signaling pathway plays a role in defending against viral and bacterial infections in F. chinensis.

Most retrotransposons from plants are believed to be either transcriptionally inactive or transcriptionally silent in somatic tissues but active during particular stages of development or under certain conditions. We have identified a bidirectionally transcriptional apple Ty1-copia-like retrotransposon called Mdoryco1-1, which was associated with apple spur mutation. Mdoryco1-1 belongs to a low-copy-number family in the sequenced apple genome. These copies were highly similar. The sense and antisense strands of the 5′-LTR (long terminal repeat) and 3′-LTR drove expression of GUS in transiently transformed tobacco leaves, activity which was dependent on the presence of the promoter function in the Mdoryco1-1 LTR. The transcriptional activity of Mdoryco1-1 was increased after ABA (abscisic acid), wounding and 2,4-D (2,4-dichlorophenoxyacetic acid) treatments, but not SA (salicylic acid) treatment, despite the predicted TCA-element (cis-acting element involved in salicylic acid responsiveness) in the Mdoryco1-1 LTR sense strand. The analysis of flanking sequences indicates that the Mdoryco1element is adjacent to genes in apple genome. The results suggested that the expression of Mdoryco1 could affect adjacent gene transcription.

Fenneropenaeus chinensis (F. chinensis) is one of the most commercially important cultured shrimps in China. The adult F. chinensis exhibit sexual dimorphism in growth and body color. In this research, we profiled the whole transcriptome of F. chinensis by using single molecule real-time-based full-length transcriptome sequencing. We further performed Illumina-based short reads RNA-seq on muscle and gonad of two sexes to detect the sex-biased expression genes. In muscle, we observed significantly more female-biased transcripts. With the differentially expressed transcripts (DETs) in muscle, some pathways related to the energy metabolism were enriched, which may be responsible for the difference of growth. We also digged out a pathway named porphyrin and chlorophyll metabolism. It was speculated to relevant to the difference of body color between the two sexes of shrimp. Interestingly, almost all DETs in these pathways were female-biased expression in muscle, which could explain the phenomenon of better growth performance and darker body color in female. In gonad, several pathways involved in reproduction were enriched. For instance, some female-biased DETs participated in the arachidonic acid metabolism, which was reported crucial in female reproduction. In conclusion, our studies identified abundant sex-biased expression transcripts and important pathways involved in sexual dimorphism by using the RNA-seq method. It provided a basis for future researches on the sexual dimorphism of F. chinensis.

The Huanghai No. 1 strain of Chinese shrimp Fenneropenaeus chinensis is an ecologically and economically important variety that is cultivated mainly in northern China. This strain has been subjected to multiple selective events in recent years. Conventional methods are insufficient to reveal the genetic background of Huanghai No. 1, especially with regard to a genome-wide transcription profile, and publicly available genomic resources for Huanghai No. 1 are limited. We obtained 907,945 original reads with an average length of 467.5 bp from muscle and haemocyte samples from Huanghai No. 1 using 454 pyrosequencing. After preprocessing, 867,245 high-quality reads were screened. Of the 64,830 assembled sequences, 34,633 of the unigenes had significant matches in the databases (similarity >30 %), including 14,138 isotigs and 20,495 singletons. A gene ontology analysis assigned 8,257 unigenes to 44 subcategories, with the majority of unigenes assigned to cellular component (28.2 %), biological process (20.5 %) and molecular function (5.0 %) categories. Pathway mapping based on information in the Kyoto Encyclopedia of Genes and Genomes database showed that 15,461 transcripts were associated with 223 pathways. A total of 14,981 putative SSRs, 72,370 SNPs, and candidate genes involved in growth, reproduction, and the immune response were identified. Our findings represent the most comprehensive transcriptomic resources currently available for Huanghai No. 1, and provide an important foundation for further genomic studies of F. chinensis.

DNA methylation plays an important role in the regulation of gene expression during biological development and tissue differentiation in eukaryotes. A methylation sensitive amplification polymorphism (MSAP) including digestion, pre-selective amplification and selective amplification was optimized to compare the levels of DNA cytosine methylation at CCGG sites in muscle, gill and hemocyte from the wild populations and the selective breeding of Huanghai No. 1 of Fenneropenaeus chinensis, respectively. Significant differences in cytosine methylation levels among three tissues in two populations were detected. The average DNA methylation ratios in muscle, gill and hemocyte of the wild population were 23.1%, 22.3% and 19.7%, while those were 21.4%, 19.6%, and 18.9% in Huanghai No. 1, respectively. The DNA methylation levels of gill from the two populations were highly significant (P 0.05). DNA polymorphic methylation of gill and hemocyte between the wild population and Huanghai No. 1 varies to some extent, while those of muscle kept in a balanced degree. Furthermore, polymorphic methylation was associated with demethylation and methylation of CCGG loci.

The aim of this study is to screen single nucleotide polymorphisms (SNPs) of the farnesoic acid O-methyltransferase gene in shrimp, Penaeus chinensis (PcFAMeT), and analyze the potential association between PcFAMeT gene polymorphisms and growth traits in a cultured population. Four SNPs (A163G, G392T, A708G, and T752C) were tested for association with six growth traits in 240 individuals using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The association analysis of SNPs of PcFAMeT gene with the six growth traits was carried out using general linear model estimation. Results indicated that SNP2 (G392T) of the PcFAMeT gene was significantly associated with body weight (P < 0.05) and carapace width (P < 0.05). The individuals of genotype GG grew faster than those of genotype GT and TT. The results would provide potential application in future shrimp breeding programs and may be used to improve the efficiency in shrimp, P. chinensis, breeding programs.

The antimicrobial sulphamethazine is widely used in aquaculture for the prevention and treatment of bacterial diseases. Residues of sulphamethazine have been detected in aquatic environments and in edible tissues of aquaculture fish and shrimps at relatively low, but detectable concentrations. Detailed information on the environmental fate and pharmacokinetics of sulphamethazine in aquaculture species is essential to predict possible ecological risks and to provide recommendations on appropriate dosages and withdrawal periods. In this study we investigated the distribution of sulphamethazine in water and sediments of Fenneropenaeus chinensis shrimp ponds treated with 50, 100 and 150 mg kg−1·bw of sulphamethazine for 5 days and measured the uptake and elimination dynamics of sulphamethazine in different shrimp organs and tissues. Results of the HPLC analysis showed highest sulphamethazine concentrations in shrimp tissues, followed by sediments and water. The rank order of the mean concentrations of sulphamethazine in shrimp tissues and organs was hepatopancreas> plasma≈ stomach> muscle≈ gill≈ intestine> carapace. The results also demonstrated a significant dose-dependent accumulation of sulphamethazine in the different biological and environmental compartments. Sulphamethazine decreased gradually with the time in all of the three compartments. The mean half-life of sulphamethazine in sediment and water was 2.15 and 2.17 days respectively. A withdrawal period of 10 days or more is proposed for F. chinensis orally treated with sulphamethazine in order to meet the current food safety standards.

In ecology the disease in the rare migratory bird population plays an important role in controlling the dynamical behavior of system. In this paper, we investigate a nonautonomous two species (migratory bird population and economical bird population) competitive model with saturation incidence and artificially harvesting and study the dynamical changes that take place due to the introduction of a disease by migratory birds. Under the reasonable assumptions, sufficient conditions for the permanence and extinction of the disease are obtained. Moreover, the global attractivity of the model is discussed by constructing a Liapunov function. To substantiate our theoretical results, extensive numerical simulations are performed for a hypothetical set of parameter values.

ATP-binding cassette (ABC) transporters are integral membrane proteins which carry diverse substrates across biological membranes. In this study, we report the cloning of a full-length, 2122-bp cDNA sequence encoding a gene of the ABC transporter subfamily G (ABCG) from the shrimp Fenneropenaeus chinensis . The deduced protein, FcABCG, contains 633 amino acid residues with a theoretical molecular weight of 70.57 kDa. Phylogenetic analysis showed that the FcABCG clusters with the shrimp Litopenaeus vannamei ABCG in the same clade and is more closely related to the insect ABC transporter/white proteins than to the ABCG2 of the fish Salmo salar . High-level mRNA expression of FcABCG was detected in the tissues of hepatopancreas and intestine using a real-time quantitative reverse transcriptase PCR. Furthermore, exposure to alkaline pH stress conditions and norfloxacin diets result in the activation of the FcABCG gene in F. chinensis . These results suggest that the ABCG transporter of F. chinensis might play a role in coping high pH stress and in exporting xenobiotics.

Caspases are a family of proteases, which play an important role in apoptosis. To evaluate the relationship between apoptosis and pH stress in crustaceans, a caspase gene (FcCasp) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length of FcCasp was 1329 bp with a 972 bp ORF, encoding a polypeptide of 323 amino acids with a calculated molecular weight and pI of 36.0 kDa and 6.27 respectively. The deduced amino acid sequence of FcCasp contained a potential active site (QACRG pentapeptide) conserved in most caspases and two profile hits (p20 and p10 domain profile). Comparison of amino acid sequences revealed that FcCasp had an overall similarity of 76–83% with other penaeid shrimp caspases. The amino acid sequence of recombinant FcCasp protein expressed in Escherichia coli was identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometer analysis. High-level expression of FcCasp in six different tissues was detected by real-time polymerase chain reaction after exposure to pH stress for 96 and 148 h. TUNEL analysis indicated that apoptosis began to appear in F. chinensis hepatopancreas exposed to extreme pH for 12 h. The amount of apoptosis seems positively correlated with the length of exposure to the pH stressor. The results suggested that FcCasp was involved in the response to environmental pH stress.

The Chinese shrimp Penaeus (Fenneropaeneus) chinensis is an important species in marine fishery and aquaculture in China. A female Chinese shrimp Penaeus (Fenneropaeneus) chinensis was captured from west coast of the Korean peninsula and mated with a “Yellow Sea No. 1” male to produce the first filial generation (F1) 100 F2 full-sib progeny from brother-sister crosses between F1 families was used for the mapping study. A genetic linkage map of the Chinese shrimp was constructed, based on 354 markers, including 300 amplified fragment length polymorphism (AFLP) markers, 42 microsatellite (SSR) markers, and 12 randomly amplified polymorphism (RAPD) markers. Forty-seven linkage groups (LGs) were identified. The total map length was 4 580.5 cM, with an average spacing of 11.3 cM, covering 75.8% of the estimated genome size. The construction of this genetic linkage map was part of a genetic breeding program. This linkage map will contribute to the discovery of genes and quantitative trait loci (QTLs) in Chinese shrimp.

A muscle cDNA library of Chinese shrimp (Fenneropenaeus chinensis) was constructed with the SMART™ cDNA Library Construction Kit. The titer of optimal primary library was 7.7×105 pfu mL−1 and that of the amplified library was 3.0×109 pfu mL−1. The percentages of the recombinant clones of primary and amplified libraries were over 98%. The insert sizes were longer than 400 bp with an average of 1000 bp. A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin I gene. This library provided a useful resource for the functional genomic research of F. chinensis.

Growth traits in 150 Chinese shrimps Fenneropenaeus chinensis were analyzed by the sequence characterized amplified regions (SCAR) technique in two populations of sixth-generation cultured shrimps with different ranges of body lengths (CP-a and CP-b) and in the wild-type population (WP). First, 240 random primers were used to screen polymorphic fragments by random amplified polymorphic DNA (RAPD). Population analysis revealed nine RAPD markers correlated with growth traits, including seven positively and two negatively correlated ones. The sequences of the RAPD markers were then used to design longer primers for SCAR. Six primer pairs were obtained, and two of these produced polymorphic fragments among the three groups. One amplified 39, 26 and 27 polymorphic fragments, with band frequencies of 78, 52 and 54% in the CP-a, CP-b and WP groups, respectively. The distributions of polymorphic fragments in the three groups were significantly different (P < 0.05) according to a χ2 test, indicating that they may be candidate markers linked to growth traits. Another primer pair amplified three alleles, resulting in six combinations of genotypes among three groups. Since allele A1 was only found in the population of shorter shrimps, it may be a negative growth marker.

This paper presents the genetic linkage map of the Chinese shrimp Penaeus (Fenneropenaeus) chinensis constructed with 472 AFLP markers. A hundred F1 progeny from an intercross between a female from the new variety “Yellow Sea No. 1” and wild caught male used for the mapping study. Two separate maps were constructed for each parent. The female linkage map consisted of 197 marker loci forming 35 linkage groups and spanned a total length of 2191.1 cM, with an average marker space of 13.5 cM. The male map consisted of 194 marker loci mapped to 36 linkage groups and spanned a total length of 1737.3 cM, with an average marker spacing of 11.0 cM. The level of segregation distortion observed in this study was 12.2%. The estimated genome length of P. chinensis was 3150.3 cM for the female and 2549.3 cM for the male, respectively. The observed genome coverage was 69.6% for the female and 68.1% for the male map. The linkage maps constructed in this study provide basic information for further linkage studies on Chinese shrimp, and more importantly, the construction of the maps are part of the work of the genetic breeding programs which will be used for growth discovered in the QTL analysis of P. chinensis.

Seven AFLP primer sets were used to investigate the genetic structure of a wild base population and three generations of marine shrimp Fenneropenaeus chinensis selected for fast growth (F5-F7). More than 500 bands were generated in total. On average, each primer set detected 33.7 polymorphic markers. The expected average heterozygosity ranged from 0.085 to 0.097. As time under selection increased, genetic diversity tended to reduce, the differentiation between generations became less, and the variation of genetic structure of the populations became smaller.

The ridgetail white prawn Exopalaemon carinicauda is one of major economic mariculture species in eastern China. The deficiency of genomic and transcriptomic data is becoming the bottleneck of further researches on its good traits. In the present study, 454 pyrosequencing was undertaken to investigate the transcriptome profiles of E. carinicauda. A collection of 1,028,710 sequence reads (459.59 Mb) obtained from cDNA prepared from eyestalk and hemocytes was assembled into 162,056 expressed sequence tags (ESTs). Of these, 29.88 % of 48,428 contigs and 70.12 % of 113,628 singlets possessed high similarities to sequences in the GenBank non-redundant database, with most significant (E value

The pathophysiology of Oriental hybrid lilies poisoning in cats was studied. Clinically normal eighteen domestic shorthair cats were orally dosed with 0, 1.5, 2.5 g wet weight of homogenate lily flower petals per kg body weight by a nasogastric tube in the study (n=3/sex/dose level). Blood and urine samples were collected before and after dosing. The cats of all treated groups presented anorexia, vomiting, lethargy and depression within 0.5 h after dosing. Serum levels of Alanine transaminase (ALT), Aspartate transaminase (AST), Alkaline phosphatase (ALP), Glutamyl transferase (GGT), Lactate dehydrogenase (LDH), Creatinine (CREA), Blood urea nitrogen (BUN) and Creatine kinase (CK) were increased in all treated groups in a dose-dependent manner. Severe hepatocellular vacuolation was present in the cats of 2.5 g/kg group. Mild and diffuse vacuolar degeneration was observed in the renal tubule epithelium of cortex and medulla in the cats of the same group. This study indicated that oriental hybrid lily is hepatotoxic to cats, associated with some effects on myocardium and kidneys.

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. In this study, a heat shock protein 90 cDNA named EcHSP90 was cloned from the hepatopancreas of ridgetail white prawn Exopalaemon carinicauda by reverse transcription polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcHSP90 was of 2695 bp, including an open reading frame (ORF) of 2163 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 82.73 kDa and an estimated isoelectric point of 4.83. BLAST analysis revealed that the EcHSP90 shared high similarity (87.6%–75.24%) with other known HSP90s. The five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in EcHSP90, which indicated that EcHSP90 should be a cytosolic member of the HSP90 family. Quantitative real-time RT-PCR analysis revealed that EcHSP90 transcript could be detected in all the tested tissues, and strongly expressed in ovary of E. carinicauda. The transcript of EcHSP90 in hepatopancreas of E. carinicauda showed different expression profiles after pH and ammonia-N stresses. The results indicated that EcHSP90 was a constitutive and inducible expressed protein and could be induced by various stresses from environment.