RESUMEN

In the context of global warming, the accelerated evaporation of seawater will lead to a continuous expansion of saline-alkali land area. As an important economic freshwater crustacean, investigation on the mechanism of damage to Eriocheir sinensis (E. sinensis) under saline-alkali environment will provide a valuable precedent for understanding the detrimental effect of climate change on crustaceans. In this study, histopathological analysis and integrative omics analysis were employed to explore the injury mechanism on the cerebral nervous system of E. sinensis exposure to saline-alkali stress. Our findings revealed that under this stress E. sinensis exhibited behavioral disorders and damage to cerebral neurosecretory cells and key organelles. Additionally, several pathways related to detoxification metabolism, neurotransmitter synthesis, and antioxidant defense were significantly down-regulated. Collectively, these results show, for the first time, that saline-alkali stress can induce neurodegenerative disease-like symptoms in E. sinensis, and provide critical information for understanding the harmful effects of saline-alkali environments.

RESUMEN

The cerebral ganglion and muscle are important regulatory tissues in Eriocheir sinensis. Therefore, it is of great significance to explore their synergistic roles in this organism's anti-stress response. In this study, proteomics, metabolomics, and combination analyses of the cerebral ganglion and muscle of E. sinensis under alkalinity stress were performed. The cerebral ganglion and muscle played a significant synergistic regulatory role in alkalinity adaptation. The key regulatory pathways involved were amino acid metabolism, energy metabolism, signal transduction, and the organismal system. They also played a modulatory role in the TCA cycle, nerve signal transduction, immune response, homeostasis maintenance, and ion channel function. In conclusion, the present study provides a theoretical reference for further research on the mechanisms regulating the growth and development of E. sinensis in saline-alkaline environments. In addition, it provides theoretical guidelines for promoting the vigorous development of the E. sinensis breeding industry in saline-alkaline environments in the future.

RESUMEN

Hepatopancreatic necrosis disease (HPND) broke out in 2015 in the Eriocheir sinensis aquaculture region of Xinghua, Jiangsu Province; however, the specific cause of HPND remains unclear. A correlation was found between HPND outbreak and the use of deltamethrin by farmers. In this study, E. sinensis specimens developed the clinical symptoms of HPND after 93 days of deltamethrin stress. The growth of E. sinensis with HPND was inhibited. Adenosine monophosphate-activated protein kinase (AMPK) is a central regulator of energy homeostasis, and its expression was up-regulated in the intestine of E. sinensis with HPND. Growth inhibitory genes (EsCabut, Es4E-BP, and EsCG6770) were also up-regulated in the intestine of E. sinensis with HPND. The expression levels of EsCabut, Es4E-BP, and EsCG6770 decreased after EsAMPK knockdown. Therefore, AMPK mediated the growth inhibition of E. sinensis with HPND. Further analysis indicated the presence of a crosstalk between the Toll and AMPK signaling pathways in E. sinensis with HPND. Multiple genes in the Toll signaling pathway were upregulated in E. sinensis under 93 days of deltamethrin stress. EsAMPK and its regulated growth inhibition genes were down-regulated after the knockdown of genes in the Toll pathway. In summary, the crosstalk between the Toll and AMPK signaling pathways mediates the growth inhibition of E. sinensis under deltamethrin stress.

Asunto(s)

Braquiuros , Piretrinas , Contaminantes Químicos del Agua , Animales , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Contaminantes Químicos del Agua/toxicidad , Piretrinas/toxicidad , Piretrinas/metabolismo , Nitrilos/toxicidad , Necrosis , Braquiuros/metabolismo

RESUMEN

Aquatic litter decomposition is highly dependent on contributions and interactions at different trophic levels. The invasion of alien aquatic organisms like the channeled apple snail (Pomacea canaliculata) might lead to changes in the decomposition process through new species interactions in the invaded wetland. However, it is not clear how aquatic macroinvertebrate predators like the Chinese mitten crab (Eriocheir sinensis) will affect the nutrient cycle in freshwater ecosystems in the face of new benthic invasion. We used the litter bag method to explore the top-down effect of crabs on the freshwater nutrient cycle with the help of soil zymography (a technology previously used in terrestrial ecosystems). The results showed significant feeding effects of crabs and snails on lotus leaf litter and cotton strips. Crabs significantly inhibited the intake of lotus litter and cotton strips and the ability to transform the environment of snails by predation. Crabs promoted the decomposition of various litter substrates by affecting the microbial community structure in the sediment. These results suggest that arthropod predators increase the complexity of detrital food webs through direct and indirect interactions, and consequently have an important impact on the material cycle and stability of freshwater ecosystems. This top-down effect makes macrobenthos play a key role in the biological control and engineering construction of freshwater ecosystems.

RESUMEN

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential "nitrite stress-Duox activation-ROS production" pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for "nitrite stress-Duox activation-ROS production-Dorsal activation-AMP synthesis" that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

Asunto(s)

Braquiuros , Penaeidae , Contaminantes Químicos del Agua , Virus del Síndrome de la Mancha Blanca 1 , Animales , Nitritos/toxicidad , Nitritos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Braquiuros/genética , Contaminantes Químicos del Agua/toxicidad , Penaeidae/metabolismo

RESUMEN

Eriocheir sinensis is one of the most important economic aquatic products in China. However, nitrite pollution has become a serious threat to the healthy culture of E. sinensis. Glutathione S-transferase (GST) is an important phase II detoxification enzyme, which plays a leading role in the cellular detoxification of exogenous substances. In this study, we obtained 15 GST genes (designated as EsGST1-15) from E. sinensis, and their expression and regulation in E. sinensis under nitrite stress were studied. EsGST1-15 belonged to different GST subclasses. EsGST1, EsGST2, EsGST3, EsGST4, and EsGST5 belonged to Delta-class GSTs; EsGST6 and EsGST7 are Theta-class GSTs; EsGST8 is a mGST-3-class GST; EsGST9 belonged to mGST-1-class GSTs; EsGST10 and EsGST11 belonged to Sigma-class GSTs; EsGST12, EsGST13, and EsGST14 are Mu-class GSTs; EsGST15 is a Kappa-class GST. Tissue distribution experiments showed that EsGSTs were widely distributed in all detected tissues. The expression level of EsGST1-15 was significantly increased in the hepatopancreas under nitrite stress, indicating that EsGSTs were involved in the detoxification of E. sinensis under nitrite stress. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a transcription factor that can activate the expression of detoxification enzyme. We detected the expression of EsGST1-15 after interfering with EsNrf2 in the hepatopancreas of E. sinensis with or without nitrite stress. Results showed that EsGST1-15 were all regulated by EsNrf2 with or without nitrite stress. Our study provides new information about the diversity, expression, and regulation of GSTs in E. sinensis under nitrite stress.

Asunto(s)

Braquiuros , Nitritos , Animales , Nitritos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Regulación de la Expresión Génica , China , Braquiuros/genética , Braquiuros/metabolismo

RESUMEN

Eriocheir sinensis is widely appreciated by the surrounding population due to its culinary delicacy and rich nutrients. The E. sinensis breeding industry is very prosperous and molting is one of the important growth characteristics. Research on the regulation of molting in E. sinensis is still in the initial stages. There is currently no relevant information on the regulatory mechanisms of heart development following molting. Comparative transcriptome analysis was used to study developmental regulation mechanisms in the heart of E. sinensis at the post-molt and inter-molt stages. The results indicated that many regulatory pathways and genes involved in regeneration, anti-oxidation, anti-aging and the immune response were significantly upregulated after molting in E. sinensis. Aside from cardiac development, the differentially expressed genes (DEGs) were relevant to myocardial movement and neuronal signal transduction. DEGs were also related to the regulation of glutathione homeostasis and biological rhythms in regard to anti-oxidation and anti-aging, and to the regulation of immune cell development and the immune response. This study provides a theoretical framework for understanding the regulation of molting in E. sinensis and in other economically important crustaceans.

RESUMEN

Eriocheir sinensis is a unique freshwater crab found in China, which is well known for its rich nutrition and sweet and delicious taste. Free amino acids in Eriocheir sinensis are not only important nutrients but also are closely related to their unique taste and aroma. Therefore, the determination of the free amino acid contents in Eriocheir sinensis is of great significance for product quality evaluation, flavor research, authenticity, and origin identification. Herein we proposed an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based method for the determination of 17 free amino acids in Eriocheir sinensis. First, 5 g of the Eriocheir sinensis sample was weighed into a 50-mL polypropylene centrifuge tube. Then, 10 mL of extraction solvents was added to the centrifuge tube, and the resultant solution was mixed well using a vortex mixer. We compared a variety of solvents and finally selected 5%(v/v) perchloric acid aqueous solution as the optimum extraction solvent. The supernatant was transferred to another polypropylene centrifuge tube after centrifuging at 8000 r/min for 5 min. The extraction procedure was repeated according to the above-mentioned steps, and the extraction solution was combined with the supernatant. The extracts were then adjusted to pH 6.5 with 1 mol/L potassium hydroxide solution, and were diluted to 50 mL with water. After filtering by both qualitative filter paper and a 0.45-µm polyether sulfone syringe filter, the extracts were determined by UHPLC-HRMS. We compared three types of mobile phases and chose 0.1%(v/v) formic acid aqueous solution mixed with acetonitrile as the optimum one. Precise parent ion and ion source parameters were also optimized. The 17 analytes, viz. aspartic acid, threonine, serine, glutamic acid, proline, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, glycine, alanine, and histidine, were separated on an XDB-C18 column (100 mm×4.6 mm, 1.7 µm) with gradient elution. The amino acids were then detected by HRMS in electrospray ionization and selected ion monitoring modes, and the analytes were quantified using external standards. The instrumental detection limit (IDL) and the instrumental quantification limit (IQL) were 0.3 mg/L and 1.0 mg/L, respectively. The linear correlation coefficients were all above 0.9990 in the concentration range of 10.0-200.0 mg/kg. Three levels of free amino acid standards were spiked into the edible parts of Eriocheir sinensis. The recoveries of the amino acids were between 78.4% and 105.3%. The intra-sample, intra-day, and inter-day repeatabilities were below 4.2%, 5.2%, and 11.4%, respectively, which were within reasonable ranges. Twenty samples of Eriocheir sinensis were tested using the proposed method. Thus, in this study, we developed an alternative method for the determination of free amino acids in Eriocheir sinensis with simple pretreatment, good selectivity, and high accuracy.

Asunto(s)

Braquiuros , Percloratos , Acetonitrilos , Alanina , Aminoácidos , Animales , Arginina , Ácido Aspártico , Cromatografía Líquida de Alta Presión , Cistina , Formiatos , Glutamatos , Glicina , Histidina , Isoleucina , Leucina , Lisina , Espectrometría de Masas , Metionina , Fenilalanina , Polipropilenos , Prolina , Serina , Solventes , Treonina , Tirosina , Valina , Agua

RESUMEN

BACKGROUND: The Chinese mitten crab, Eriocheir sinensis (E. sinensis), is a popular crab species in both domestic and foreign markets. Trash fish are essential for E. sinensis breeding, but have caused serious water pollution. The municipal party committee for the main production areas of E. sinensis implemented a ban on feeding on trash fish since 2020. METHODS: In this study, we performed a culture experiment of E. sinensis feeding on trash fish and formulated feed, with comparative transcriptome analysis on hepatopancreas of E. sinensis. RESULTS: The results indicate that formulated feed causes no significant difference in growth, survival rate or content of amino acids in the muscles of adult E. sinensis. Formulated feed caused a slight downregulation of pathways involved in amino acid metabolism, development, energy metabolism and homeostasis maintenance. CONCLUSIONS: On the whole, formulated feed can serve as an undifferentiated substitution for trash fish. This study provides a theoretical foundation for optimizing research on E. sinensis feed.

Asunto(s)

Perfilación de la Expresión Génica , Hepatopáncreas , Animales , Metabolismo Energético , Hepatopáncreas/metabolismo , Transcriptoma

RESUMEN

There has been a recent emphasis on production of large-sized Eriocheir sinensis broodstock. In China, aquaculturists generally prefer wild-caught (WC) crabs from the Yangtze River as broodstock because offspring performance is superior to that of pond-reared (PR) broodstock. Currently, however, there is a ban on fishing in the Yangtze River, and effects on E. sinensis breeding have not been ascertained. There was comparison in the present study of reproductive performance and semen characteristics of male broodstock of PR and WC groups. After copulation, sperm quantity in the vas deferens of crabs in specimens of both groups was large, although there was a consistent decrease in vaso-somatic index. Although sperm density of PR crabs was less, that of WC specimens remained relatively constant. Specimens of neither group, however, had changes in the hepatopancreas index or condition factor, and sperm survival was close to 100%. Although the acrosome reaction was detected in response to cold-temperature induction, there were differences in extent of reaction to cold temperatures. Importantly, in as many as 98% of sperm from female spermathecae, the reaction was completed, which was considerably greater than 15% for sperm of males post-mating. It is concluded there was no difference between PR and WC crabs with respect to reproductive performance or semen characteristics, and, notably, sperm from PR crabs were of sufficient quality for use in E. sinensis aquaculture enterprises. Accordingly, it is predicted the Yangtze River fishing ban would only have a limited effect on supply of male E. sinensis broodstock.

Asunto(s)

Crianza de Animales Domésticos , Braquiuros/fisiología , Animales , Animales Salvajes , Tamaño Corporal , Masculino , Reproducción/fisiología , Semen , Análisis de Semen

RESUMEN

Formulated diet (FD) and iced trash fish (ITF) are common diets during E. sinensis farming. However, whether FD can completely replace ITF during long-term E. sinensis farming is still unclear. Thus this study was conducted to compare the differences in amino acid metabolism and intestinal microbiota of the E. sinensis fed on different diets. The crabs were randomly divided into three groups fed on FD, ITF and mixed diet (MD, FD: ITF = 1:1), respectively. The results showed that there were no significant differences in amino acid composition among FD, MD and ITF groups. The activities of AST and ALT, and the mRNA levels of amino acid metabolism-related genes were significantly up-regulated in FD or/and MD groups compared with ITF group. The diversity of intestinal bacterial community was similar between the FD and ITF groups, but the relative abundance of dominant taxa showed marked differences between the two groups. At the phylum level, the relative abundance of Firmicutes was significantly higher, but the relative abundance of Proteobacteria was significantly lower in the FD group than that in ITF group. Meanwhile, at genus level, the relative abundance of Candidatus_Hepatoplasma in FD was higher than that in ITF group. Data related to functional prediction demonstrated that the significantly differenced pathways between the two groups were observed in metabolism (Pyrimidine metabolism, Glycolysis/Gluconeogenesis and Citrate cycle) and environmental information processing (transporters). The overall results indicated that replacement of ITF by FD did not affect amino acid composition, but altered amino acid metabolism and the relative abundance of intestinal microbiota. Our data provided a valuable reference for FD application replacing ITF during long-term E. sinensis farming.

Asunto(s)

Microbioma Gastrointestinal , Aminoácidos , Animales , China , Dieta , Hielo

RESUMEN

c-Jun NH2-terminal kinase (JNK) is a member of mitogen-activated protein kinases (MAPKs) that participates in the regulation of various physiological and pathological processes. In this study, we identified a novel JNK (EsJNK) and determined the cDNA sequence of its isoform (EsJNK-a) from the Chinese mitten crab Eriocheir sinensis. The open reading frame (ORF) of EsJNK was predicted to encode 421 peptides with a serine/threonine protein kinase, a catalytic (S_TKc) domain, and a low complexity region. The ORF of EsJNK-a was 1380 bp encoding a protein with 459 amino acids, which was 38 amino acids more than that of EsJNK. The predicted tertiary structure of EsJNK was conserved and contained 15 α-helices and 10 ß-sheets. Phylogenetic tree analysis revealed that EsJNK was clustered with the JNK homologs of other crustaceans. Quantitative real-time PCR assays showed that EsJNK was expressed in all the tissues examined, but it was relatively higher in hemocytes, muscles, and intestines. The expression of EsJNK mRNA in the hemocytes was upregulated by lipopolysaccharides and peptidoglycans, as well as by Staphylococcus aureus or Vibrio parahaemolyticus challenge. Functionally, after silencing EsJNK by siRNA in crabs, the expression levels of two antimicrobial peptides (AMPs), namely, anti-lipopolysaccharide factor and crustin, were significantly inhibited. The purified recombinant EsJNK protein with His-tag accelerated the elimination of the aforementioned bacteria in vivo. However, knockdown of EsJNK had an opposite effect. These findings suggested that EsJNK might be involved in the antibacterial immune defense of crabs by regulating the transcription of AMPs.

Asunto(s)

Proteínas de Artrópodos/inmunología , Braquiuros/inmunología , Inmunidad Innata/inmunología , MAP Quinasa Quinasa 4/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Animales , Proteínas de Artrópodos/genética , Braquiuros/enzimología , Braquiuros/genética , Hemocitos/inmunología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Filogenia

RESUMEN

Eriocheir sinensis is an important euryhaline catadromous crustacean of the Yangtze River and an important commercial species for breeding in China. However, wild E. sinensis have suffered serious damage attributed to overfishing, climate change, etc. The Ministry of Agriculture of China issued a notice banning the commercial fishing of wild E. sinensis. E. sinensis megalopa migrates upriver into fresh water for growth and fattening, which creates optimal conditions to experimentally explore its hyposaline osmoregulation mechanism. We performed comparative transcriptome analyses of E. sinensis megalopae under hyposaline stress. The results suggest that KEGG pathways and genes related to genetic information processing, developmental regulation, immune and anti-stress responses were differentially expressed. The present study reveals the most significantly enriched pathways and functional gene groups, and explores the hyposaline osmoregulation mode of E. sinensis megalopae. This study lays a theoretical foundation for further studies on the osmoregulation and developmental mechanisms of E. sinensis.

Asunto(s)

Braquiuros/genética , Osmorregulación/genética , Animales , Braquiuros/crecimiento & desarrollo , Braquiuros/inmunología , Braquiuros/metabolismo , Presión Osmótica , RNA-Seq/estadística & datos numéricos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma

RESUMEN

Precise regulation of methyl farnesoate (MF) titer is of prime importance throughout the crustacean life-cycle. Although the synthetic pathway of MF is well-documented, little is known about its degradation and recycling in crustaceans. Juvenile hormone esterase-like (JHE-like) carboxylesterase (CXE) is a key enzyme in MF degradation, thus playing a significant role in regulating the MF titer. We identified and characterized two cDNAs, Es-CXE1 and Es-CXE2, encoding JHE-like CXEs in Chinese mitten crab. Full-length cDNAs of Es-CXE1 and Es-CXE2 encode proteins composed of 584 and 597 amino acids, respectively, both of which contain a typical carboxylesterase domain. Alignment and phylogenetic analyses revealed that the Es-CXEs are highly similar to those of other crustaceans. To further validate their functions, we evaluated the mRNA expression patterns of the Es-CXEs in various tissues and in different physiological conditions. Tissue-specific expression analysis showed that the two Es-CXEs were predominantly expressed in the hepatopancreas and ovaries, which are the major tissues for MF metabolism. Es-CXE2 expression levels in the hepatopancreas and ovaries were about 100 and 25-fold higher, than the respective Es-CXE1 expressions. During ovarian rapid development stage, the global expressions of Es-CXEs were up-regulated in the hepatopancreas and down-regulated in the ovaries. After eyestalk ablation (ESA), the mRNA expressions of the two Es-CXEs were up-regulated in the hepatopancreas, further indicating their potential in degrading MF. Taken together, our results suggest that Es-CXEs, the key component of the juvenile hormone degradation pathway, may play vital roles in the development and reproduction of the Chinese mitten crab.

Asunto(s)

Braquiuros/enzimología , Braquiuros/genética , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica , Técnicas de Ablación , Secuencia de Aminoácidos , Animales , Braquiuros/crecimiento & desarrollo , Hidrolasas de Éster Carboxílico/química , Clonación Molecular , Femenino , Ovario/crecimiento & desarrollo , Filogenia , Transporte de Proteínas , Alineación de Secuencia , Relación Estructura-Actividad

RESUMEN

Chinese mitten crabs that reach maturity 1 year earlier than normal crabs are known as precocious juvenile crabs. The molecular mechanisms underlying the precocity of the Chinese mitten crab are poorly understood. To identify the genes that may be involved in the control of precocity in Chinese mitten crab, we measured the expression profile of eyestalk genes in precocious and normally developed juvenile crabs using high-throughput sequencing on an Illumina HiSeq 2500 platform. We obtained 56,446,284 raw reads from the precocious crabs and 58,029,476 raw reads from the normally developed juvenile crabs. Reads from the two libraries were combined into a single data set. De novo assembly of the combined read set yielded 78,777 unigenes with an average length of 1563 bp. A total of 41,405 unigenes with predicted ORFs were selected for functional annotation. Among these genes, we identified three neuropeptide genes belonging to the crustacean hyperglycemic hormone family and two neuropeptide genes encoding the chromatophorotropic hormones. Transcriptome comparison between the two libraries revealed 42 genes that exhibited significant differential expression, of which 29 genes were up-regulated and 13 genes were down-regulated in the precocious crabs. To confirm the sequencing data, six differentially expressed genes with functional annotations were selected and validated by qRT-PCR. In conclusion, we obtained the comprehensive transcriptome of the eyestalk tissues of precocious juvenile crabs. The sequencing results may provide new insights into the biomolecular basis of precocity in the Chinese mitten crab.

Asunto(s)

Proteínas de Artrópodos/genética , Braquiuros/genética , Neuropéptidos/genética , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/metabolismo , Braquiuros/química , Ojo/química , Ojo/metabolismo , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Neuropéptidos/química , Neuropéptidos/metabolismo

RESUMEN

Myostatin (Mstn), a member of the transforming growth factor ß superfamily, plays an inhibiting role in mammalian muscle growth. Mammals like human, cattle, mouse, sheep, and dog carrying null alleles of Mstn display a double-muscle phenotype. Mstn is conserved in fish; however, little is known whether the fish with mutated mstn display a similar phenotype to mammals because of the lack of mutant fish with mstn null alleles. Previously, we knocked out one of the duplicated copies of myostatin gene (mstna) in yellow catfish using zinc-finger nucleases. In this study, we report the identification of the second myostatin gene (mstnb) and knockout of mstnb in yellow catfish. The gene comprises three exons. It is predicted to encode 373 amino acid residues. The predicted protein exhibits 59.3% identity with yellow catfish Mstna and 57.3% identity with human MSTN. Employing TALEN (transcription activator-like effector nucleases) technology, we obtained two founders (from four randomly selected founders) of yellow catfish carrying the mutated mstnb gene in their germ cells. Totally, six mutated alleles of mstnb were obtained from the founders. Among the six alleles, four are nonframeshift and two are frameshift mutation. The frameshift mutated alleles include mstnb(nju22), an 8 bp deletion, and mstnb(nju24), a complex type of mutation comprising a 7 bp deletion and a 12 bp insertion. They are predicted to encode function null Mstnb. Our results will help to understand the roles of mstn genes in fish growth.

Asunto(s)

Bagres/genética , Endonucleasas/metabolismo , Proteínas de Peces/genética , Miostatina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bagres/embriología , Bagres/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , Embrión no Mamífero/metabolismo , Femenino , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Técnicas de Inactivación de Genes , Marcación de Gen , Patrón de Herencia , Masculino , Datos de Secuencia Molecular , Miostatina/química , Miostatina/metabolismo , Filogenia

RESUMEN

Channel catfish (Ictalurus spp.) is an economically important species in freshwater aquaculture around the world and occupies a prominent position in the aquaculture industry of the United States. MicroRNAs (miRNAs) play important roles in the regulation of almost every biological process in eukaryotes; however, there is little information available concerning miRNAs in channel catfish. In this study, a small-RNA cDNA library was constructed from 10 tissues of channel catfish, and Solexa sequencing technology was used to perform high-throughput sequencing of the library. A total of 14,919,026 raw reads, representing 161,288 unique sequences, were obtained from the small-cDNA library. After comparing the small RNA sequences with the RFam database, 4,542,396 reads that represent 25,538 unique sequences were mapped to the genome sequence of zebrafish to perform distribution analysis and to screen for candidate miRNA genes. Subsequent bioinformatic analysis identified 237 conserved miRNAs and 45 novel miRNAs in the channel catfish. Stem-loop RT-PCR was applied to validate and profile the expression of the novel miRNAs in 10 tissues. Some novel miRNAs, such as ipu-miR-129b, ipu-miR-7562 and ipu-miR-7553, were expressed in all tissues examined. However, some novel miRNAs appear to be tissue specific. Ipu-miR-7575 is predominantly expressed in stomach. Ipu-miR-7147 and ipu-miR-203c are highly expressed in heart, but are relatively weakly expressed in other tissues. Based on sequence complementarity between miRNAs and mRNA targets, potential target sequences for the 45 novel miRNAs were identified by searching for antisense hits in the reference RNA sequences of the channel catfish. These potential target sequences are involved in immune regulation, transcriptional regulation, metabolism and many other biological functions. The discovery of miRNAs in the channel catfish genome by this study contributes to a better understanding of the role miRNAs play in regulating diverse biological processes in fish and vertebrates.

Asunto(s)

Ictaluridae/genética , MicroARNs/genética , Animales , Secuenciación de Nucleótidos de Alto Rendimiento/métodos

RESUMEN

Catfish (Ictalurus spp.) is an important aquaculture species around the world, accounting for over 60 % of the domestic aquaculture output in the United States. However, little information is available about I. punctatus miRNAs which play an important role in the regulation of almost every biological process. In the present studies, we applied a bioinformatic strategy to identify 16 miRNAs which represent 12 miRNA families in I. punctatus by searching both expressed sequence tags and genome survey sequences databases. The A + U contents of the candidate pre-miRNA sequence range from 51 to 63 %, and the pre-miRNA sequences vary from 55 to 63 bp in length. To verify the predicted miRNAs, real-time PCR was used to profile the expression of 16 miRNAs with different tissues of I. punctatus. All the miRNA candidates were detectable in five tissues except for ipu-miR-9-3p. Based on sequence complementarity between miRNAs and their mRNA targets, potential targets for I. punctatus miRNAs were predicted. Due to the limited information for the I. punctatus transcripts, only one sequence targeted by ipu-miR-135 was identified to be an I. punctatus EB1 mRNA. Bioinformatic analyses indicated that the 3' untranslated region (3'-UTR) of EB1 mRNA contains an ipu-miR-135 target site, which are perfectly complementary to the seed region (positions 2-8) of the mature ipu-miR-135. I. punctatus miRNAs characterized in this study may provide useful information for the miRNAs research in I. punctatus and other aquaculture species.

Asunto(s)

Ictaluridae/genética , MicroARNs/genética , Animales , Secuencia de Bases , Biología Computacional , Secuencia Conservada , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Secuencias Invertidas Repetidas , MicroARNs/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos , Pliegue del ARN , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Termodinámica , Transcriptoma

RESUMEN

Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of ß-actin gene in yellow catfish. The sequence is 1,017 bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own ß-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.

Asunto(s)

Actinas/metabolismo , Animales Modificados Genéticamente/metabolismo , Proteínas Bacterianas/metabolismo , Bagres/metabolismo , Proteínas Luminiscentes/metabolismo , Regiones Promotoras Genéticas , Actinas/genética , Animales , Animales Modificados Genéticamente/genética , Proteínas Bacterianas/genética , Tamaño Corporal , Bagres/genética , Clonación Molecular , Codón Iniciador/genética , Codón Iniciador/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Ingeniería Genética/métodos , Proteínas Luminiscentes/genética , Microinyecciones , Transgenes , Pez Cebra/genética , Pez Cebra/metabolismo

RESUMEN

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstn(nju6)) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstn(nju7)), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstn(nju6/+) and 14 mstn(nju7/+) yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish.

Asunto(s)

Bagres/genética , Endonucleasas/metabolismo , Marcación de Gen , Ingeniería Genética , Patrón de Herencia/genética , Miostatina/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión no Mamífero/metabolismo , Silenciador del Gen , Datos de Secuencia Molecular , Pez Cebra/embriología