Genome-wide identification and differentially expression analysis of lncRNAs in tilapia.

BACKGROUND: Long noncoding RNAs (LncRNAs) play important roles in fundamental biological processes. However, knowledge about the genome-wide distribution and stress-related expression of lncRNAs in tilapia is still limited. RESULTS: Genome-wide identification of lncRNAs in the tilapia genome was carried out in this study using bioinformatics tools. 103 RNAseq datasets that generated in our laboratory or collected from NCBI database were analyzed. In total, 72,276 high-confidence lncRNAs were identified. The averaged positive correlation coefficient (r_mean = 0.286) between overlapped lncRNA and mRNA pairs showed significant differences with the values for all lncRNA-mRNA pairs (r_mean = 0.176, z statistics = - 2.45, p value = 0.00071) and mRNA-mRNA pairs (r_mean = 0.186, z statistics = - 2.23, p value = 0.0129). Weighted correlation network analysis of the lncRNA and mRNA datasets from 12 tissues identified 21 modules and many interesting mRNA genes that clustered with lncRNAs. Overrepresentation test indicated that these mRNAs enriched in many biological processes, such as meiosis (p = 0.00164), DNA replication (p = 0.00246), metabolic process (p = 0.000838) and in molecular function, e.g., helicase activity (p = 0.000102) and catalytic activity (p = 0.0000612). Differential expression (DE) analysis identified 99 stress-related lncRNA genes and 1955 tissue-specific DE lncRNA genes. MiRNA-lncRNA interaction analysis detected 72,267 lncRNAs containing motifs with sequence complementary to 458 miRNAs. CONCLUSIONS: This study provides an invaluable resource for further studies on molecular bases of lncRNAs in tilapia genomes. Further function analysis of the lncRNAs will help to elucidate their roles in regulating stress-related adaptation in tilapia.

Age-related changes in the mitochondrial, synthesis of steroids, and cellular homeostasis of the chicken ovary.

In poultry reproduction, the decline of ovarian function due to aging is related to dysfunction of mitochondria exacerbated by a reduction in antioxidant capacity, ultimately leading to follicle atresia and decreased egg production. However, the mechanisms of mitochondrial dysfunction in the chicken ovary in aging have remained to be understood. Hence, this study aims to investigate the effects of aging on mitochondrial function and cellular homeostasis. We collect ovarian tissue, small white follicles (SWF), large white follicles (LWF), and small yellow follicles (SYF) from three different laying periods of hens. The transmission electron microscopy (TEM) results showed that mitochondrial damage occurred in ovarian tissue during the late laying period (LP), characterized by structural swelling, scattered mitochondrial cristae, and an increase in the vacuoles. At the same time, with age, the synthesis of steroid hormones in the ovaries and follicular tissues is reduced. The levels of autophagy and cell apoptosis in ovarian tissues were both increased in the LP. In addition, aging adversely impacts mitochondrial function, leading to a decrease in mitochondrial unfolded protein response (UPRmt) functions. This study will expand the knowledge about regressing ovarian aging in hens and increasing egg production in older layers for poultry production.

Effect of immunization against OPN5 on the reproductive performance in Shan Partridge ducks under different photoperiods.

Photoperiod is an important environmental factor that influences seasonal reproduction behavior in birds. Birds translate photoperiodic information into neuroendocrine signals through deep brain photoreceptors (DBPs). OPN5 has been considered candidate DBPs involved in regulating seasonal reproduction in birds. We found that OPN5 could mediate light to regulate the follicle development in ducks. In this study, we further verified the effect of OPN5 on follicular development in Shan Partridge ducks by immunizing against the extracellular domain (ECD) of OPN5. We investigated the specific regulatory mechanism of photoperiod mediated by OPN5 on the reproductive activity of ducks. The trial randomly divided 120 Shan Partridge ducks into 3 groups with different treatments: the immunization of OPN5 group was done at d0, d15, d30, and d40 with 1 mL of vaccine containing OPN5 protein (thus containing 1, 1, 0.5, and 0.5 mg of OPN5-KLH protein), and the control group (CS and CL groups) was injected at the same time with the same dose of OPN5-uncontained blank vaccine. The group of CS (900 lux), OPN5 (600 lux), and CL (600 lux) lasted for 40 d in 12 L:12 D photoperiods, respectively. Then, the groups of CS, OPN5, and CL subsequently received 12 L:12 D, 12 L:12 D, and 17 L:7 D light treatments for 33 d, respectively. The ducks were caged in 3 constant rooms with the same feeding conditions for each group, free water, and limited feeding (150 g per duck each day). Duck serum and tissue samples were collected at d 40, d 62, and d 73 (n = 12). It was found that before prolonged light, the group of immunization (group OPN5) and the group of strong light intensity (group CS) were higher than the group of CL in egg production. Subsequent to prolonged light, the group CL in egg production rose about the same as the group immunization, while the strong light group (group CS) was lower. Group OPN5 increased the ovarian index of ducks, and both the immunization of group OPN5 and group CL (extended light) increased the thickness of the granular layer and promoted the secretion of E2, P4, LH, and PRL hormones. Compared with group CS, group CL and OPN5 increased the mRNA level and protein expression of OPN5 in the hypothalamus on d 62 and d 73 (P < 0.05). The gene or protein expression patterns of GnRH, TRH, TSHß, DIO2, THRß, VIP, and PRL were positively correlated with OPN5, whereas the gene expression patterns of GnIH and DIO3 were negatively correlated with OPN5. The results showed that immunization against OPN5 could activate the corresponding transmembrane receptors to promote the expression of OPN5, up-regulate the expression of TSHß and DIO2, and then regulate the HPG axis-related genes to facilitate the follicular development of Shan Partridge ducks. In addition, in this experiment, prolonging the photoperiod or enhancing the light intensity could also enhance follicle development, but the effect was not as significant as immunizing against OPN5. Our results will offer beneficial data and more supportive shreds of evidence in favor of elucidating the role of OPN5 in relation to photoperiods and reproduction.

Effects of stocking density on ovarian development and maturation during the rearing period in Shan-ma ducks.

Stocking density critically affects the growth and subsequent performance of animals in modern poultry production. This study investigated the effects of stocking density on ovarian development, ovarian maturation, and the mRNA expression of key genes in the reproductive axis during the rearing period of Shan-ma ducks. The experiments involved 180 healthy 7-wk-old Shan-ma ducks and randomly divided into low stocking density (LSD; n = 30, density = 5 birds/m2), medium stocking density (MSD; n = 60, density = 10 birds/m2) and high stocking density groups (HSD; n = 90, density = 15 birds/m2), for rearing. After examining ovarian development and measuring hormone levels in the plasma and expression levels of key regulatory genes in the reproductive axis at 19 wk of rearing, analysis of the gonad index analysis, reflecting stocking density, uncovered statistically significant differences. The gonad index of the LSD group was significantly higher than those of the MSD and HSD groups (P < 0.01), while no significant difference was observed between the MSD and HSD groups. pre-ovulatory follicles (POFs) and small yellow follicles (SYFs) development was only apparent in the LSD group, with the large white follicles (LWFs) number of this group being significantly higher than that of the MSD group (P < 0.05). The blood levels of E2 (estradiol), P4 (progesterone), and T (testosterone) were significantly higher in the LSD group than in the MSD and HSD groups (P < 0.05 or 0.01). Also, the levels of both P4 and T were significantly higher in the MSD group than in the HSD group (P < 0.01). The gene expression levels of GnRHR, FSH, AMHR, and FSHR were significantly increased in the LSD group compared to the MSD and HSD groups (P < 0.05 or 0.01), while the expression levels of GnIHR and GDF9 were significantly decreased in the LSD and MSD groups compared to the HSD group (P < 0.05 or 0.01). Steroid biosynthesis pathway genes such as StAR, CYP11A1, 3ß-HSD, CYP19A1, and BMP15 were significantly downregulated at greater stocking densities (P < 0.05 or 0.01). Likewise, the protein expression of StAR, 3ß-HSD, and CYP19A1 was also significantly decreased (P < 0.05 or 0.01). These results demonstrate that both medium and high stocking densities suppressed the expression of the key reproduction-promoting factors, while the expression level of the key reproductive inhibitory factors was enhanced. Therefore, rates of ovarian development and maturation could be reduced by a high stocking density leading to a delay in reproduction performance during the rearing period of Shan-ma ducks.

Long-day photoperiods affect expression of OPN5 and the TSH-DIO2/DIO3 pathway in Magang goose ganders.

This study sought to understand the regulation mechanism of OPN5 through the TSH-DIO2/DIO3 pathway mediated photoperiod on the breeding activity of short-day breeding birds. In this study, the reproductive activity of Magang goose was regulated by artificial light, and the reproductive activity of the ganders were determined according to the daily laying rate of female geese. The testicular development and the serum reproductive hormone concentrations of ganders were measured during the reproductive period (d 0), the reproductive degeneration period (d 13 and 27) and the resting period (d 45). The mRNA and protein expression patterns of OPN5, the HPG axis reproductive genes, and TSH-DIO2/DIO3 pathway related genes were examined. Results showed that the laying rate of geese and the gonadal indices (GSI) decreased gradually after the photoperiod increased. Histological observation found that the spermatogenic function of the testis was normal on d 0 and 13, while degeneration occurred by d 27 and 45. Serum testosterone, FSH, and LH concentration showed a slight increase on d 13, followed by a sharp decrease on d 27 and 45 (P < 0.01), while PRL concentrations were low on d 0 and 13, and increased rapidly on d 27 and 45 (P < 0.01).The expression pattern of GnRH, FSH, LH, and THRß mRNA were similar, with high levels on d 0 and 13 and a decreasing trend on d 27 and 45 (P < 0.05 or P < 0.01); and GnRHR mRNA levels were higher on d 13 (P < 0.05), but then had decreased by d 27 and 45 (P < 0.01). The expression pattern of GnIH and GnIHR was similar, which was opposite to that of GnRHR. VIP, PRL, and PRLR increased gradually and peaked on d 45 (P < 0.01). The expression trend of TRH, TSHß, and DIO2 was similar to that of GnRHR, and the expression abundance increased on d 13, and then decreased on d 27 and 45. GnRH protein expression was significantly higher than during the other 3 periods (P < 0.01) while the GnIH protein levels were extremely low on d 0, had gradually increased by d 13, and significantly increased by d 27 and 45 (P < 0.01). The protein expression trends of THR and DIO2 were similar to that of GNIH. DIO3 protein expression was low on d 0 and 13, and increased by d 27 and 45. These results suggest that when the photoperiod increased, the hypothalamus OPN5 gene and protein were upregulated and the pituitary TSHß, TSHR, and hypothalamus THRß, TRH, and DIO2 were downregulated, and thus the reproductive activity of geese was inhibited.

Effects of melatonin on testicular function in adult male mice under different photoperiods.

Photoperiod is an important environmental factor affecting animal physiological function. Melatonin is an endogenous hormone that plays an important role in circadian and seasonal (or cyclical) rhythms and seasonal reproduction in mammals. To investigate the effects of melatonin on the reproductive performance of adult male mice under different photoperiods, sixty mice were randomly allotted to six groups: control (Light Dark, 12 L:12 D), control plus melatonin (MLD, 12 L:12 D), 24-hour continuous light (LL, 24 L:0 D), 24-hour continuous light plus melatonin (MLL 24 L:0 D), constant darkness (DD, 0 L:24 D), and constant darkness plus melatonin (MDD, 0 L:24 D). Normal saline (100 µL) was injected into the LD, LL, and DD groups at noon each day; the MLD, MLL, and MDD groups were injected with melatonin (1 mg/mL; 2 mg/kg·body weigh). After 24 hours of prolonged light exposure, testis morphology decreased, convoluted seminiferous tubules became sparse, the diameter of convoluted seminiferous tubules decreased, and the level of sex hormones decreased. After the administration of exogenous melatonin, testicular morphology and sex hormone levels decreased in the MLD group under normal light conditions. In the MLL group, the testicular tissue morphology returned to normal, the diameter of convoluted tubules increased, the hormone levels of LH (Luteinizing hormone) and MTL (melatonin) significantly increased (P<0.05), and th0e gene expressions of LHß and Mtnr1A (Melatonin receptors 1A) increased. There was almost no difference in the MDD group under continuous darkness. In conclusion, melatonin can damage the reproductive performance of male mice under normal light conditions, while exogenous melatonin can alleviate and protect the testicular injury of male mice under continuous light conditions.

Differential Transcriptomic and Metabolomic Responses in the Liver of Nile Tilapia (Oreochromis niloticus) Exposed to Acute Ammonia.

Ammonia is toxic to aquatic animal. Currently, only limited works were reported on the responses of aquatic animals after ammonia exposure using "omics" technologies. Tilapia suffers from the stress of ammonia-nitrogen during intensive recirculating aquaculture. Optimizing ammonia stress tolerance has become an important issue in tilapia breeding. The molecular and biochemical mechanisms of ammonia-nitrogen toxicity have not been understood comprehensively in tilapia yet. In this study, using RNA-seq and gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer (GC-TOF-MS) techniques, we investigated differential expressed genes (DEGs) and metabolomes in the liver at 6 h post-challenges (6 hpc) and 24 h post-challenges (24 hpc) under high concentration of ammonia-nitrogen treatment. We detected 2258 DEGs at 6 hpc and 315 DEGs at 24 hpc. Functional enrichment analysis indicated that DEGs were significantly associated with cholesterol biosynthesis, steroid and lipid metabolism, energy conservation, and mitochondrial tissue organization. Metabolomic analysis detected 31 and 36 metabolites showing significant responses to ammonia-nitrogen stress at 6 and 24 hpc, respectively. D-(Glycerol 1-phosphate), fumaric acid, and L-malic acid were found significantly down-regulated at both 6 and 24 hpc. The integrative analysis of transcriptomics and metabolomics suggested considerable alterations and precise control of gene expression at both physiological and molecular levels in response to the stress of ammonia-nitrogen in tilapia.

Identifying a Long QTL Cluster Across chrLG18 Associated with Salt Tolerance in Tilapia Using GWAS and QTL-seq.

Understanding the genetic mechanism of osmoregulation is important for the improvement of salt tolerance in tilapia. In our previous study, we have identified a major quantitative trait locus (QTL) region located at 23.0 Mb of chrLG18 in a Nile tilapia line by QTL-seq. However, the conservation of these QTLs in other tilapia populations or species is not clear. In this study, we successfully investigated the QTLs associated with salt tolerance in a mass cross population from the GIFT line of Nile tilapia (Oreochromis niloticus) using a ddRAD-seq-based genome-wide association study (GWAS) and in a full-sib family from the Malaysia red tilapia strain (Oreochromis spp) using QTL-seq. Our study confirmed the major QTL interval that is located at nearly 23.0 Mb of chrLG18 in Nile tilapia and revealed a long QTL cluster across chrLG18 controlling for the salt-tolerant trait in both red tilapia and Nile tilapia. This is the first GWAS analysis on salt tolerance in tilapia. Our finding provides important insights into the genetic architecture of salinity tolerance in tilapia and supplies a basis for fine mapping QTLs, marker-assisted selection, and further detailed functional analysis of the underlying genes for salt tolerance in tilapia.

Rapid and sensitive detection of goose parvovirus and duck-origin novel goose parvovirus by recombinase polymerase amplification combined with a vertical flow visualization strip.

Goose parvovirus (GPV) is one of the most serious viral pathogens in goslings. Recently, a new pathogen to the Chinese mainland-duck-origin novel goose parvovirus (N-GPV)-was found to be 90.8-94.6% identical to the nucleotide sequence of GPV, and typically causes growth disorders and high infection rates in meat ducks. The spread of both of these viruses hinders the healthy development of the waterfowl breeding industry. In this study, recombinase polymerase amplification (RPA) was combined with a vertical flow (VF) visualization strip to develop a universal assay for the rapid detection of GPV and N-GPV. A set of specific primers and probes were designed to target the VP3 gene. Detection was possible at a constant temperature of 37 °C within 5-10 min. The assay successfully detected GPV and N-GPV with high-specificity and did not exhibit cross-reactivity with other waterfowl viruses and bacteria. The analytical sensitivity of the GPV-RPA-VF assay was 2 × 102 copies of GPV plasmid. Validation of the GPV-RPA-VF assay-using 60 samples from the field--confirmed 100% similarity between the results of GPV-RPA-VF and conventional qPCR. The results indicate that the GPV-RPA-VF assay was accurate, sensitive, and specific. This assay can be performed with minimal equipment and training to rapidly detect GPV and N-GPV during the early phase of an outbreak, especially when timely veterinary diagnoses are needed in the field and in rural areas.

Identification of Goose PKR Gene: Structure, Expression Profiling, and Antiviral Activity Against Newcastle Disease Virus.

Double-stranded RNA-dependent protein kinase (PKR) is an important antiviral IFN-stimulated gene (ISGs) that recognizes double-stranded RNA (dsRNA) and mediates inhibition of translation initiation and protein synthesis in various types of viral infection. In this study, the complete coding sequence (CDS) of goose PKR (goPKR) is identified and characterized. The open reading frame (ORF) of goPKR is 1668 bp, which encodes a polypeptide of 555 amino acids. The sequence identity results demonstrate that the goose PKR is most closely related to duck PKR gene, with nucleotide identities of 91.6%, whereas nucleotide identity of the goose PKR to chicken, human, and mouse PKR is 76.4%, 51.9%, and 52.0%, respectively. Interestingly, the deduced amino acid sequence of goose PKR contains 3 main structure domains, including 2 double-strand RNA-binding motif (dsRBM) domains and one serine/threonine protein kinase domain. This is similar to the chicken and mammals, whereas it is different from duck PKR protein, which contains only one dsRBM1 domain and one serine/threonine protein kinase domain. Quantitative real-time PCR analysis indicates that goose PKR mRNA is widely expressed in all sampled tissues. It is highly expressed in the blood, spleen, lung, and bursa of Fabricius and jejunum and is slightly expressed in heart, muscle, trachea, and brain. The results of confocal microscopy suggest that PKR-EGFP is mainly localized in the cytoplasm, and overexpression of goPKR protein significantly reduces Newcastle disease virus (NDV) replication (viral copies and viral titer) in goose embryo fibroblasts. These findings show that goose PKR is an important antiviral ISG, involved in the antiviral innate immune defense to NDV in geese.

Identifying a Major QTL Associated with Salinity Tolerance in Nile Tilapia Using QTL-Seq.

Selection of new lines with high salinity tolerance allows for economically feasible production of tilapias in brackish water areas. Mapping QTLs and identifying the markers linked to salinity-tolerant traits are the first steps in the improvement of the tolerance in tilapia through marker-assisted selection techniques. By using QTL-seq strategy and linkage-based analysis, two significant QTL intervals (chrLG4 and chrLG18) on salinity-tolerant traits were firstly identified in the Nile tilapia. Fine mapping with microsatellite and SNP markers suggested a major QTL region that located at 23.0 Mb of chrLG18 and explained 79% of phenotypic variation with a LOD value of 95. Expression analysis indicated that at least 10 genes (e.g., LACTB2, KINH, NCOA2, DIP2C, LARP4B, PEX5R, and KCNJ9) near or within the QTL interval were significantly differentially expressed in intestines, brains, or gills under 10, 15, or 20 ppt challenges. Our findings suggest that QTL-seq can be effectively utilized in QTL mapping of salinity-tolerant traits in fish. The identified major QTL is a promising locus to improve our knowledge on the genetic mechanism of salinity tolerance in tilapia.