Transcriptome of miRNA during inhibition of Bombyx mori nuclear polyhedrosis virus by geldanamycin in BmN cells.

Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of several viruses that cause great harm to the sericulture industry, and its pathogenic mechanism is still being explored. Geldanamycin (GA), a kind of HSP90 inhibitor, has been verified to suppress BmNPV proliferation. However, the molecular mechanism by which GA inhibits BmNPV is unclear. MicroRNAs (miRNAs) have been shown to play a key role in regulating virus proliferation and host-pathogen interactions. In this study, BmN cells infected with BmNPV were treated by GA and DMSO for 72 h, respectively, then transcriptome analysis of miRNA was performed from the GA group and the control group. As a result, a total of 29 miRNAs were differentially expressed (DE), with 13 upregulated and 16 downregulated. Using bioinformatics analysis, it was found that the target genes of DEmiRNAs were involved in ubiquitin-mediated proteolysis, phagosome, proteasome, endocytosis pathways, and so on. Six DEmiRNAs were verified by quantitative reverse-transcription polymerase chain reaction. DElong noncoding RNA (DElncRNA)-DEmiRNA-messenger RNA (mRNA) regulatory networks involved in apoptosis and immune pathways were constructed in GA-treated BmN cells, which included 12 DEmiRNA, 132 DElncRNA, and 69 mRNAs. This regulatory network enriched the functional role of miRNA in the BmNPV-silkworm interactions and improved our understanding of the molecular mechanism of HSP90 inhibitors on BmNPV proliferation.

Identification of long noncoding RNAs in silkworm larvae infected with Bombyx mori cypovirus.

Bombyx mori cypovirus (BmCPV) is one of the most important pathogens causing severe disease to silkworm. Emerging evidence indicates that long noncoding RNAs (lncRNAs) play importantly regulatory roles in virus infection and host immune response. To better understand the interaction between silkworm, Bombyx mori and BmCPV, we performed a comparative transcriptome analysis on lncRNAs and mRNAs between the virus-infected and noninfected silkworm larvae midgut at two time points postinoculation. A total of 16,753 genes and 1845 candidate lncRNAs were identified, among which 356 messenger RNA (mRNAs) and 41 lncRNAs were differentially expressed (DE). Target gene prediction revealed that most of DEmRNAs (123) were coexpressed with 28 DElncRNAs, suggesting that the expression of mRNA is mainly affected through trans- regulation by BmCPV-induced lncRNAs, and a regulatory network of DElncRNAs and DEmRNAs was then constructed. According to the network, many genes involved in apoptosis, autophagy, and antiviral response, such as ATG3, PDCD6, IBP2, and MFB1, could be targeted by different DElncRNAs, implying the essential roles of these genes and lncRNAs in BmCPV infection. In all, our studies revealed for the first time the alteration of lncRNA expression in BmCPV-infected larvae and its potential influence on BmCPV replication, providing a new perspective for host-cypovirus interaction studies.

Integrative analysis of circRNA/miRNA/mRNA regulatory network reveals the potential immune function of circRNAs in the Bombyx mori fat body.

Bombyx mori nucleopolyhedrosis virus (BmNPV) is one of the greatest threats to sustainable development of the sericulture industry. Circular RNA (circRNA), a type of non-coding RNA, has been shown to play important roles in gene expression regulation, immune response, and diseases. The fat body is a tissue with both metabolic and immune functions. To explore the potential immune function of circRNAs, we analyzed differentially expressed (DE)circRNAs, microRNAs(miRNAs), and mRNAs in the B. mori fat body in response to BmNPV infection using high-throughput RNA sequencing. A total of 77 DEcircRNAs, 32 DEmiRNAs, and 730 DEmRNAs that are associated with BmNPV infection were identified. We constructed a DEcircRNA/DEmiRNA/DEmRNA and DEcircRNA/DEmiRNA/BmNPV gene regulatory network and validated the differential expression of circ_0001432 and its corresponding miRNA (miR-2774c and miR-3406-5p) and mRNA (778467 and 101745232) in the network. Tissue-specific expression of circ_0001432 and its expression at different time points were also examined. KEGG pathway analysis of DEmRNAs, target genes of DEmiRNAs, and host genes of DEcircRNAs in the network showed that these genes were enriched in several metabolic pathways and signaling pathways, which could play important roles in insect immune responses. Our results suggest that circRNA could be involved in immune responses of the B. mori fat body and help in understanding the molecular mechanisms underlying silkworm-pathogen interactions.

Genome-wide association study provides insights into the genetic architecture of bone size and mass in chickens.

Bone size is an important trait for chickens because of its association with osteoporosis in layers and meat production in broilers. Here, we employed high density genotyping platforms to detect candidate genes for bone traits. Estimates of the narrow heritabilities ranged from 0.37 ± 0.04 for shank length to 0.59 ± 0.04 for tibia length. The dominance heritability was 0.12 ± 0.04 for shank length. Using a linear mixed model approach, we identified a promising locus within NCAPG on chromosome 4, which was associated with tibia length and mass, femur length and area, and shank length. In addition, three other loci were associated with bone size or mass at a Bonferroni-corrected genome-wide significance threshold of 1%. One region on chicken chromosome 1 between 168.38 and 171.82 Mb harbored HTR2A, LPAR6, CAB39L, and TRPC4. A second region that accounted for 2.2% of the phenotypic variance was located around WNT9A on chromosome 2, where allele substitution was predicted to be associated with tibia length. Four candidate genes identified on chromosome 27 comprising SPOP, NGFR, GIP, and HOXB3 were associated with tibia length and mass, femur length and area, and shank length. Genome partitioning analysis indicated that the variance explained by each chromosome was proportional to its length.

Expression profile analysis of circular RNAs in BmN cells (Bombyx mori) upon BmNPV infection.

The disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV) has always been difficult to control, resulting in tremendous economic losses in the sericulture industry. Although much has been learned about the impact of noncoding RNAs on pathogenesis, the role of circular RNA (circRNA) in insect immunity remains unclear. To explore circRNA regulation involved in BmNPV infection, we used transcriptome analysis of BmN cells with or without BmNPV infection to generate circRNA data set. A total of 444 novel circRNAs were identified in BmN cells, with 198 pervasively distributed both in the control group and BmNPV-infection group. The host genes were enriched inMAPK signaling pathway, dorso-ventral axis formation, and ECM-receptor interaction, which were required for the normal larval growth. A total of 75 circRNAs were differentially expressed (DE) on BmNPV infection. Six downregulated circRNAs were validated by Sanger sequencing and qRT-PCR. DEcircRNA-miRNA-DEmRNA network was constructed based on the six validated circRNAs. Pathway analysis indicated that the predicted target genes were mainly enriched in the metabolic pathway and immune-related signaling pathway. Our results may provide a basis for further studies on circRNA function in BmN cells challenged by BmNPV infection and offer an insight into the molecular mechanism on silkworm-virus interaction.

Analysis of lncRNA-mediated gene regulatory network of Bombyx mori in response to BmNPV infection.

Bombyx mori nucleopolyhedrosis virus (BmNPV) has always been a great challenge to the development and stability of the sericulture industry. LncRNAs have been reported to play important roles in gene expression regulation, development and immune response but the roles of lncRNAs in BmNPV infection and silkworm-BmNPV interaction are not clear. We used a genome-wide transcriptome analysis to identify the lncRNAs in Bombyx mori cells (BmN cells) and analyzed the differentially expressed lncRNAs, microRNAs and protein-coding genes in silkworm cells with or without BmNPV infection. A total of 13,159 candidate lncRNAs were identified in the BmN cells, among which 4450 lncRNAs were differentially expressed (DE) with 2837 up-regulated and 1613 down-regulated. In addition, 66 differentially expressed miRNAs (DEmiRNAs) and 7448 differentially expressed mRNAs (DEmRNAs) were identified, and DElncRNA-DEmiRNA-DEmRNA regulatory network was constructed. Gene expression was variable in 4973 of predicted lncRNA cis target genes in BmNPV infected cells. KEGG pathway analysis indicated that the target genes of DElncRNAs are enriched in ubiquitin mediated proteolysis, endocytosis and lysosome pathways. qRT-PCR validated the differential expression of several lncRNAs and miRNAs. Our results suggested that DElncRNAs participate in host response to BmNPV infection via interactions with their target genes and miRNAs. Our results will help us to improve our understanding of lncRNA-mediated regulatory roles in BmNPV infection and provide new insights into silkworm-pathogen interactions.

Genetic evaluation of eggshell color based on additive and dominance models in laying hens.

OBJECTIVE: Eggshells with a uniform color and intensity are important for egg production because many consumers assess the quality of an egg according to the shell color. In the present study, we evaluated the influence of dominant effects on the variations in eggshell color after 32 weeks in a crossbred population. METHODS: This study was conducted using 7,878 eggshell records from 2,626 hens. Heritability was estimated using a univariate animal model, which included inbreeding coefficients as a fixed effect and animal additive genetic, dominant genetic, and residuals as random effects. Genetic correlations were obtained using a bivariate animal model. The optimal diagnostic criteria identified in this study were: L* value (lightness) using a dominance model, and a* (redness), and b* (yellowness) value using an additive model. RESULTS: The estimated heritabilities were 0.65 for shell lightness, 0.42 for redness, and 0.60 for yellowness. The dominance heritability was 0.23 for lightness. The estimated genetic correlations were 0.61 between lightness and redness, -0.84 between lightness and yellowness, and -0.39 between redness and yellowness. CONCLUSION: These results indicate that dominant genetic effects could help to explain the phenotypic variance in eggshell color, especially based on data from blue-shelled chickens. Considering the dominant genetic variation identified for shell color, this variation should be employed to produce blue eggs for commercial purposes using a planned mating system.

Dynamic expression and functional analysis of circRNA in granulosa cells during follicular development in chicken.

BACKGROUND: Circular RNA (circRNA) is a type of noncoding RNA involved in a variety of biological processes, especially in post-transcriptional regulation. The granulosa cells of follicles play a determining role in ovarian development. However, the function of circRNA in chicken follicles is unclear. To better understand the molecular mechanism underlying follicular development and granulosa cell function, we performed a strategy of second-generation sequencing and linear RNA depletion for granulosa cells from small yellow follicles (SYF, 5-8 mm), the smallest hierarchal follicles (F6, 9-12 mm), and the largest hierarchal follicles (F1, ~ 40 mm). RESULTS: We predicted a total of 11,642 circRNAs that distributed on almost all chromosomes. The majority of the splice lengths of circRNAs were 200-500 nt and mainly produced from intron and CDS regions. During follicle growth, differentially expressed (DE) circRNAs showed dynamic changes which were tissue- and stage-specific. The host genes of DE circRNAs were functionally enriched in GTPase activity and several pathways involved in reproduction. Moreover, bioinformatic prediction analysis for circRalGPS2 demonstrated that circRNAs from the same genes may share common miRNA to act as a sponge. The predicted target genes were enriched in various biological processes including cognition, cell communication, and regulation of signaling, and several pathways related to reproduction such as tight junction, oocyte meiosis, progesterone-mediated oocyte maturation, and GnRH signaling. CONCLUSIONS: This study provides a starting point for further experimental investigations into chicken circRNAs and casts a light on the understanding of follicle development.

Genetic architecture and candidate genes detected for chicken internal organ weight with a 600 K single nucleotide polymorphism array.

OBJECTIVE: Internal organs indirectly affect economic performance and well-being of animals. Study of internal organs during later layer period will allow full utilization of layer hens. Hence, we conducted a genome-wide association study (GWAS) to identify potential quantitative trait loci or genes that potentially contribute to internal organ weight. METHODS: A total of 1,512 chickens originating from White Leghorn and Dongxiang Blue-Shelled chickens were genotyped using high-density Affymetrix 600 K single nucleotide polymorphism (SNP) array. We conducted a GWAS, linkage disequilibrium analysis, and heritability estimated based on SNP information by using GEMMA, Haploview and GCTA software. RESULTS: Our results displayed that internal organ weights show moderate to high (0.283 to 0.640) heritability. Variance partitioned across chromosomes and chromosome lengths had a linear relationship for liver weight and gizzard weight (R2 = 0.493, 0.753). A total of 23 highly significant SNPs that associated with all internal organ weights were mainly located on Gallus gallus autosome (GGA) 1 and GGA4. Six SNPs on GGA2 affected heart weight. After the final analysis, five top SNPs were in or near genes 5-Hydroxytryptamine receptor 2A, general transcription factor IIF polypeptide 2, WD repeat and FYVE domain containing 2, non-SMC condensin I complex subunit G, and sonic hedgehog, which were considered as candidate genes having a pervasive role in internal organ weights. CONCLUSION: Our findings provide an understanding of the underlying genetic architecture of internal organs and are beneficial in the selection of chickens.

Genome-wide association study revealed a promising region and candidate genes for eggshell quality in an F2 resource population.

BACKGROUND: Eggshell is subject to quality loss with aging process of laying hens, and damaged eggshells result in economic losses of eggs. However, the genetic architecture underlying the dynamic eggshell quality remains elusive. Here, we measured eggshell quality traits, including eggshell weight (ESW), eggshell thickness (EST) and eggshell strength (ESS) at 11 time points from onset of laying to 72 weeks of age and conducted comprehensive genome-wide association studies (GWAS) in 1534 F2 hens derived from reciprocal crosses between White Leghorn (WL) and Dongxiang chickens (DX). RESULTS: ESWs at all ages exhibited moderate SNP-based heritability estimates (0.30 ~ 0.46), while the estimates for EST (0.21 ~ 0.31) and ESS (0.20 ~ 0.27) were relatively low. Eleven independent univariate genome-wide screens for each trait totally identified 1059, 1026 and 1356 significant associations with ESW, EST and ESS, respectively. Most significant loci were in a region spanning from 57.3 to 71.4 Mb of chromosome 1 (GGA1), which together account for 8.4 ~ 16.5% of the phenotypic variance for ESW from 32 to 72 weeks of age, 4.1 ~ 6.9% and 2.95 ~ 16.1% for EST and ESS from 40 to 72 weeks of age. According to linkage disequilibrium (LD) and conditional analysis, the significant SNPs in this region were in extremely strong linkage disequilibrium status. Ultimately, two missense SNPs in GGA1 and one in GGA4 were considered as promising loci on three independent genes including ITPR2, PIK3C2G, and NCAPG. The homozygotes of advantageously effective alleles on PIK3C2G and ITPR2 possessed the best eggshell quality and could partly counteract the negative effect of aging process. NCAPG had certain effect on eggshell quality for young hens. CONCLUSIONS: Identification of the promising region as well as potential candidate genes will greatly advance our understanding of the genetic basis underlying dynamic eggshell quality and has the practical significance in breeding program for the improvement of eggshell quality, especially at the later part of laying cycle.

Genome-wide association study dissects genetic architecture underlying longitudinal egg weights in chickens.

BACKGROUND: As a major economic trait in chickens, egg weight (EW) receives widespread interests in breeding, production and consumption. However, limited information is available for underlying genetic architecture of longitudinal trend in EW. Herein, we measured EWs at nine time points from onset of laying to 60 week of age, and conducted comprehensive genome-wide association studies (GWAS) in 1,534 F2 hens derived from reciprocal crosses between White Leghorn and Dongxiang chickens. RESULTS: Egg weights at all ages except the first egg weight (FEW) exhibited high SNP-based heritability estimates (0.47~0.60). Strong pair-wise genetic correlations (0.77~1.00) were found among all EWs. Nine separate univariate genome-wide screens suggested 73 signals showing significant associations with longitudinal EWs. After multivariate and conditional analyses, four variants on three chromosomes remained independent contributions. The minor alleles at two loci exerted consistent and positive substitution effects on EWs, and other two were negative. The four loci together accounted for 3.84 % of the phenotypic variance for FEW and 7.29~11.06 % for EWs from 32 to 60 week of age. We obtained five candidate genes, of which NCAPG harbors a non-synonymous SNP (rs14491030) causing a valine-to-alanine amino-acid substitution. Genome partitioning analysis indicated a strong linear correlation between the variance explained by each chromosome and its length, which provided evidence that EW follows a highly polygenic nature of inheritance. CONCLUSIONS: Identification of significant genetic causes that together implicate EWs at different ages will greatly advance our understanding of the genetic basis behind longitudinal EWs, and would be helpful to illuminate the future breeding direction on how to select desired egg size.

Genome-wide association studies for feed intake and efficiency in two laying periods of chickens.

BACKGROUND: Feed contributes to over 60 % of the total production costs in the poultry industry. Increasing feed costs prompt geneticists to include feed intake and efficiency as selection goals in breeding programs. In the present study, we used an F2 chicken population in a genome-wide association study (GWAS) to detect potential genetic variants and candidate genes associated with daily feed intake (FI) and feed efficiency, including residual feed intake (RFI) and feed conversion ratio (FCR). METHODS: A total of 1534 F2 hens from a White Leghorn and Dongxiang reciprocal cross were phenotyped for feed intake and efficiency between 37 and 40 weeks (FI1, RFI1, and FCR1) and between 57 and 60 weeks (FI2, RFI2, and FCR2), and genotyped using the chicken 600 K single nucleotide polymorphism (SNP) genotyping array. Univariate, bivariate, and conditional genome-wide association studies (GWAS) were performed with GEMMA, a genome-wide efficient mixed model association algorithm. The statistical significance threshold for association was inferred by the simpleM method. RESULTS: We identified eight genomic regions that each contained at least one genetic variant that showed a significant association with FI. Genomic regions on Gallus gallus (GGA) chromosome 4 coincided with known quantitative trait loci (QTL) that affect feed intake of layers. Of particular interest, eight SNPs on GGA1 in the region between 169.23 and 171.55 Mb were consistently associated with FI in both univariate and bivariate GWAS, which explained 3.72 and 2.57 % of the phenotypic variance of FI1 and FI2, respectively. The CAB39L gene can be considered as a promising candidate for FI1. For RFI, a haplotype block on GGA27 harbored a significant SNP associated with RFI2. The major allele of rs315135692 was favorable for a lower RFI, with a phenotypic difference of 3.35 g/day between opposite homozygous genotypes. Strong signals on GGA1 were detected in the bivariate GWAS for FCR. CONCLUSIONS: The results demonstrated the polygenic nature of feed intake. GWAS identified novel variants and confirmed a QTL that was previously reported for feed intake in chickens. Genetic variants associated with feed efficiency may be used in genomic breeding programs to select more efficient layers.

Genetic parameters of feed efficiency traits in laying period of chickens.

Laying records on 1,534 F2 hens, derived from a reciprocal cross between White Leghorns and Dongxiang blue-shelled chickens, were used to estimate genetic parameters for residual feed intake (RFI), feed conversion ratio (FCR), daily feed intake (FI), metabolic BW (MBW), BW gain (BWG), and daily egg mass (EM) at 37 to 40 (T1) and 57 to 60 wk age (T2), respectively. Genetic analysis was subsequently conducted with the AI-REML method using an animal model. Estimates for heritability of RFI, FCR, and FI were 0.21, 0.19, and 0.20 in T1, and 0.29, 0.13, and 0.26 in T2, respectively. In T1 and T2, RFI showed high and positive genetic correlations with FCR (0.51, 0.43) and FI (0.72, 0.84), whereas the genetic correlation between FI and FCR was very low (-0.09, 0.11). Genetically, negative correlations were found between RFI and its component traits (-0.01 to -0.47). In addition, high genetic correlations, from 0.76 to 0.94, were observed between T1 and T2 for RFI, FCR, and FI, suggesting that feed efficiency traits in the 2 stages had a similar genetic background. The results indicate that selection for low RFI could reduce FI without significant changes in EM, while selection on FCR will increase EM. The present study lays the foundation for genetic improvement of feed efficiency during the laying period of chickens.

microRNA transcriptome analysis of granulosa cells predicts that the Notch and insulin pathways affect follicular development in chickens.

MicroRNAs (miRNAs) have been documented to play critical roles in chicken reproduction. Granulosa cell (GC) development of the follicle is closely related to hierarchical follicle ordering, making it an important factor in determining laying performance. Thus, it is meaningful to mine follicular development-related miRNAs. To identify regulatory miRNAs and the biological mechanisms by which they control follicular development, we conducted small RNA sequencing of GCs isolated from prehierarchical follicles named small yellow follicle (SYFG), the smallest hierarchical follicle (F6G), and the largest hierarchical follicle (F1G). A total of 99, 196, and 110 differentially expressed miRNAs (DEMs) were identified in SYFG.vs.F6G, SYFG.vs.F1G, and F6G.vs.F1G, respectively. Of these, 22 miRNAs, including miR-223, miR-103a, miR-449c-3p, and miR-203a, were ubiquitously identified as DEMs in three stages. Target gene prediction suggested that these miRNAs are associated with the MAPK, TGF-ß, and Wnt signaling pathways, which are all associated with follicular development. The Notch and insulin signaling pathways were commonly enriched in all three comparisons. RT-qPCR analysis further indicated that the expression levels of PSEN2, which encodes an essential factor regulating Notch and insulin signaling, was significantly changed in SYFG, F6G, and F1G. The current study provides basic data and offers a new foundation for further exploration of the roles of miRNAs in follicular development in chickens.

Effects of quercetin on granulosa cells from prehierarchical follicles by modulating MAPK signaling pathway in chicken.

Quercetin (Que), widely found in a huge variety of plants, plays important roles in ovarian function. However, to data, there have been no reports about Que regulating granulosa cells (GCs) in prehierarchical follicles in chicken. Herein, GCs from follicles diameter from 4 to 8 mm in chicken were treated by Que in vitro culture to investigate how Que exerts its effect on follicular development. GCs treated by Que in concentrations of 10, 100, and 1,000 ng/mL were tested for cell proliferation and progesterone secretion. Eight cDNA libraries were constructed from GCs (4 samples per group) to explore transcriptome expression changes. The role of the MAPK/ERK signaling pathway was validated in this process. Treatment with 100 and 1,000 ng/mL levels of Que significantly promoted cell proliferation and progesterone secretion (P < 0.05). RNA-seq analysis data showed that 402 and 263 differentially expressed genes (DEGs) were up- and down-regulated, respectively. Functional enrichment analysis that the pathways related to follicular development included biosynthesis of amino acids, MAPK signaling pathway, and calcium signaling pathway. Notably, the function exerted in GCs of the different levels of Que was associated with the suppression of the MAPK pathway. In conclusion, our results proved that low levels of Que could promote MAPK signaling pathway, but high levels of Que inhibit MAPK signaling pathway in GCs from the prehierarchical follicles, promote cell proliferation and progesterone secretion, and benefit follicle selection.

The Effects of 1-Deoxynojirimycin from Mulberry on Oxidative Stress and Inflammation in Laying Hens and the Direct Effects on Intestine Epithelium Cells In Vitro.

The intestine is highly vulnerable to various factors and has been proposed as a promising determinant for poultry health. Phytogenic or plant-derived feed additives can be used to help improve intestinal health. In this study, we aimed to investigate the effects of DNJ on the antioxidative parameters, including malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and inflammatory cytokines (IL-6, IL-1ß, and TNF-α), in plasma and intestinal tissues using layers supplemented with or without the DNJ extract of mulberry leaves (DNJ-E) via the ELISA method. A total of 192 healthy Hy-Line Brown layers, aged 47 weeks old, were used to conduct a 56-day study. All hens were randomly separated into four groups as follows: a basal diet containing 0 mg/kg DNJ-E(CON), 50 mg/kg, 100 mg/kg, and 150 mg/kg DNJ-E. Furthermore, the potential mechanism by which DNJ influences intestinal function was also investigated in in vitro cultured intestinal epithelium cells (IEC) with quantification methods including the use of a cell counting kit-8 (CCK8), ELISA, qRT-PCR, and ROS detection. The results showed that CAT in plasma significantly increased following 50 mg/kg DNJ-E supplementation. Moreover, 50 mg/kg DNJ-E supplementation was associated with increases in T-SOD in the jejunum and ileum. However, there was no significant difference in inflammatory cytokines between groups in in vivo experiments. Subsequent in vitro IEC studies revealed that cell viability increased significantly following 5 µM and 10 µM DNJ treatments while decreasing significantly following 20 µM DNJ treatment. Antioxidative parameters improved at 5 µM and 10 µM DNJ concentrations. However, there were no ameliorative effects on antioxidant parameters observed under 20 µM DNJ treatment. The expression levels of Nrf2 mRNA increased significantly under DNJ treatment. DNJ treatment was associated with significant changes in the expression of genes of inflammatory cytokines. In conclusion, our study revealed that DNJ could improve oxidative stress and inflammation responses in the chicken intestine. These findings provide a theoretical reference for the development of functional feed additives that regulate intestinal health and lay the foundation for systematically revealing the mechanism of DNJ.

A cypovirus encoded microRNA negatively regulates the NF-κB pathway to enhance viral multiplication in Silkworm, Bombyx mori.

MicroRNAs (miRNAs) are small non-coding RNAs that function as novel gene expression regulators at the post-transcriptional level. Not with standing that the biogenesis and function of miRNAs are well-understood in eukaryotes, little is known about RNA virus-encoded miRNAs. Bombyx mori cypovirus (BmCPV) is a double-stranded RNA virus with a segmented genome that causes cytoplasmic polyhedrosis disease in silkworm larvae. To date, the interaction between BmCPV and silkworm remains largely unclear. 22 candidate BmCPV-encoded miRNAs were identified in this study through small RNA sequencing, stem-loop RT-PCR and qRT-PCR. Then, generation and function analyses were conducted on one of the candidate miRNAs, BmCPV-miR-1, in the BmN cells and the silkworm larvae by RNA interference, quantitative PCR, dual-luciferase assay. Our results revealed that BmCPV-miR-1 was encoded by BmCPV genome RNA rather than the degraded fragments of the viral genome. Its generation depended on Dicer-1 and might also be correlated with Dicer-2, Argonaute-1 and Argonaute-2. Moreover, BmCPV-miR-1 could suppress the expression of the target gene, B. mori inhibitor of nuclear factor kappa-B kinase subunit beta (BmIKKß), via binding to the target mRNA 3'-untranslated region, which fine-tuned the host NF-κB signaling pathway and consequently enhanced viral replication. Our results provide new evidence supporting the hypothesis that RNA viruses could generate miRNAs to modulate antiviral host defense.

The anti-Müllerian hormone gene's second exon is associated with the reproductive performance of Jinghai Yellow chickens.

Anti-Müllerian hormone (AMH), a member of the transforming growth factor- ß superfamily, plays important regulatory roles in follicular development and sex differentiation. Although much has been learned about the impact of polymorphisms of AMH on reproduction in animals, the effect on chicken reproduction is not well explored. In this study, the polymorphism of five exons of AMH gene and its effect on the reproductive performance of Jinghai Yellow chickens were studied. Primers for the amplification of AMH exons were designed, and Sanger sequencing was performed. Finally, only the polymorphism in the second exon of the AMH gene was found in the present population. Polymorphisms in the second exon of the AMH gene in 246 Jinghai Yellow hens and their associations with reproductive traits were analyzed. In total, four single nucleotide polymorphism (SNP) mutations were detected in the second exon of the AMH gene: g.1868A > C (AA, aa and Aa); g.1883G > A (BB, bb and Bb); g.1987G > A (CC, cc and Cc); and g.1996A > G (DD, dd and Dd). Only the mutation of g.1996A > G affected the reproductive traits: the age of laying first egg (AFE) of dd genotype was significantly ( p < 0.01 ) earlier than that in the DD and Dd hens. Moreover, the egg number by 300 d old (EN300) of dd individuals was significantly higher than that of DD and Dd individuals ( p < 0.01 ). Thus, we inferred that the dd genotype is the beneficial genotype. Additionally, AFE and EN300 showed significantly better performance in both the H2H2 and H7H7 diplotypes compared with other diplotype individuals ( p < 0.01 ). Thus, the H2H2 and H7H7 genotype had the best combination of AFE and EN300. Our study may allow for molecular marker section in poultry breeding.

Two Putative Cypovirus-Encoded miRNAs Co-regulate the Host Gene of GTP-Binding Nuclear Protein Ran and Facilitate Virus Replication.

microRNA (miRNA) plays important roles in regulating various biological processes, including host-pathogen interaction. Recent studies have demonstrated that virus-encoded miRNAs can manipulate host gene expression to ensure viral effective multiplication. Bombyx mori cypovirus (BmCPV), a double-stranded RNA virus with a segmented genome, is one of the important pathogens for the economically important insect silkworm. Our present study indicated that two putative miRNAs encoded by BmCPV could promote viral replication by inhibiting the gene expression of B. mori GTP-binding nuclear protein Ran (BmRan), an essential component of the exportin-5-mediated nucleocytoplasmic transport of small RNAs. BmCPV-miR-1 and BmCPV-miR-3 are two of the BmCPV-encoded miRNAs identified in our previous studies. BmRan is a common target gene of them with binding sites all located in the 3'-untranslated region (3'-UTR) of its mRNA. The expression levels of the two miRNAs in the midgut of larvae infected with BmCPV gradually increased with the advance of infection, while the expression of the target gene BmRan decreased gradually. The miRNAs and the recombinant target gene consisting of reporter gene mCherry and 3'-UTR of BmRan mRNA were expressed in HEK293T cells for validating the interaction between the miRNAs and the target gene. qRT-PCR results revealed that BmCPV-miR-1 and BmCPV-miR-3 negatively regulate target gene expression not only separately but also cooperatively by binding to the 3'-UTR of BmRan mRNA. By transfecting miRNA mimics into BmN cells and injecting the mimics into the body of silkworm larvae, it was indicated that both BmCPV-miR-1 and BmCPV-miR-3 could repress the expression of BmRan in BmN cells and in the silkworm, and the cooperative action of the two miRNAs could enhance the repression of BmRan expression. Furthermore, the repression of BmRan could facilitate the replication of BmCPV genomic RNAs. It is speculated that BmCPV-miR-1 and BmCPV-miR-3 might reduce the generation of host miRNAs by inhibiting expression of BmRan, thus creating a favorable intracellular environment for virus replication. Our results are helpful to better understand the pathogenic mechanism of BmCPV to the silkworm, and provide insights into one of the evasion strategies used by viruses to counter the host defense for their effective multiplication.

Effects of Energy-Restricted Feeding during Rearing on the Performance, Uniformity, and Development of Rugao Layer Breeders at the Initiation of the Laying Period.

The aim of this study was to assess the effects of energy-restricted feeding during rearing on the performance, uniformity, and development of layer breeders at the initiation of the laying period. A total of 2400 8-week-old Rugao layer breeders were randomly assigned to one of five groups (480 pullets per group) with eight replicates and were fed one of five diets that were nutritionally equal with the exception of apparent metabolizable energy corrected for nitrogen (AMEn) content (2850, 2750, 2650, 2550, and 2450 kcal AMEn/kg) from 8 to 18 weeks of age. The daily amount of feed was restricted to the absolute quantity of the diet consumed by laying hens fed 2850 kcal AMEn per kg diet ad libitum (control). From 18 to 21 weeks of age, all hens were fed a basal diet ad libitum. The body weight (BW) of the laying pullets decreased linearly with increasing energy restriction (p < 0.001) but recovered within 3 weeks of ad libitum feeding (p = 0.290). A gradual increase in the degree of energy restriction resulted in a gradual decrease in average daily weight gain (ADG) and a gradual increase in the feed conversion ratio (FCR) and energy conversion ratio (ECR) from 8 to 18 weeks of age (p < 0.001, p < 0.001, p = 0.008). In contrast, the ADG and ADFI (p < 0.001, p < 0.001) gradually increased, while the FCR and ECR (p < 0.001, p < 0.001) gradually improved from 18 to 21 weeks of age. From 8 to 21 weeks of age, ECR improved (p = 0.005) with an increasing degree of energy restriction. The energy-restricted feeding for 6 weeks to the end of the trial improved BW uniformity (p < 0.05). The relative length and circumference of tarsus (p < 0.001, p < 0.001), and the relative weights and lengths of the small intestine, duodenum, jejunum, ileum, and caeca increased linearly (p < 0.001, p = 0.012, p < 0.007, p = 0.012, p = 0.040; p < 0.001, p = 0.003, p = 0.032, p = 0.029, p = 0.040) with increasing energy restriction at 18 weeks of age. After switching to ad libitum feeding for 3 weeks, the relative weights and lengths of the small intestine, duodenum, and jejunum of laying pullets increased linearly with increasing energy restriction (p < 0.001, p = 0.016, p = 0.011; p = 0.009, p = 0.028, p = 0.032). In conclusion, moderate energy restriction (85.97%, 2450 vs. 2850 kcal AMEn/kg) from 8 to 18 weeks of age and switching to ad libitum feeding from 18 to 21 weeks of age can be used to improve BW uniformity and stimulate the development of the duodenum and jejunum of native layer breeders at the initiation of the laying period without compromising BW.