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The excavation and utilization of dormancy loci in breeding are effective endeavors for enhancing the resistance to pre-harvest sprouting (PHS) of wheat varieties. CH1539 is a wheat breeding line with high-level seed dormancy. To clarify the dormant loci carried by CH1539 and obtain linked molecular markers, in this study, a recombinant inbred line (RIL) population derived from the cross of weak dormant SY95-71 and strong dormant CH1539 was genotyped using the Wheat17K single-nucleotide polymorphism (SNP) array, and a high-density genetic map covering 21 chromosomes and consisting of 2437 SNP markers was constructed. Then, the germination percentage (GP) and germination index (GI) of the seeds from each RIL were estimated. Two QTLs for GP on chromosomes 5A and 6B, and four QTLs for GI on chromosomes 5A, 6B, 6D and 7A were identified. Among them, the QTL on chromosomes 6B controlling both GP and GI, temporarily named QGp/Gi.sxau-6B, is a major QTL for seed dormancy with the maximum phenotypic variance explained of 17.66~34.11%. One PCR-based diagnostic marker Ger6B-3 for QGp/Gi.sxau-6B was developed, and the genetic effect of QGp/Gi.sxau-6B on the RIL population and a set of wheat germplasm comprising 97 accessions was successfully confirmed. QGp/Gi.sxau-6B located in the 28.7~30.9 Mbp physical position is different from all the known dormancy loci on chromosomes 6B, and within the interval, there are 30 high-confidence annotated genes. Our results revealed a novel QTL QGp/Gi.sxau-6B whose CH1539 allele had a strong and broad effect on seed dormancy, which will be useful in further PHS-resistant wheat breeding.

Natural products derived from medicinal plants offer convenience and therapeutic potential and have inspired the development of antimicrobial agents. Thus, it is worth exploring the combination of nanotechnology and natural products. In this study, silver nanoparticles (AgNPs) were synthesized from the leaf extract of Ginkgo biloba (Gb), having abundant flavonoid compounds. The reaction conditions and the colloidal stability were assessed using ultraviolet–visible spectroscopy. X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy (FTIR) were used to characterize the AgNPs. AgNPs exhibited a spherical morphology, uniform dispersion, and diameter ranging from ~8 to 9 nm. The FTIR data indicated that phytoconstituents, such as polyphenols, flavonoids, and terpenoids, could potentially serve as reducing and capping agents. The antibacterial activity of the synthesized AgNPs was assessed using broth dilution and agar well diffusion assays. The results demonstrate antibacterial effects against both Gram-positive and Gram-negative strains at low AgNP concentrations. The cytotoxicity of AgNPs was examined in vitro using the CCK-8 method, which showed that low concentrations of AgNPs are noncytotoxic to normal cells and promote cell growth. In conclusion, an environmentally friendly approach for synthesizing AgNPs from Gb leaves yielded antibacterial AgNPs with minimal toxicity, holding promise for future applications in the field of biomedicine.

Leucine-rich repeat (LRR) is a structural motif has important recognition function in immune receptors, such as Tolls and NOD-like receptors (NLRs). The immune-related LRR proteins can be divided into two categories, LRR-containing proteins and LRR-only proteins. The latter contain LRR motifs while they are without other functional domains. However, the functional mechanisms of the LRR-only proteins were still unclear in invertebrates. Here, we identified a gene encoding a secretory LRR-only protein, which possessed similarity with vertebrate CD14 and was designated as LvCD14L, from the Pacific whiteleg shrimp Litopenaeus vannamei. Its transcripts in shrimp hemocytes were apparently responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvCD14L with dsRNA resulted in significant increase of the viable bacteria in the hepatopancreas of shrimp upon V. parahaemolyticus infection. Further functional studies revealed that LvCD14L could bind to microorganisms’ PAMPs, showed interaction with LvToll1 and LvToll2, and regulated the expression of LvDorsal and LvALF2 in hemocytes. These results suggest that LvCD14L functions as a pattern recognition receptor and activates the NF-κB pathway through interaction with LvTolls. The present study reveals a shrimp LvCD14L-Tolls-NF-κB signaling pathway like the CD14/TLR4/NF-κB signaling pathway in mammalians, which enriches the functional mechanism of secretory LRR-only immune receptors during pathogens infection in invertebrates.

The Pacific white shrimp Litopenaeus vannamei is the most economically important crustacean in the world. The growth and development of shrimp muscle has always been the focus of attention. Myocyte Enhancer Factor 2 (MEF2), a member of MADS transcription factor, has an essential influence on various growth and development programs, including myogenesis. In this study, based on the genome and transcriptome data of L. vannamei, the gene structure and expression profiles of MEF2 were characterized. We found that the LvMEF2 was widely expressed in various tissues, mainly in the Oka organ, brain, intestine, heart, and muscle. Moreover, LvMEF2 has a large number of splice variants, and the main forms are the mutually exclusive exon and alternative 5′ splice site. The expression profiles of the LvMEF2 splice variants varied under different conditions. Interestingly, some splice variants have tissue or developmental expression specificity. After RNA interference into LvMEF2, the increment in the body length and weight decreased significantly and even caused death, suggesting that LvMEF2 can affect the growth and survival of L. vannamei. Transcriptome analysis showed that after LvMEF2 was knocked down, the protein synthesis and immune-related pathways were affected, and the associated muscle protein synthesis decreased, indicating that LvMEF2 affected muscle formation and the immune system. The results provide an important basis for future studies of the MEF2 gene and the mechanism of muscle growth and development in shrimp.

Studies have demonstrated that autoantibodies to tumor-associated antigens (TAAs) may be used as efficient biomarkers with low-cost and highly sensitive characteristics. In this study, an enzyme-linked immunosorbent assay (ELISA) was conducted to analyze autoantibodies to paired box protein Pax-5 (PAX5), protein patched homolog 1 (PTCH1), and guanine nucleotide-binding protein subunit alpha-11 (GNA11) in sera from Hispanic Americans including hepatocellular carcinoma (HCC) patients, patients with liver cirrhosis (LC), patients with chronic hepatitis (CH), as well as normal controls. Meanwhile, 33 serial sera from eight HCC patients before and after diagnosis were used to explore the potential of these three autoantibodies as early biomarkers. In addition, an independent non-Hispanic cohort was used to evaluate the specificity of these three autoantibodies. In the Hispanic cohort, at the 95.0% specificity for healthy controls, 52.0%, 44.0%, and 44.0% of HCC patients showed significantly elevated levels of autoantibodies to PAX5, PTCH1, and GNA11, respectively. Among patients with LC, the frequencies for autoantibodies to PAX5, PTCH1, and GNA11 were 32.1%, 35.7%, and 25.0%, respectively. The area under the ROC curves (AUCs) of autoantibodies to PAX5, PTCH1, and GNA11 for identifying HCC from healthy controls were 0.908, 0.924, and 0.913, respectively. When these three autoantibodies were combined as a panel, the sensitivity could be improved to 68%. The prevalence of PAX5, PTCH1, and GNA11 autoantibodies has already occurred in 62.5%, 62.5%, or 75.0% of patients before clinical diagnosis, respectively. In the non-Hispanic cohort, autoantibodies to PTCH1 showed no significant difference; however, autoantibodies to PAX5, PTCH1, and GNA11 showed potential value as biomarkers for early detection of HCC in the Hispanic population and they may monitor the transition of patients with high-risk (LC, CH) to HCC. Using a panel of the three anti-TAA autoantibodies may enhance the detection of HCC.

Insulin-like peptide (ILP) has been identified in various crustaceans, but whether it has a similar function in regulating hemolymph glucose as vertebrate insulin is unclear. We analyzed the components of hemolymph sugar in the Pacific white shrimp, Litopenaeus vannamei, and investigated the changes of hemolymph glucose concentration and the expressions of ILP and glucose metabolism genes under different treatments. We found glucose was a major component of hemolymph sugar in shrimp. Starvation caused hemolymph glucose to rise first and then decline, and the raised hemolymph glucose after exogenous glucose injection returned to basal levels within a short time, indicating that shrimp have a regulatory mechanism to maintain hemolymph glucose homeostasis. In addition, injections of bovine insulin and recombinant LvILP protein both resulted in a fast decline in hemolymph glucose. Notably, RNA interference of LvILP did not significantly affect hemolymph glucose levels, but it inhibited exogenous glucose clearance. Based on the detection of glucose metabolism genes, we found LvILP might maintain hemolymph glucose stability by regulating the expression of these genes. These results suggest that ILP has a conserved function in shrimp similar to insulin in vertebrates and plays an important role in maintaining hemolymph glucose homeostasis.

The insulin signaling (IIS) pathway plays an important role in the metabolism, growth, development, reproduction, and longevity of an organism. As a key member of the IIS pathway, insulin-like growth factor binding proteins (IGFBPs) are widely distributed a family in invertebrates and vertebrates that are critical in various aspects of physiology. As an important mariculture species, the growth of Pacific white shrimp, Litopenaeus vannamei, is one of the most concerning characteristics in this area of study. In this study, we identified three IGFBP genes in the genome of L. vannamei and analyzed their gene structures, phylogenetics, and expression profiles. LvIGFBP1 was found to contain three domains (the insulin growth factor binding (IB) domain, the Kazal-type serine proteinase inhibitor (Kazal) domain, and the immunoglobulin C-2 (IGc2) domain), while LvIGFBP2 and LvIGFBP3 only contained a single IB domain. LvIGFBP1 exhibited high expression in most tissues and different developmental stages, while LvIGFBP2 and LvIGFBP3 were only slightly expressed in hemocytes. The RNA interference of LvIGFBP1 resulted in a significantly smaller increment of body weight than that of control groups. These results will improve our understanding of the conservative structure and function of IGFBPs and show potential applications for the growth of shrimp.

The sesquiterpenoid methyl farnesoate (MF), a juvenile hormone (JH) analog, plays important roles in many physiological processes of crustaceans, such as morphogenesis, molting and reproduction. Juvenile hormone esterase-like (JHE-like) carboxylesterase (CXE) is a key enzyme in MF degradation, playing a significant role in regulating MF titer. However, its function is barely known in shrimp. In this study, a total of 21 JHE-like CXEs (LvCXEs) were characterized in Pacific white shrimp Litopenaeus vannamei, based on the full genome and multi-transcriptomic data. LvCXE has a conserved triplet catalytic site (Ser-Glu-His) and a characteristic GxSxG motif. Most LvCXEs were highly expressed in the hepatopancreas, which was the main site for MF degradation. LvCXEs containing a GESAG motif showed a specific expansion in the L. vannamei genome. Those GESAG-containing LvCXEs presented differential expressions at different larvae stages and different molting stages of L. vannamei, which suggested their potential functions in development and molting. Additionally, when the transcription level of CXEs was inhibited, it could lead to failed molt and death of L. vannamei. When we further detected the expression levels of the key ecdysone responsive transcription factors including LvE75, LvBr-C, LvHr3 and LvFtz-f1 after the CXE inhibitor was injected into L. vannamei, they all showed apparent down-regulation. These results suggested that the expansion of LvCXEs in the L. vannamei genome should contribute to the regulation of metamorphosis at larvae stages and frequent molting during the growth of L. vannamei.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a globally serious disease adversely affecting wheat production. The Bgt-resistant wheat breeding line CH09W89 was derived after backcrossing a Bgt resistant wheat-Thinopyrum intermedium partial amphiploid TAI7045 with susceptible wheat cultivars. At the seedling stage, CH09W89 exhibited immunity or high resistance to Bgt pathotypes E09, E20, E21, E23, E26, Bg1, and Bg2, similar to its donor line TAI7045 and Th. intermedium. No Th. intermedium chromatin was detected based on genomic in situ hybridization of mitotic chromosomes. To determine the mode of inheritance of the Bgt resistance and the chromosomal location of the resistance gene, CH09W89 was crossed with two susceptible wheat cultivars. The results of the genetic analysis showed that the adult resistance to Bgt E09 in CH09W89 was controlled by a single recessive gene, which was tentatively designated as pmCH89. Two polymorphic SSR markers, Xwmc310 and Xwmc125, were linked to the resistance gene with genetic distances 3.1 and 2.7 cM, respectively. Using the Chinese Spring aneuploid and deletion lines, the resistance gene and its linked markers were assigned to chromosome arm 4BL in the bin 0.68–0.78. Due to its unique position on chromosome 4BL, pmCH89 appears to be a new locus for resistance to powdery mildew. These results will be of benefit for improving powdery mildew resistance in wheat breeding programs.

A new wheat-Thinopyrum translocation line CH13-21 was selected from the progenies derived from a cross between wheat-Th. intermedium partial amphiploid TAI7047 and wheat line Mianyang11. CH13-21 was characterized by using genomic in situ hybridization (GISH), multicolor-GISH (mc-GISH), multicolor-fluorescence in situ hybridization (mc-FISH) and chromosome-specific molecular markers. When inoculated with stripe rust and powdery mildew isolates, CH13-21 displayed novel resistance to powdery mildew and stripe rust which inherited from its Thinopyrum parent. The chromosomal counting analyses indicated that CH13-21 has 42 chromosomes, with normal bivalent pairing at metaphase I of meiosis. GISH probed by Th. intermedium genomic DNA showed that CH13-21 contained a pair of wheat-Th. intermedium translocated chromosomes. Sequential mc-FISH analyses probed by pSc119.2 and pAs1 clearly revealed that chromosome arm 6BS of CH13-21 was replaced by Thinopyrum chromatin in the translocation chromosome. The molecular markers analysis further confirmed that the introduced Th. intermedium chromatin in CH13-21 belonged to the long arm of homoeologous group 6 chromosome. Therefore, CH13-21 was a new T6BS.6Ai#1L compensating Robertsonian translocation line. It concludes that CH13-21 is a new genetic resource for wheat breeding programs providing novel variation for disease resistances.

In order to rapidly and efficiently excavate antihypertensive ingredients in Todarodes pacificus, its myosin heavy chain was hydrolyzed in silico and the angiotensin-converting enzyme (ACE) inhibitory peptides were predicted using integrated bioinformatics tools. The results showed the degree of hydrolysis (DH) theoretically achieved 56.8% when digested with papain, ficin, and prolyl endopeptidase (PREP), producing 126 ACE inhibitory peptides. By predicting the toxicity, allergenicity, gastrointestinal stability, and intestinal epithelial permeability, 30 peptides were finally screened, of which 21 had been reported and 9 were new. Moreover, the newly discovered peptides were synthesized to evaluate their in vitro ACE inhibition, showing Ile-Ile-Tyr and Asn-Pro-Pro-Lys had strong effects with a pIC50 of 4.58 and 4.41, respectively. Further, their interaction mechanisms and bonding configurations with ACE were explored by molecular simulation. The preferred conformation of Ile-Ile-Tyr and Asn-Pro-Pro-Lys located in ACE were successfully predicted using the appropriate docking parameters. The molecular dynamics (MD) result indicated that they bound tightly to the active site of ACE by means of coordination with Zn(II) and hydrogen bonding and hydrophobic interaction with the residues in the pockets of S1 and S2, resulting in stable complexes. In summary, this work proposed a strategy for screening and identifying antihypertensive peptides from Todarodes pacificus.

Vibrio spp. are major causes of mortality in white shrimp (Litopenaeus vannamei) which is lacking adaptive immunity. Passive immunization with a specific egg yolk antibody (IgY) is a potential method for the protection of shrimp against vibriosis. In this study, immune effects of the specific egg yolk powders (IgY) against both V. harveyi and V. parahaemolyticus on white shrimp were evaluated. The egg yolk powders against V. harveyi and V. parahaemolyticus for passive immunization of white shrimp were prepared, while a tube agglutination assay and an indirect enzyme-linked immunosorbent assay (ELISA) were used for detection of IgY titer. Anti-Vibrio egg yolk was encapsulated by β-cyclodextrin, which could keep the activity of the antibody in the gastrointestinal tract of shrimp. The results showed that the anti-Vibrio egg powders had an inhibiting effect on V. harveyi and V. parahaemolyticus in vitro. Lower mortality of infected zoeae, mysis, and postlarva was observed in groups fed with anti-Vibrio egg powders, compared with those fed with normal egg powders. The bacterial load in postlarva fed with specific egg powders in seeding ponds was significantly lower than those fed with normal egg powders in seeding ponds. These results show that passive immunization by oral administration with specific egg yolk powders (IgY) may provide a valuable protection of vibrio infections in white shrimp.

Aeromonas veronii is a kind of opportunistic pathogen to fish and humans, significantly impending aquaculture production. Recently, we isolated two A. veronii strains, named GYC1 and GYC2, from diseased Gibel carp (Carassius gibelio) in China. Based on gyrB (DNA gyrase B subunit) genes of GYC1 and GYC2, the constructed phylogenetic tree showed that the two strains were clustered with A. veronii. Sixteen virulence genes related to the pathogenicity of Aeromonas spp. were subjected to PCR assay. The genes of ompAI, ompAII, lafA, act, aer, fla, gcaT and acg were detected in the two strains, while genes of hly, ahp, lip, ast and alt were not detected. Additionally, genes eprCAI, ela and exu were only detected in the strain GYC1. Furthermore, the results of extracellular enzyme analysis revealed that the two isolates can produce hemolysin, caseinase, esterase, amylase and lecithinase, which were closely related to the pathogenicity of the two strains. However, the results showed that there was no gelatinase activity in either strain. According to the antibiotic resistant assay, the two strains were sensitive to cephalosporins and aminoglycosides, while they were resistant to penicillins and quinolones. Through this study, the virulence characteristics, including virulence genes and extracellular enzymes, the pathogenicity of A. veronii was clarified, enhancing the understanding about this pathogenic bacterium and providing the theoretical basis in disease control.