Widely targeted metabolomic analysis reveals that volatile metabolites in cigar tobacco leaves dynamically change during fermentation.

Changes in volatile metabolites during cigar tobacco leaves fermentation as well as the metabolic pathways of metabolites with significant differences were investigated to determine the influence of cigar tobacco leaves fermentation on its flavor. The volatile substances in cigar tobacco leaves at different stages were detected by headspace-solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS), and the main differences in volatile substances in cigar tobacco leaves at different fermentation stages of Yunxue1 in Yuxi production area were analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The results show that in the process of cigar tobacco leaves fermentation (YXF0, YXF1, YXF2, YXF3, YXF4, YXF5), a total of 613 volatile metabolites were detected, and a significant difference was found in 263 kinds of metabolites. Among them, the main upregulated differential metabolites were 1,3,6,10-Cyclotetradecatetraene, 3,7,11-trimethyl-14-(1-methylethyl)-, [S-(E,Z,E,E)]-, Benzoic acid, Benzaldehyde, etc. While the main downregulated differential metabolites included beta.-Myrcene, trans-Farnesol, etc. The metabolites with significant differences are mainly concentrated in the biosynthesis of monoterpenes, diterpenes, sesquiterpenes and triterpenes, the degradation metabolism of amino acids, such as valine, leucine and isoleucine, and the biosynthesis of phenylpropyl. There were 8 different metabolites in 5 groups, including 4- (1-methylethyl) -1-cyclohexene-1-formaldehyde、2, 4-dihydroxyacetophenone、2-methylbutyl 3-methylbutyrate and methylpyrazine, all of which showed upregulation trend during fermentation. In the fermentation process, volatile metabolites participate in various synthesis and degradation pathways. The biosynthesis pathway of terpenes and amino acid synthesis and degradation pathway are connected to produce various terpenes, aldehydes and other substances, such as 1,3,6,10-Cyclotetradecatetraene, 3,7,11-trimethyl-14-(1-methylethyl)-, [S-(E,Z,E,E)]-、benzaldehyde and 4-hydroxybenzaldehyde, which are conducive to the overall flavor and quality of cigar tobacco leaves.

The ARM repeat domain of hemocyanin interacts with MKK4 to modulate antimicrobial peptides expression.

The mitogen-activated protein kinase (MAPK) intracellular signaling pathway mediates numerous biological processes, including antimicrobial immune response by inducing antimicrobial peptides (AMPs) production. Although MAPK signaling cascade proteins have been identified in penaeid shrimp, their modulation via the MKK4-p38-c-Jun cascade and effect on AMPs production is unknown. Here, we show that hemocyanin (PvHMC), antimicrobial peptides (anti-lipopolysaccharide factor, crustin, and penaeidins), and MKK4-p38-c-Jun cascade proteins are simultaneously induced by pathogens (Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus) in Penaeus vannamei. Intriguingly, knockdown of PvHMC with or without pathogen challenge attenuated the expression of MKK4-p38-c-Jun cascade proteins and their phosphorylation level, which consequently decreased AMPs expression. Further analysis revealed that PvHMC interacts via its armadillo (ARM) repeat domain with PvMKK4 to modulate the p38 MAPK signaling pathway. Thus, the ARM repeat domain enables penaeid shrimp hemocyanin to modulate AMPs expression during antimicrobial response by activating the p38 MAPK signaling pathway.

The sterol regulatory element binding protein homolog of Penaeus vannamei modulates fatty acid metabolism and immune response.

The sterol regulatory element binding proteins (SREBPs) transcription factors family, which regulate the expression of genes involved in cellular lipid metabolism and homeostasis, have recently been implicated in various physiological and pathophysiological processes such as immune regulation and inflammation in vertebrates. Consistent with other invertebrates, we identified a single SREBP ortholog in Penaeus vannamei (designated PvSREBP) with transcripts ubiquitously expressed in tissues and induced by lipopolysaccharide (LPS), Vibrio parahaemolyticus and Streptococcus iniae. In vivo RNA interference (RNAi) of PvSREBP attenuated the expression of several fatty acid metabolism-related genes (i.e., cyclooxygenase (PvCOX), lipoxygenase (PvLOX), fatty acid binding protein (PvFABP) and fatty acid synthase (PvFASN)), which consequently decreased the levels of total polyunsaturated fatty acids (ΣPUFAs). In addition, PvSREBP silencing decreased transcript levels of several immune-related genes such as hemocyanin (PvHMC) and trypsin (PvTrypsin), as well as genes encoding for heat-shock proteins (i.e., PvHSP60, PvHSP70 and PvHSP90). Moreover, in silico analysis revealed the presence of SREBP binding motifs on the promoters of most of the dysregulated genes, while shrimp depleted of PvSREBP were more susceptible to V. parahaemolyticus infection. Collectively, we demonstrated the involvement of shrimp SREBP in fatty acids metabolism and immune response, and propose that PvSREBP and PvHMC modulate each other through a feedback mechanism to establish homeostasis. The current study is the first to show the dual role of SREBP in fatty acid metabolism and immune response in invertebrates and crustaceans.

N-terminal diversity of Litopenaeus vannamei hemocyanin and immunity.

Hemocyanin is primarily a respiratory copper-containing glycoprotein present in the hemolymph of mollusks and arthropods. Recently, hemocyanin has attracted huge research interest due to its multifunctionality and polymorphism. Most previous immune-related studies on shrimp hemocyanin have focused on the C-terminal. Moreover, we previously reported that the C-terminal domain of Litopenaeus vannamei hemocyanin possesses single nucleotide polymorphisms (SNPs), but little is known about the molecular diversity of the N-terminal domain. In the current study, diversity within the N-terminal domain of L. vannamei hemocyanin (LvHMC-N) was explored using bioinformatics and molecular biology techniques as well as immune challenge. Twenty-five LvHMC-N variants were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing, with multiple sequence alignment showing that the 25 variants shared 87%-99 % sequence homology with LvHMC (AJ250830.1). In different shrimp individuals and different shrimp tissues (i.e., hemocytes, stomach, muscle and hepatopancreas), the LvHMC-N variants were expressed differently. Pathogen challenge could modulate the molecular diversity of LvHMC-N, as three LvHMC-Nr variants (LvHMC-Nr1, LvHMC-Nr2 and LvHMC-Nr3) were identified by sequencing following Vibrio parahaemolyticus challenge. Most importantly, recombinant proteins of these three variants (rLvHMC-Nr1, rLvHMC-Nr2 and rLvHMC- Nr3) had relatively high in vitro agglutinative activities against V. parahaemolyticus, Vibrio alginolyticus and Streptoccocus iniae. Our present data indicates that the N-terminus of L. vannamei hemocyanin also possess molecular diversity, which seems to be associated with immune resistance to pathogenic infections.

Spp38 MAPK participates in maintaining the homeostasis of hemolymph microbiota in Scylla paramamosain.

The p38 mitogen-activated protein kinases (MAPKs) are evolutionally conserved from yeasts to mammals, and are involved in the regulation of cells response to various extracellular stimuli. In this study, the p38 MAPK gene (designated as Spp38) of mud crab (Scylla paramamosain) was identified and studied. Spp38 contained the conserved Thr-Gly-Tyr (TGY) motif and a Ala-Thr-Arg-Trp (ATRW) substrate-binding site. Spp38 transcript was ubiquitously expressed in all tissues examined, with the highest expression found in muscle and hepatopancras. Quantitative real-time PCR revealed that Spp38 was upregulated in hemocytes and hepatopancras after infection with Vibrio parahemolyticus and Lipopolysaccharides (LPS). Reporter gene assays indicated that Spp38 activated the expression of anti-lipopolysaccharides (SpALF1 - SpALF6) in S. paramamosian. RNA interference (RNAi)-mediated knockdown of Spp38 or inhibition of Spp38 by SB203580 decreased the expression levels of SpALF1-6 and dual oxidase (SpDuox1 and SpDuox2) in S. paramamosian, which consequently reduced reactive oxygen species (ROS) production thereby significantly increasing the bacterial count in the hemolymph of mud crabs. Similarly, there was a significant reduction in bacterial clearance ability of hemolymph after Spp38 knockdown followed by V. parahemolyticus infection. Taken together, the current data indicated that Spp38 could play a vital role in maintaining the homeostasis of hemolymph microbiota in S. paramamosain.