Naringin ameliorates liver fibrosis in zebrafish by modulating IDO1-mediated lipid metabolism and inflammatory infiltration.

Liver fibrosis (LF) is an important reparative process in response to acute or chronic hepatic injury, which has the potential to advance towards cirrhosis and hepatocellular carcinoma. Dietary naringin consumption contributes to protection against LF in animal studies, while the exact protective mechanism of naringin remains unclear. This study aimed to investigate the molecular mechanisms behind the potential protective effect of naringin against TAA-induced LF in zebrafish. In this study, we utilized zebrafish to create the LF model and investigate the therapeutic mechanism of naringin. Firstly, we evaluated the changes in hepatic fibrosis and lipid accumulation in the liver following naringin treatment with oil red O, Nile red, and Sirius red and immunohistochemistry. In addition, we employed an ROS probe to directly measure oxidative stress and monitor inflammatory cell migration in a zebrafish transgenic line. Morpholino was used in the knockdown of IDO1 in order to verify its vital role in LF. Our findings demonstrated that naringin exhibited anti-inflammatory and anti-fibrotic action in conjunction with a reversal in lipid accumulation, oxidative stress and suppression of macrophage infiltration and activation of hepatic stellate cells. Furthermore, the results showed that the antifibrotic effect of naringin was removed upon IDO1 knockdown, proving that naringin exerts a protective effect by regulating IDO1. Naringin demonstrates remarkable protective effects against LF, effectively counteracting inflammation and hepatic steatosis in zebrafish liver. These findings suggest that naringin may function as an effective IDO1 inhibitor, holding the potential for clinical translation as a therapeutic agent for the treatment of LF.

Functional Characterization of Two RNA Methyltransferase Genes METTL3 and METTL14 Uncovers the Roles of m6A in Mediating Adaptation of Plutella xylostella to Host Plants.

N6-methyladenosine (m6A) is one of the major epigenetic modifications in eukaryotes. Although increasing functions of m6A have been identified in insects, its role in Plutella xylostella L. for host plant adaptation remains unclear. In the current study, we show that the m6A content of P. xylostella was relatively low in different developmental stages and tissues, with no significant differences. Two RNA methyltransferase genes, PxMETTL3 (methyltransferase-like 3) and PxMETTL14 (methyltransferase-like 14), were identified and characterized. PxMETTL3 could be transcribed into two transcripts, and PxMETTL14 had only one transcript; both of these genes were highly expressed in egg and adult stages and reproductive tissues. The CRISPR/Cas9-mediated knockout of PxMETTL3 (ΔPxMETTL3-2) or PxMETTL14 (ΔPxMETTL14-14) confirmed their function in m6A installation into RNA. Furthermore, upon transfer from an artificial diet to the host plant, the mutant strains were affected in terms of larval and pupal weight or adult emergence rate, while the wildtype (WT) strain did not exhibit any difference. In addition, the fecundity and egg hatching rate of the WT strain decreased significantly, whereas only the ΔPxMETTL14-14 mutant strain displayed significantly decreased fecundity. There seemed to be a tradeoff between the stress adaptation and reproduction in P. xylostella mediated by m6A modification. During host transfer, the expression of PxMETTL14 was consistent with the change in m6A content, which implied that PxMETTL14 could respond to host plant defense effectively, and may regulate m6A content. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed transcripts with changes in m6A levels revealed that the potential functions of m6A-related genes may be involved in steroid biosynthesis for larval performance and metabolic pathways for adult reproduction. Overall, our work reveals an epigenetic regulation mechanism for the rapid adaptation of P. xylostella to variations in the host environment.

Fitness comparison of Plutella xylostella on original and marginal hosts using age-stage, two-sex life tables.

The diamondback moth, Plutella xylostella, is an important agricultural pest that severely damages cruciferous vegetables. Although previously considered a threat only to Brassica species, P. xylostella has been observed to feed on noncruciferous vegetables. Here, we established a population of P. xylostella on the pea Pisum sativum (PxP population). We compared this PxP population's performance on the pea host plant to a population (PxR) reared on the original host plant radish (Raphanus sativus) for several generations using an age-stage, two-sex life table and analyzed the correlations between different fitness parameters. In the 1st generation of the PxP population, survival rate of immature stage was 17%, while the survival rate of PxR was 68%; the duration of the 4th larval instar (5.30 d) and mortality (25%) of this generation were significantly longer (2.8 d) and higher (1%) than that of PxR, respectively (both p < .001). Upon long-term acclimation, the PxP fitness improved significantly, especially that the survival rate of immature stages increased to approximately 60% in the 15th, 30th, and 45th generations. However, PxP feeding on pea exhibited poorer fitness with longer larval developmental time, shorter total life span, lighter pupa, and lower fecundity in different generations compared with PxP feeding on radish. PxP feeding on pea also showed a significantly lower intrinsic rate of increase (r), net reproduction rate (R 0), finite increase rate (λ), and longer mean generation time (T) than PxP feeding on radish in all generations tested. Significant positive correlations were observed between pupal weight and female fecundity in pea-fed populations, and between female longevity and female fecundity in pea-fed and radish-fed populations. Our findings suggest that P. xylostella adaptation to pea does not improve overall fitness compared with the original host radish, making pea a marginal host for P. xylostella.

Differential Profiles of Gut Microbiota and Metabolites Associated with Host Shift of Plutella xylostella.

Evolutionary and ecological forces are important factors that shape gut microbial profiles in hosts, which can help insects adapt to different environments through modulating their metabolites. However, little is known about how gut microbes and metabolites are altered when lepidopteran pest species switch hosts. In the present study, using 16S-rDNA sequencing and mass spectrometry-based metabolomics, we analyzed the gut microbiota and metabolites of three populations of Plutella xylostella: one feeding on radish (PxR) and two feeding on peas (PxP; with PxP-1 and PxP-17 being the first and 17th generations after host shift from radish to peas, respectively). We found that the diversity of gut microbes in PxP-17 was significantly lower than those in PxR and PxP-1, which indicates a distinct change in gut microbiota after host shift. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the functions of energy metabolism, signal transduction, and xenobiotics biodegradation and metabolism were increased in PxP-17, suggesting their potential roles in host adaptation. Metabolic profiling showed a significant difference in the abundance of gut metabolites between PxR and PxP-17, and significant correlations of gut bacteria with gut metabolites. These findings shed light on the interaction among plants, herbivores, and symbionts, and advance our understanding of host adaptation associated with gut bacteria and metabolic activities in P. xylostella.

CRISPR/Cas9-induced vitellogenin knockout lead to incomplete embryonic development in Plutella xylostella.

Vitellogenin (Vg) is important for insect egg maturation and embryo development. In the present study, we characterized the molecular structure and expression profile of Vg gene, and analyzed its reproductive functions in diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops, using CRISPR/Cas9 system. The P. xylostella Vg (PxVg) included all conserved domains and motifs that were commonly found in most insect Vgs except for the polyserine tract. PxVg gene was highly expressed in female pupae and adults. PxVg protein was detected in eggs and female adults. PxVg was mainly expressed in the fat body and its protein was detected in most tissues, except in the midgut. CRISPR/Cas9-induced PxVg knockout successfully constructed a homozygous mutant strain with a 5-base pair nucleotide deletion. No PxVg protein was found in the mutant individuals and in their ovaries. There were no significant differences between wild (WT) and mutant (Mut-5) types of P. xylostella in terms of ovariole length and the number of fully developed oocytes in newly emerged females. No significant difference was observed in the number of eggs laid within two days, but there was a lower egg hatchability (84% for WT vs. 47% for Mut-5). This is the first study presenting the functions of Vg in ovary development, egg maturation, oviposition and embryonic development of P. xylostella. Our results suggest that the reproductive functions of Vg may be species-specific in insects. It is possible that Vg may not be the major egg yolk protein precursor in P. xylostella. Other "functional Vgs" closely involved in the yolk formation and oogenesis would need to be further explored in P. xylostella.

Mass spectrometry imaging: An emerging technology for the analysis of metabolites in insects.

Mass spectrometry imaging (MSI) can visualize the composition, abundance, and spatial distribution of molecules in tissues or cells, which has been widely used in the research of life science. Insects, especially the agricultural pests, have received a great deal of interests from the scientists in biodiversity and food security. This review introduces the major characteristics of MSI, summarizes its application to the investigation of insect endogenous metabolites, exogenous metabolites, and the spatiotemporal changes of metabolites between insects and plants, and discusses its shortfalls and perspectives. The significance of these concerns is beneficial for future insect research such as physiology and metabolism.

Host Plant-Derived miRNAs Potentially Modulate the Development of a Cosmopolitan Insect Pest, Plutella xylostella.

Plant microRNAs (miRNAs) have recently been reported to be involved in the cross-kingdom regulation of specific cellular and physiological processes in animals. However, little of this phenomenon is known for the communication between host plant and insect herbivore. In this study, the plant-derived miRNAs in the hemolymph of a cruciferous specialist Plutella xylostella were identified by small RNAs sequencing. A total of 39 miRNAs with typical characteristics of plant miRNAs were detected, of which 24 had read counts ≥ 2 in each library. Three plant-derived miRNAs with the highest read counts were validated, and all of them were predicted to target the hemocyanin domains-containing genes of P. xylostella. The luciferase assays in the Drosophila S2 cell demonstrated that miR159a and novel-7703-5p could target BJHSP1 and PPO2 respectively, possibly in an incomplete complementary pairing mode. We further found that treatment with agomir-7703-5p significantly influenced the pupal development and egg-hatching rate when reared on the artificial diet. The developments of both pupae and adults were severely affected upon their transfer to Arabidopsis thaliana, but this might be independent of the cross-kingdom regulation of the three plant-derived miRNAs on their target genes in P. xylostella, based on expression analysis. Taken together, our work reveals that the plant-derived miRNAs could break the barrier of the insect mid-gut to enter the circulatory system, and potentially regulate the development of P. xylostella. Our findings provide new insights into the co-evolution of insect herbivore and host plant, and novel direction for pest control using plant-derived miRNAs.

Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes.

Six new cell lines were established from embryonic tissues of the diamondback moth, Plutella xylostella (L.). The cell lines showed differential characteristics, including growth in attachment or in suspension, susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxification pathway in P. xylostella larvae. Five of the cell lines grew attached to the culture flask and one cell line grew unattached as a suspension cell line. The cell lines had population doubling times ranging from 18 to 23 h. Among five of the P. xylostella cell lines examined for infection of a nucleopolyhedrovirus from Autographa californica, AcMNPV, four cell lines were highly susceptible to AcMNPV infection, but one was only semi-permissive to AcMNPV infection. The production of two recombinant proteins, a ß-galactosidase of bacterial origin and a secreted alkaline phosphatase of eukaryotic origin, in the P. xylostella cell lines was examined in comparison with that in the cell line Sf9 which is commonly used for recombinant protein production. In the P. xylostella cell lines, expression of three important midgut genes involved in the glucosinolate detoxification pathway, including the glucosinolate sulfatase genes GSS1 and GSS2 and the sulfatase modifying factor gene SUMF1, was detected. The P. xylostella cell lines developed in this study could be useful in in vitro research systems for studying insec-virus interactions and complex molecular mechanisms in glucosinolate detoxification and insect-plant interactions.

CRISPR/Cas9-Mediated Vitellogenin Receptor Knockout Leads to Functional Deficiency in the Reproductive Development of Plutella xylostella.

The vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) gene superfamily and plays an indispensable role in Vg transport, yolk deposition, and oocyte development. For this reason, it has become a promising target for pest control. The involvement of VgR in Vg transport and reproductive functions remains unclear in diamondback moths, Plutella xylostella (L.), a destructive pest of cruciferous crops. Here, we cloned and identified the complete cDNA sequence of P. xylostella VgR, which encoded 1805 amino acid residues and contained four conserved domains of LDLR superfamily. PxVgR was mainly expressed in female adults, more specifically in the ovary. PxVgR protein also showed the similar expression profile with the PxVgR transcript. CRISPR/Cas9-mediated PxVgR knockout created a homozygous mutant of P. xylostella with 5-bp-nucleotide deletion in the PxVgR. The expression deficiency of PxVgR protein was detected in the ovaries and eggs of mutant individuals. Vg protein was still detected in the eggs of the mutant individuals, but with a decreased expression level. However, PxVg transcripts were not significantly affected by the PxVgR knockout. Knockout of PxVgR resulted in shorter ovarioles of newly emerged females. No significant difference was detected between wild and mutant individuals in terms of the number of eggs laid in the first 3 days after mating. The loss of PxVgR gene resulted in smaller and whiter eggs and lower egg hatching rate. This study represents the first report on the functions of VgR in Vg transport, ovary development, oviposition, and embryonic development of P. xylostella using CRISPR/Cas9 technology. This study lays the foundation for understanding molecular mechanisms of P. xylostella reproduction, and for making use of VgR as a potential genetic-based molecular target for better control of the P. xylostella.

Structure and expression of sulfatase and sulfatase modifying factor genes in the diamondback moth, Plutella xylostella.

The diamondback moth, Plutella xylostella (L.), uses sulfatases (SULF) to counteract the glucosinolate-myrosinase defensive system that cruciferous plants have evolved to deter insect feeding. Sulfatase activity is regulated by post-translational modification of a cysteine residue by sulfatase modifying factor 1 (SUMF1). We identified 12 SULF genes (PxylSulfs) and two SUMF1 genes (PxylSumf1s) in the P. xylostella genome. Phylogenetic analysis of SULFs and SUMFs from P. xylostella, Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens showed that the SULFs were clustered into five groups, and the SUMFs could be divided into two groups. Profiling of the expression of PxylSulfs and PxylSumfs by RNA-seq and by quantitative real-time polymerase chain reaction showed that two glucosinolate sulfatase genes (GSS), PxylSulf2 and PxylSulf3, were primarily expressed in the midgut of 3rd- and 4th-instar larvae. Moreover, expression of sulfatases PxylSulf2, PxylSulf3 and PxylSulf4 were correlated with expression of the sulfatases modifying factor PxylSumf1a. The findings from this study provide new insights into the structure and expression of SUMF1 and PxylSulf genes that are considered to be key factors for the evolutionary success of P. xylostella as a specialist herbivore of cruciferous plants.

Transcriptome profiling of the Plutella xylostella (Lepidoptera: Plutellidae) ovary reveals genes involved in oogenesis.

BACKGROUND: As a specialized organ, the insect ovary performs valuable functions by ensuring fecundity and population survival. Oogenesis is the complex physiological process resulting in the production of mature eggs, which are involved in epigenetic programming, germ cell behavior, cell cycle regulation, etc. Identification of the genes involved in ovary development and oogenesis is critical to better understand the reproductive biology and screening for the potential molecular targets in Plutella xylostella, a worldwide destructive pest of economically major crops. RESULTS: Based on transcriptome sequencing, a total of 7.88Gb clean nucleotides was obtained, with 19,934 genes and 1861 new transcripts being identified. Expression profiling indicated that 61.7% of the genes were expressed (FPKM≥1) in the P. xylostella ovary. GO annotation showed that the pathways of multicellular organism reproduction and multicellular organism reproduction process, as well as gamete generation and chorion were significantly enriched. Processes that were most likely relevant to reproduction included the spliceosome, ubiquitin mediated proteolysis, endocytosis, PI3K-Akt signaling pathway, insulin signaling pathway, cAMP signaling pathway, and focal adhesion were identified in the top 20 'highly represented' KEGG pathways. Functional genes involved in oogenesis were further analyzed and validated by qRT-PCR to show their potential predominant roles in P. xylostella reproduction. CONCLUSIONS: Our newly developed P. xylostella ovary transcriptome provides an overview of the gene expression profiling in this specialized tissue and the functional gene network closely related to the ovary development and oogenesis. This is the first genome-wide transcriptome dataset of P. xylostella ovary that includes a subset of functionally activated genes. This global approach will be the basis for further studies on molecular mechanisms of P. xylostella reproduction aimed at screening potential molecular targets for integrated pest management.

Binding of human recombinant mutant soluble ectodomain of FGFR2IIIc to c subtype of FGFRs: implications for anticancer activity.

FGFRs are considered essential targets for cancer therapy. We previously reported that msFGFR2c, a Ser252Trp mutant soluble ectodomain of FGFR2IIIc, inhibited tumor growth by blocking FGF signaling pathway. However, the underlying molecular mechanism is still obscure. In this study, we reported that msFGFR2c but not wild-type soluble ectodomain of FGFR2IIIc (wsFGFR2c) could selectively bind to c subtype of FGFRs in the presence of FGF-2. Thermodynamic analysis demonstrated that msFGFR2c bound to wsFGFR2c in the presence of FGF-2 with a K value of 6.61 × 105 M-1. Molecular dynamics simulations revealed that the mutated residue Trp252 of msFGFR2c preferred a π-π interaction with His254 of wsFGFR2c. Concomitantly, Arg255 of msFGFR2c and Glu250 of wsFGFR2c adjusted their conformations and formed three H-bonds. These two interactions therefore stabilized the final structure of wsFGFR2c and msFGFR2c heterocomplex. In FGFR2IIIc-positive/high FGF-2-secreted BT-549 cells, msFGFR2c significantly inhibited the proliferation and induced apoptosis by the blockage of FGF-2-activated FGFRs phosphorylation, also the growth and angiogenesis of its xenograft tumors implanted in chick embryo chorioallantoic membrane model. While weaker the above inhibitory effects of msFGFR2c were observed on FGFR2IIIc-negative/low FGF-2-secreted MCF-7 and MDA-MB-231 cell lines in vitro and in vivo. Moreover, msFGFR2c significantly inhibited the proliferation of FGFR1IIIc-positive NCI-H1299 lung cancer cells by the suppression of FGF-2-induced FGFR1 activation and suppressed the growth of NCI-H1299 transplanted tumors in nude mice. In sum, msFGFR2c is a potential anti-tumor agent targeting FGFR2c/FGFR1c-positive tumor cells. These findings also provide a molecular basis for msFGFR2c to disrupt the activation of FGF signaling.