Alternative splicing of chitin deacetylase 2 regulates chitin and fatty acid metabolism in Asian citrus psyllid, Diaphorina citri.

Chitin plays an important role in the development and molting of insects. The key genes involved in chitin metabolism were considered promising targets for pest control. In this study, two splice variants of chitin deacetylase 2 (CDA2) from Diaphorina citri were identified, including DcCDA2a and DcCDA2b. Bioinformatics analysis revealed that DcCDA2a and DcCDA2b encoded 550 and 544 amino acid residues with a signal peptide, respectively. Spatio-temporal expression patterns analysis showed that DcCDA2a and DcCDA2b were highly expressed in D. citri wing and nymph stages. Moreover, DcCDA2a and DcCDA2b expression levels were induced by 20-hydroxyecdysone (20E). Silencing DcCDA2a by RNA interference (RNAi) significantly disrupted the D. citri molting and increased D. citri mortality and malformation rate, whereas inhibition of DcCDA2b resulted in a semimolting phenotype. Furthermore, silencing DcCDA2a and DcCDA2b significantly suppressed D. citri chitin and fatty acid metabolism. Our results indicated that DcCDA2 might play crucial roles in regulating D. citri chitin and fatty acid metabolism, and it could be used as a potential target for controlling D. citri.

Quantitative ubiquitylome crosstalk with proteome analysis revealed cytoskeleton proteins influence CLas pathogen infection in Diaphorina citri.

Huanglongbing (HLB), also known as citrus greening disease, is caused by Candidatus Liberbacter asiaticus (CLas) and transmitted by Diaphorina citri. Previous studies reported that CLas infection significantly influences the structure of the D. citri cytoskeleton. However, the mechanisms through which CLas manipulates cytoskeleton-related proteins remain unclear. In this study, we performed quantitative ubiquitylome crosstalk with the proteome to reveal the roles of cytoskeleton-related proteins during the infection of D. citri by CLas. Western blotting revealed a significant difference in ubiquitination levels between the CLas-free and CLas-infected groups. According to ubiquitylome and 4D label-free proteome analysis, 343 quantified lysine ubiquitination (Kub) sites and 666 differentially expressed proteins (DEPs) were identified in CLas-infected groups compared with CLas-free groups. A total of 53 sites in 51 DEPs were upregulated, while 290 sites in 192 DEPs were downregulated. Furthermore, functional enrichment analysis indicated that 18 DEPs and 21 lysine ubiquitinated proteins were associated with the cytoskeleton, showing an obvious interaction. Ubiquitination of D. citri tropomyosin was confirmed by immunoprecipitation, Western blotting, and LC-MS/MS. RNAi-mediated knockdown of tropomyosin significantly increased CLas bacterial content in D. citri. In summary, we provided the most comprehensive lysine ubiquitinome analysis of the D. citri response to CLas infection, thus furthering our understanding of the role of the ubiquitination of cytoskeleton proteins in CLas infection.

Silencing of Glycogen Synthase Kinase 3 Significantly Inhibits Chitin and Fatty Acid Metabolism in Asian Citrus Psyllid, Diaphorina citri.

Glycogen is a predominant carbohydrate reserve in various organisms, which provides energy for different life activities. Glycogen synthase kinase 3 (GSK3) is a central player that catalyzes glucose and converts it into glycogen. In this study, a GSK3 gene was identified from the D. citri genome database and named DcGSK3. A reverse transcription quantitative PCR (RT-qPCR) analysis showed that DcGSK3 was expressed at a high level in the head and egg. The silencing of DcGSK3 by RNA interference (RNAi) led to a loss-of-function phenotype. In addition, DcGSK3 knockdown decreased trehalase activity, glycogen, trehalose, glucose and free fatty acid content. Moreover, the expression levels of the genes associated with chitin and fatty acid synthesis were significantly downregulated after the silencing of DcGSK3. According to a comparative transcriptomics analysis, 991 differentially expressed genes (DEGs) were identified in dsDcGSK3 groups compared with dsGFP groups. A KEGG enrichment analysis suggested that these DEGs were primarily involved in carbon and fatty acid metabolism. The clustering analysis of DEGs further confirmed that chitin and fatty acid metabolism-related DEGs were upregulated at 24 h and were downregulated at 48 h. Our results suggest that DcGSK3 plays an important role in regulating the chitin and fatty acid metabolism of D. citri.

Bombyx mori ferritin heavy-chain homolog facilitates BmNPV proliferation by inhibiting reactive oxygen species-mediated apoptosis.

Ferritin heavy-chain homolog (FerHCH), an iron-binding protein, plays an important role in the host defense against oxidative stress and pathogen infections. In our previous research, Bombyx mori native ferritin had an interaction with B. mori nucleopolyhedrovirus (BmNPV). However, the underlying molecular mechanism of single ferritin homolog responses to BmNPV infection remains unclear. In this study, we found that BmNPV titer and B. mori FerHCH (BmFerHCH) expression were positively correlated with the ferric iron concentration. We performed RNA interference (RNAi) and overexpression experiments to investigate the effects of BmFerHCH on BmNPV proliferation. BmFerHCH knockdown suppressed BmNPV proliferation in vivo and in vitro, whereas BmFerHCH overexpression facilitated BmNPV proliferation. In addition, the oxidative stress level was increased significantly in BmN cells after budded virus infection, while BmFerHCH could neutralize the increased ROS production induced by BmNPV. Of note, we found that ROS was involved in BmNPV-induced apoptosis. Through inhibiting ROS, apoptosis was suppressed by BmFerHCH, whereas BmFerHCH knockdown facilitated apoptosis. Therefore, we hypothesize that BmFerHCH-mediated inhibition of virus-induced apoptosis depends on suppressing ROS accumulation and, thereby, facilitates virus replication. These results suggest that BmFerHCH plays an important role in facilitating BmNPV proliferation and modulating BmFerHCH is potential strategy for studying host-pathogen interactions.

Functional analysis of a peptidoglycan recognition protein involved in the immune response in the common cutworm, Spodoptera litura.

Peptidoglycan recognition proteins (GRPs) are family of pattern recognition receptors (PRRs), which can recognize the peptidoglycan and trigger the innate immune system against the microorganisms in insects. In this study, we identified a GRP-LB from Spodoptera litura genome database and named SlGRP-LB, which contained a complete open reading frame (ORF) of 639 bp, encoding a protein of 212 amino acids with a signal peptide and GRP domain. Phylogenetic tree analysis suggested that the SlGRP-LB has a close relationship with Helicoverpa armigera GRP-LB (HaGRP-LB). Tissue expression analysis revealed that SlGRP-LB had a high expression level in the fat body. The expression levels of SlGRP-LB were significantly upregulated in the hemolymph, fat body, and midgut from 3 to 12 h after injection of Escherichia coli and Staphylococcus aureus, while the expression levels were not downregulated at 24 h postinfection. Knockdown of SlGRP-LB expression by RNA interference reduced the expression of antibacterial peptide-related genes in the fat body and midgut, while their expression levels were upregulated in the hemolymph. In addition, the recombinant SlGRP-LB was expressed by using E. coli expression system, and it exhibited binding activity to E. coli. Taken together, the data suggest that S. litura GRP-LB might play a crucial role in regulating immune response in S. litura.

Validamycin treatment significantly inhibits the glycometabolism and chitin synthesis in the common cutworm, Spodoptera litura.

Validamycin, as a broadly applied antibiotic, has been used to control rice sheath blight disease. Furthermore, validamycin was considered as an insecticide to control agricultural pests. Insight into the mechanism of validamycin's action on insects can provide molecular targets for the control of agricultural pests. In this study, a toxicological test analysis revealed that Spodoptera litura larval growth and development was significantly inhibited and the pupation rate was significantly reduced with the increase of the concentration of validamycin. According to the NMR-based metabolomic analysis, a total of 15 metabolites involved in glycolysis and tricarboxylic acid cycle (TCA) pathways were identified. Additionally, trehalase activities, glucose and chitin contents were significantly downregulated, but the trehalose content was upregulated after exposure to validamycin. Reverse transcription quantitative PCR analysis revealed that the expression level of genes involved in glycolysis, TCA and chitin synthesis were upregulated after treating with validamycin. Further chitin staining also confirmed that chitin content was downregulated at 12 h after validamycin treatment. Our results indicated that validamycin worked via two different molecular mechanisms, one through inhibiting glycometabolism and the other by inhibiting chitin synthesis in S. litura. The information lays a theoretical foundation for further control of S. litura.

Functional Characterization of a Trehalose-6-Phosphate Synthase in Diaphorina citri Revealed by RNA Interference and Transcriptome Sequencing.

Trehalose-6-phosphate synthase (TPS) plays an important role in the synthesis of trehalose. In the current study, a TPS gene was obtained from Diaphorina citri, and named as DcTPS1 which encoded a protein of 833 amino acid residues. Real-time quantitative PCR (qPCR) analysis revealed that DcTPS1 had the highest expression level in the midgut and fifth-instar nymph stage. Knockdown of DcTPS1 by RNA interference (RNAi) induced an abnormal phenotype and increased mortality and malformation rate with a decreased molting rate. In addition, silencing of DcTPS1 significantly inhibited D. citri chitin metabolism and fatty acid metabolism, while the expression levels of fatty acid decomposition-related genes were downregulated. Furthermore, comparative transcriptomics analysis revealed that 791 differentially expressed genes (DEGs) were upregulated and 678 DEGs were downregulated when comparing dsDcTPS1 groups with dsGFP groups. Bioinformatics analysis showed that upregulated DEGs were mainly involved in oxidative phosphorylation, whereas downregulated DEGs were mainly attributed to the lysosome and ribosome. These results indicated that DcTPS1 played an important role in the growth and development of D. citri.

An odorant-binding protein of Asian citrus psyllid, Diaphorina citri, participates in the response of host plant volatiles.

BACKGROUND: Odorant-binding proteins (OBPs) in insects contribute to the sensitivity of the olfactory system and connect external odorants to olfactory receptor neurons. Determination of the chemosensory functions in Diaphorina citri, a vector of the citrus Huanglongbing pathogen, may help in developing a potential target for pest management. RESULTS: Diaphorina citri showed dose-dependent electroantennogram recording (EAG) responses to 12 host plant volatiles. A two-choice behavioral trap experiment showed that four compounds (methyl salicylate, linalool, citral and R-(+)-limonene) that elicited high EAG responses also had significant attraction to adults. The expression profiles induced by these compounds were detected in nine OBP genes, DcitOBP1-9. DcitOBP3, DcitOBP6 and DcitOBP7 commonly showed significant upregulation or downregulation compared with the control. Microscale thermophoresis (MST) showed that the recombinant protein DcitOBP7 had high in vitro binding affinities (Kd < 10 µm) to methyl salicylate, linalool and R-(+)-limonene, and moderate binding affinity to citral with a Kd value of 15.95 µm. Furthermore, RNA interference (RNAi)-suppressed messenger RNA (mRNA) expression of DcitOBP7 resulted in a significant reduction in EAG activity and in adult D. citri behavioral responses to tested volatiles and the preferred host, Murraya paniculata. The hydrophilic residue Arg107 of DcitOBP7 may have a key role in binding odorants via formation of hydrogen bonds. CONCLUSION: These results show that DcitOBP7 plays an important role in the olfactory response. This finding may provide new insight into the functions of OBP families in D. citri and aid in the development of safe strategies for managing D. citri populations. © 2021 Society of Chemical Industry.

Inhibition of trehalase affects the trehalose and chitin metabolism pathways in Diaphorina citri (Hemiptera: Psyllidae).

The Asian citrus psyllid, Diaphorina citri is the principal vector of huanglongbing, which transmits Candidatus Liberibacter asiaticus. Trehalase is a key enzyme involved in trehalose hydrolysis and plays an important role in insect growth and development. The specific functions of this enzyme in D. citri have not been determined. In this study, three trehalase genes (DcTre1-1, DcTre1-2, and DcTre2) were identified based on the D. citri genome database. Bioinformatic analysis showed that DcTre1-1 and DcTre1-2 are related to soluble trehalase, whereas DcTre2 is associated with membrane-bound trehalase. Spatiotemporal expression analysis indicated that DcTre1-1 and DcTre1-2 had the highest expression levels in the head and wing, respectively, and DcTre2 had high expression levels in the fat body. Furthermore, DcTre1-1 and DcTre1-2 expression levels were induced by 20-hydroxyecdysone and juvenile hormone Ⅲ, but DcTre2 was unaffected. The expression levels of DcTre1-1, DcTre1-2, and DcTre2 were significantly upregulated, which resulted in high mortality after treatment with validamycin. Trehalase activities and glucose contents were downregulated, but the trehalose content increased after treatment with validamycin. In addition, the expression levels of chitin metabolism-related genes significantly decreased at 24 and 48 h after treatment with validamycin. Furthermore, silencing of DcTre1-1, DcTre1-2, and DcTre2 reduced the expression levels of chitin metabolism-related genes and led to a malformed phenotype of D. citri. These results indicate that D. citri trehalase plays an essential role in regulating chitin metabolism and provides a new target for control of D. citri.

Integrated transcriptome sequencing and RNA interference reveals molecular changes in Diaphorina citri after exposure to validamycin.

Validamycin has been widely used as a specific competitive inhibitor of trehalase. In our previous research, validamycin significantly inhibited trehalase activity and chitin synthesis in Diaphorina citri, resulting in abnormal phenotypes. However, the mechanism of validamycin's action on D. citri remains unclear. Here, using a comparative transcriptome analysis, 464 differentially expressed genes (DEGs) in D. citri were identified after validamycin treatment. A Gene Ontology enrichment analysis revealed that these DEGs were mainly involved in "small molecule process", "structural molecule activity" and "transition metal ion binding". DEGs involved in chitin metabolism, cuticle synthesis and insecticide detoxification were validated by reverse transcription quantitative polymerase chain reaction. The RNA interference of D. citri chitinase-like protein ENO3 and D. citri cuticle protein 7 genes significantly affected D. citri molting. Moreover, the recombinant chitinase-like protein ENO3 exhibited a chitin-binding property, and an antimicrobial activity against Bacillus subtilis. This study provides a first insight into the molecular changes in D. citri after exposure to validamycin and identifies two effective RNA interference targets for D. citri control.

Identification and Functional Analysis of Two Chitin Synthase Genes in the Common Cutworm, Spodoptera litura.

Chitin is one the main components of the insect cuticle, and chitin synthase (CHS) is an important enzyme required for chitin formation. CHS has been characterized in various insect species, but the structure and biochemical properties in Spodoptera litura have not been determined. In this study, we identified two CHS genes, SlCHS1 and SlCHS2, which encode proteins with 1565 and 1520 amino acid residues, respectively. Transcriptional analysis suggested that SlCHS1 has a high expression level in the integument whereas SlCHS2 showed the highest expression level in the midgut. During S. litura growth and development, SlCHS1 and SlCHS2 were both predominantly expressed in the fourth-instar larval stage. In addition, the expression of SlCHS1 and SlCHS2 could be induced by 20-hydroxyecdysone (20E). Silencing of SlCHS1 by RNA interference significantly inhibited the pupation and molting of S. litura larvae (RNAi), while knockdown of SlCHS2 had no significant effects on the S. litura phenotype. These results may provide a new molecular target for control of S. litura.

Transcriptome Analyses of Diaphorina citri Midgut Responses to Candidatus Liberibacter Asiaticus Infection.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important transmission vector of the citrus greening disease Candidatus Liberibacter asiaticus (CLas). The D. citri midgut exhibits an important tissue barrier against CLas infection. However, the molecular mechanism of the midgut response to CLas infection has not been comprehensively elucidated. In this study, we identified 778 differentially expressed genes (DEGs) in the midgut upon CLas infection, by comparative transcriptome analyses, including 499 upregulated DEGs and 279 downregulated DEGs. Functional annotation analysis showed that these DEGs were associated with ubiquitination, the immune response, the ribosome, endocytosis, the cytoskeleton and insecticide resistance. KEGG enrichment analysis revealed that most of the DEGs were primarily involved in endocytosis and the ribosome. A total of fourteen DEG functions were further validated by reverse transcription quantitative PCR (RT-qPCR). This study will contribute to our understanding of the molecular interaction between CLas and D. citri.

Identification of FerLCH, isolation of ferritin and functional analysis related to interaction with pathogens in Eri-silkworm, Samia cynthia ricini.

Ferritin is a ubiquitous and conserved iron storage protein that plays a significant role in host detoxification, iron storage, and immune response. Although ferritin has been studied in many species, little is known about its role in the Eri-silkworm (Samia cynthia ricini). In this study, the ferritin light-chain subunit gene, named ScFerLCH, was identified from S. c. ricini. The full-length gene, ScFerLCH, was 1,155 bp and encoded a protein consisting of 231 amino acids with a deduced molecular weight of 26.38 kDa. Higher ScFerLCH expression levels were found in the midgut, silk gland, and fat body by quantitative reverse-transcription polymerase chain reaction and western blot analysis. Injection of Staphylococcus aureus and Pseudomonas aeruginosa could induce upregulation of ScFerLCH in the hemolymph, fat body, and midgut, indicating that ScFerLCH may contribute to the host defense against invading pathogens. In addition, the native ferritin protein was isolated from S. c. ricini by native polyacrylamide gel electrophoresis and its two subunits, ferritin heavy-chain subunit (ScFerHCH) and ferritin light-chain subunit (ScFerLCH), were identified by mass spectrometry. Specifically, we found that recombinant ferritin subunits could self-assemble into a protein complex in vitro; moreover, both recombinant subunits and the protein complex were found to bind different bacteria, including Escherichia coli, P. aeruginosa, S. aureus, and Bacillus subtilis. However, bactericidal tests showed that the protein complex could not inhibit the growth of bacteria directly. Taken together, our results suggest that ScFerritin might play an important role in mediating molecular interaction with pathogens.

Immune Functional Analysis of Chitin Deacetylase 3 from the Asian Citrus Psyllid Diaphorina citri.

Chitin deacetylase (CDA) is a chitin degradation enzyme that strictly catalyzes the deacetylation of chitin to form chitosan, which plays an important role in regulating growth and development, as well as the immune response. In this study, a chitin deacetylase 3 gene (CDA3) was identified with a complete open reading frame (ORF) of 1362 bp from the genome database of Diaphorina citri, encoding a protein of 453 amino acids. Spatiotemporal expression analysis suggested that D. citri CDA3 (DcCDA3) had the highest expression level in the integument and third-instar nymph stage. Furthermore, DcCDA3 expression level can be induced by 20-hydroxyecdysone (20E). Injection of Escherichia coli and Staphylococcus aureus induced the upregulation of DcCDA3 in the midgut, while DcCDA3 was downregulated in the fat body. After silencing DcCDA3 by RNA interference, there was no influence on the D. citri phenotype. In addition, bactericidal tests showed that recombinant DcCDA3 inhibited gram-positive bacteria, including S. aureus and Bacillus subtilis (B. subtilis). In conclusion, our results suggest that DcCDA3 might play an important role in the immune response of D. citri.

Silencing of the Chitin Synthase Gene Is Lethal to the Asian Citrus Psyllid, Diaphorina citri.

Chitin synthase is a critical enzyme that catalyzes N-acetylglucosamine to form chitin, which plays an important role in the growth and development of insects. In this study, we identified a chitin synthase gene (CHS) with a complete open reading frame (ORF) of 3180 bp from the genome database of Diaphorina citri, encoding a protein of 1059 amino acid residues with the appropriate signature motifs (EDR and QRRRW). Reverse transcription-quantitative PCR (RT-qPCR) analysis suggested that D. citri CHS (DcCHS) was expressed throughout all developmental stages and all tissues. DcCHS had the highest expression level in the integument and fifth-instar nymph stage. Furthermore, the effects of diflubenzuron (DFB) on D. citri mortality and DcCHS expression level were investigated using fifth-instar nymph through leaf dip bioassay, and the results revealed that the nymph exposed to DFB had the highest mortality compared with control group (Triton-100). Silencing of DcCHS by RNA interference resulted in malformed phenotypes and increased mortality with decreased molting rate. In addition, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) also revealed corresponding ultrastructural defects. Our results suggest that DcCHS might play an important role in the development of D. citri and can be used as a potential target for psyllid control.

Identification and functional analysis of an iron-binding protein, ferritin heavy chain subunit, from the swallowtail butterfly, Papilio xuthus.

Ferritin, which is ubiquitous among all living organisms, plays a crucial role in maintaining iron homeostasis, immune response, and detoxification. In the present research, we identified an iron-binding protein, ferritin heavy chain subunit, from Papilio xuthus and named PxFerHCH. The complete complementary DNA of PxFerHCH was 1,252 bp encoding a sequence of 211 amino acids, which includes an iron-responsive element. Phylogenetic analysis showed that PxFerHCH is clustered with Manduca sexta and Galleria mellonella ferritin heavy chain subunits. Expression levels of PxFerHCH in various tissues were analyzed by reverse transcription quantitative polymerase chain reaction, and the results exhibited that PxFerHCH was expressed in all tissues with the highest expression in the fat body. The relative expression level of PxFerHCH in response to bacterial (Escherichia coli and Staphylococcus aureus) challenges sharply increased by about 12 hr postinfection (hpi) and then decreased at 24 hpi. In addition, the iron-binding capacity and antioxidation activity of recombinant PxFerHCH protein were also investigated. These results reveal that PxFerHCH might play an important role in defense against bacterial infection.

Potential roles of insect Tropomyosin1-X1 isoform in the process of Candidatus Liberibacter asiaticus infection of Diaphorina citri.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the transmitting vector of Candidatus Liberibacter asiaticus (CLas), which causes citrus disease Huanglongbing (HLB). In recent years, control of HLB has been achieved by reducing the vector population. In the present study, we identified an isoform of D. citri tropomyosin (herein designated as DcTm1-X1). DcTm1-X1 was down-regulated in CLas-infected ACPs compared with uninfected ACPs. Bioinformatics analysis revealed that the full-length DcTm1-X1 is 2955 bp and encodes a protein of 284 amino acids with a deduced molecular weight of 32.15 kDa. Phylogenetic tree analysis suggested that DcTm1-X1 shares a high amino acid identity with its homolog in Acyrthosiphon pisum. Higher DcTm1-X1 expression levels were found in the leg of the psyllid by reverse transcription quantitative PCR (RT-qPCR). According to Blue Native PAGE analysis and mass spectrometric analysis, DcTm1-X1 interacts with citrate synthase (CS) and V-type proton ATPase subunit B-like (VAT). In addition, knockdown of DcTm1-X1 by RNA interference (RNAi) significantly increased the mortality rate of nymphs and the infection rate of CLas at different time points. Taken together, our results show that DcTm1-X1 might play an important role in response to CLas, but also lay a foundation for further research on the functions of DcTm1-X1.

Molecular Characterization of Two Mitogen-Activated Protein Kinases: p38 MAP Kinase and Ribosomal S6 Kinase From Bombyx mori (Lepidoptera: Bombycidae), and Insight Into Their Roles in Response to BmNPV Infection.

Proteins p38 map kinase and ribosomal S6 kinase (S6K) as members of mitogen-activated protein kinases (MAPKs) play important roles against pathogens. In this study, Bmp38 and BmS6K were identified as differentially expressed proteins from iTRAQ database. Bmp38 and BmS6K were expressed, and recombinant proteins were purified. The bioinformatics analysis showed that both proteins have serine/threonine-protein kinases, catalytic domain (S_TKc) with 360 and 753 amino acids, respectively. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that Bmp38 and BmS6K had high expression in the midgut and hemolymph. The comparative expression level of Bmp38 and BmS6K in BC9 was upregulated than in P50 in the midgut after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Western bolt results showed a positive correlation between RT-qPCR and iTRAQ data for Bmp38, but BmS6K data showed partial correlation with iTRAQ. Injection of anti-Bmp38 and anti-BmS6K serum suggested that Bmp38 may be involved against BmNPV infection, whereas BmS6K may require phosphorylation modification to inhibit BmNPV infection. Taken together, our results suggest that Bmp38 and BmS6k might play an important role in innate immunity of silkworm against BmNPV.

Molecular characterisation of Apolipophorin-III gene in Samia cynthia ricini and its roles in response to bacterial infection.

Apolipophorin-III (ApoLp-III) is an abundant hemolymph protein mainly involved in lipid transport and innate immunity in insects. In the present study, the gene Samia cynthia ricini ApoLp-III (ScApoLp-III) was identified from a transcriptome database, and contained 790 nucleotides with a putative open reading frame (ORF) of 561 bp encoding 186 amino acid residues. Phylogenetic analysis revealed that ScApoLp-III had significant homology with ApoLp-III protein from Antheraea pernyi. Higher ScApoLp-III expression levels were found in the fat body and silk gland by reverse transcription quantitative PCR (RT-qPCR). Injection of Staphylococcus aureus induced up-regulation of ScApoLp-III in the midgut, fat body and hemocytes. However, ScApoLp-III was down-regulated in the midgut and fat body after Pseudomonas aeruginosa injection, indicating that ScApoLp-III may contribute to the host's defense against invading pathogens. Additionally, recombinant ScApoLp-III was found to bind different bacteria, including E. coli, P. aeruginosa, S. aureus and B. subtilis. Bactericidal tests showed that recombinant ScApoLp-III strongly inhibited Gram-negative bacteria, including Escherichia coli and P. aeruginosa. However, it had no obvious influence on Gram-positive bacteria. Taken together, our results suggest that the ScApoLp-III might play an important role in the innate immunity of S. c. ricini.

Long non-coding RNA TUG1 promotes the proliferation of colorectal cancer cells through regulating Wnt/ß-catenin pathway.

The long non-coding RNA taurine up-regulated gene 1 (TUG1) has been shown to be dysregulated in various types of malignant cancer; however, its underlying mechanism of action has not been fully elucidated. The present study aimed to investigate the biological role and clinical significance of TUG1 in the progression of colorectal cancer (CRC). A reverse transcription-quantitative polymerase chain reaction assay was used to evaluate TUG1 expression in tissues from patients with CRC. The effect of TUG1 on cell viability of CRC cells using MTT assay. The influence of TUG1 on tumorigenesis was monitored using an in vivo xenograft model. The status of the Wnt/ß-catenin signaling pathway was evaluated using immunofluorescence, western blotting and luciferase reporter assays. The results demonstrated that the expression of TUG1 was positively associated with the pathological grade and clinical stage of CRC patients. Knockdown of TUG1 inhibited the proliferation of CRC cells and attenuated the activity of Wnt/ß-catenin pathway in CRC cells. In addition, TUG1 knockdown inhibited the tumorigenicity in the in vivo CRC xenograft model, as well as the nuclear localization of ß-catenin and downstream gene transcription. Taken together, the data of the present study highlighted the pivotal role of the TUG1-Wnt/ß-catenin signaling pathway in CRC, which could be targeted to improve the therapeutic efficacy of CRC.