Expression profiles of tyrosine metabolic pathway genes and functional analysis of DOPA decarboxylase in puparium tanning of Bactrocera dorsalis (Hendel).

BACKGROUND: Tanning is an important physiological process with critical roles in cuticle pigmentation and sclerotization. Previous studies have shown that insect cuticle tanning is closely associated with the tyrosine metabolism pathway, which consists of a series of enzymes. RESULTS: In this study, 24 tyrosine metabolism pathway genes were identified in the oriental fruit fly Bactrocera dorsalis (Hendel) genome. Gene expression profiles throughout 15 developmental stages of B. dorsalis were established based on our previous RNA sequencing data, and we found that 13 enzyme genes could be involved in the process of pupariation. Accordingly, a tyrosine-mediated tanning pathway during the pupariation of B. dorsalis was predicted and a critical enzyme, 3,4-dihydroxyphenylalanine (DOPA) decarboxylase (DDC), was used to explore its possible roles in formation of the puparium. First, a real-time quantitative polymerase chain reaction confirmed that BdDDC had an epidermis-specific expression pattern, and was highly expressed during larval metamorphosis in B. dorsalis. Subsequent disruption of BdDDC by feeding 5-day-old larvae with DDC inhibitor (l-α-methyl-DOPA) could lead to: (i) a significant decrease in BdDDC enzyme activity and dopamine concentration; (ii) defects in puparium pigmentation; (iii) impairment of the morphology and less thickness of the puparium; and (iv) lower pupal weight and obstacles to eclosion. CONCLUSION: This study provided a potential tyrosine metabolic pathway that was responsible for insect tanning during pupariation, and the BdDDC enzyme has been shown to have crucial roles in larval-pupal tanning of B. dorsalis. © 2021 Society of Chemical Industry.

Association between Poststroke Depression and Psychological Crisis: A Retrospective Cross-Sectional Study.

OBJECTIVE: To investigate the association between poststroke depression (PSD) and psychological crisis in patients who have experienced a stroke within 6 months. METHODS: This was a retrospective cross-sectional study that enrolled patients within 6 months after stroke onset. The investigators reviewed medical charts to obtain patients' baseline characteristics, and a psychologist evaluated each patient for depression using the Taiwanese Depression Questionnaire (TDQ) and for psychological crisis using the Triage Assessment System (TAS). A generalized linear model (GLM) was used to analyze the association between the results of the TDQ and TAS. RESULTS: Ninety-seven patients with stroke were included. Age (p = 0.003), time since onset of stroke (p = 0.041), diabetes mellitus (p = 0.004), hypertension (p = 0.016), heart disease (p = 0.005), and TDQ score were significantly different between the hemorrhagic stroke group and the ischemic stroke group. The TDQ score was significantly lower in the hemorrhagic stroke group (p = 0.012). The TDQ score was associated with the TAS total score and each domain score, and the presence of heart disease was associated with poorer TAS score in the behavioral domain (p = 0.016). CONCLUSION: PSD is likely an important component of psychological crisis in stroke patients. For clinicians, a comprehensive psychologic evaluation is necessary to optimize treatment.

Structure-function analysis of PGRP-S1 from the oriental armyworm, Mythimna separata.

Peptidoglycan recognition proteins (PGRPs) are well known for their abilities to recognize or hydrolyze peptidoglycan (PGN), one of the major bacterial cell wall components. However, much less is known about their antifungal activities. PGRP-S1 was previously identified from a crop pest, Mythimna separata (Walker) (Lepidoptera: Noctuidae). PGRP-S1 showed bacteriolytic activities against Gram-positive and Gram-negative bacteria. In this study, tissue expression analysis showed that PGRP-S1 was mainly expressed in the midgut of naïve larvae. The induction analysis showed that it was significantly induced in the larval midgut 12 h post the injection of Beauveria bassiana conidia. To identify the key residues that are related to its microbicidal activities, the structure of PGPR-S1 was predicted for structural comparison and molecular docking analysis. Six residues (H61, H62, Y97, H171, T175, and C179) were mutated to Ala individually by site-directed mutagenesis. The recombinant wild-type (WT) and mutant proteins were expressed and purified. The recombinant proteins bound to different polysaccharides, PGNs, and bacteria. H61A, Y97A, H171A, and C179A lost amidase activity. Accordingly, antibacterial assay and scanning electron microscopy confirmed that only H62A and T175A retained bacteriolytic activities. The germination of B. bassiana conidia was significantly inhibited by WT, H61A, Y97A, T175A, and C179A mutants. Electron microscopy showed that some conidia became ruptured after treatment. The growth of hyphae was inhibited by the WT, H61A, H62A, and T175A. In summary, our data showed that different residues of PGRP-S1 are involved in the antibacterial and antifungal activities.

Molecular Characterization and Expression Profiling of Nuclear Receptor Gene Families in Oriental Fruit Fly, Bactrocera Dorsalis (Hendel).

The oriental fruit fly (Bactrocera dorsalis) is a pest that causes large economic losses in the fruit and vegetable industry, so its control is a major challenge. Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors that directly combine with DNA to regulate the expression of downstream target genes. NRs are closely associated with multiple physiological processes such as metabolism, reproduction, and development. Through sequence searches and analysis, we identified 21 B. dorsalis NR genes, all of which contained at least one of the two characteristic binding domains. On the basis of the conserved sequences and phylogenetic relationships, we divided the 21 NR genes into seven subfamilies. All members of the NR0 subfamily and BdHR83, which belonged to the NR2E group, lacked ligand-binding domains. The BdDSF and BdHR51, which also belonged to the NR2Egroup, and BdE78 (which belonged to the NR1E group) all lacked DNA-binding domains. The BdDSF and BdHR83 sequences were incomplete, and were not successfully amplified. Development- and tissue-specific expression profiling demonstrated that the transcript levels of the 19 NR genes varied considerably among eggs, larva, pupae, and adults, as well as among larval and adult male and female tissues. Our results will contribute to a better understanding of NR evolution and expand our knowledge of B. dorsalis physiology.

Characterization of PGRP-S1 from the oriental armyworm, Mythimna separata.

Peptidoglycan is the key component forming the backbone of bacterial cell wall. It can be recognized by a group of pattern recognition receptors, known as peptidoglycan recognition proteins (PGRPs) in insects and higher animals. PGRPs may serve as immune receptors or N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). Here, we report the characterization of a short PGRP, PGRP-S1, from the oriental armyworm, Mythimna separata. MsePGRP-S1 cDNA encodes a protein of 197 amino acids (aa) with a PGRP domain of about 150 aa. MsePGRP-S1 was expressed in several tissues of naïve larvae, including hemocytes, midgut, fat body and epidermis. Bacterial challenges caused variable changes in different tissues at the mRNA level. The recombinant protein bound strongly to Staphylococcus aureus and purified peptidoglycans from Staphylococcus aureus and Bacillus subtilis. It can inhibit the growth of gram-negative and gram-positive bacteria by disrupting bacterial surface. It can degrade peptidoglycans from Escherichia coli and Staphylococcus aureus. Taken together, these data demonstrate that M. separata PGRP-S1 is involved in defending against bacteria.

Antimicrobial activities of a proline-rich proprotein from Spodoptera litura.

Antimicrobial peptides (AMPs) are produced by the stimulated humoral immune system. Most mature AMPs contain less than 50 amino acid residues. Some of them are generated from proproteins upon microbial challenges. Here, we report the antimicrobial activities of a proline-rich proprotein, named SlLebocin1 (SlLeb1), from the tobacco cutworm Spodoptera litura. SlLebocin1 cDNA contains a 477-bp open reading frame (ORF). It is mainly expressed in hemocytes and the midgut in naïve larvae. The transcript level was significantly induced in hemocytes but repressed in the midgut and fat body by bacterial challenges. The proprotein contains 158 amino acids with 3 RXXR motifs that are characteristic of some Lepidopteral lebocin proproteins. Four peptides corresponding to the predicted processed fragments were synthesized chemically, and their antimicrobial activities against two Gram-negative and two Gram-positive bacterial strains were analyzed. The peptides showed differential antimicrobial activities. For Escherichia coli and Bacillus subtilis, only the C-terminal fragment (124-158) showed strong inhibitory effects. For Staphylococcus aureus, all peptides showed partial inhibitions. None of them inhibited Serratia marcescens. Bacterial morphologies were examined by the scanning electron microscopy and confocal laser scanning microscopy. The antimicrobial peptides either disrupted cellular membrane or inhibited cell division and caused elongated/enlarged morphologies. The results may provide ideas for designing novel antibiotics.

Corazonin Signaling Is Required in the Male for Sperm Transfer in the Oriental Fruit Fly Bactrocera dorsalis.

Corazonin (Crz) is a widely distributed neuropeptide (or neurohormone) in insects with diverse physiological functions. The present study aimed to reveal the functions of Crz and its receptor (CrzR) in the regulation of sexual behavior and fertility in male Bactrocera dorsalis. Tissue-specific expression analyses showed that the BdCrz transcript was most abundant in the central nervous system (CNS), and the BdCrzR transcript was most abundant in both the fat body and CNS. Immunochemical localization confirmed that three pairs of Crz-immunoreactive neurons are located in the dorsolateral protocerebrum region of male adult brain. Importantly, RNAi-mediated Crz knockdown lengthened mating duration in males, and knockdown of Crz or CrzR strongly decreased male fertility in the following 3 days, while the courtship behavior and mating efficiency were not affected. The reduced number of sperm in the reproductive organs of mated females indicated that Crz knockdown in males reduced sperm transfer. The findings of this study indicate that Crz contributes to the reproductive physiology of the oriental fruit fly B. dorsalis by regulating sperm transfer in male adults.

Genome-wide annotation of cuticular proteins in the oriental fruit fly (Bactrocera dorsalis), changes during pupariation and expression analysis of CPAP3 protein genes in response to environmental stresses.

Cuticular proteins (CPs) are essential components of the insect cuticle as they create a structural and protective shield and may have a role in insect development. In this paper, we studied the CPs in the oriental fruit fly (Bactrocera dorsalis), one of the most economically important pests in the Tephritidae family around the world. The availability of a complete genome sequence (NCBI Assembly: ASM78921v2) allowed the identification of 164 CP genes in B. dorsalis. Comparative analysis of the CPs in B. dorsalis with those in the model insect Drosophila melanogaster and the closely related Ceratitis capitata, and CPs from mosquitoes, Lepidoptera, Hymenoptera and Coleoptera identified Diptera-specific genes and cuticle development patterns. Analysis of their evolutionary relationship revealed that some CP families had evolved according to the phylogeny of the different insect species, while others shared a closer relationship based on domain architecture. Subsequently, transcriptome analysis showed that while most of the CPs (60-100% of the family members) are expressed in the epidermis, some were also present in internal organs such as the fat body and the reproductive organs. Furthermore, the study of the expression profiles throughout development revealed a profound change in the expression of CPs during the formation of the puparium (pupariation). Further analysis of the expression profiles of the CPAP3 genes under various environmental stresses revealed them to be involved in the response to pesticides and arid and extreme temperatures conditions. In conclusion, the data provide a particular overview of CPs and their evolutionary and transcriptional dynamics, and in turn they lay a molecular foundation to explore their roles in the unique developmental process of insect metamorphosis and stress responses.

Tyrosine hydroxylase coordinates larval-pupal tanning and immunity in oriental fruit fly (Bactrocera dorsalis).

BACKGROUND: The oriental fruit fly Bactrocera dorsalis (Hendel), a notorious world pest infesting fruits and vegetables, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH) that is required for cuticle tanning (sclerotization and pigmentation) in many insects, could be a potential target in controlling B. dorsalis. RESULTS: We cloned TH cDNA (BdTH) of B. dorsalis. The complete open reading frame of BdTH (KY911196) was 1737 bp in length, encoding a protein of 578 amino acids. Quantitative real-time PCR confirmed that BdTH was highly expressed in the epidermis of 3rd instar larvae, and its expression increased prior to pupation, suggesting a role in larval-pupal cuticle tanning. When we injected dsBdTH or 3-iodo-tyrosine (3-IT) as a TH inhibitor or fed insect diet supplemented with 3-IT, there was significant impairment of larval-pupal cuticle tanning and a severe obstacle to eclosion in adults followed by death in most. Furthermore, injection of Escherichia coli into larvae fed 3-IT resulted in 92% mortality and the expressions of four antimicrobial peptide genes were significantly downregulated. CONCLUSION: These results suggest that BdTH might play a critical role in larval-pupal tanning and immunity of B. dorsalis, and could be used as a potential novel target for pest control. © 2017 Society of Chemical Industry.

Molecular cloning and analysis of PGRP-L1 and IMD from silkworm Bombyx mori.

Peptidoglycan is one of the major components of bacterial cell wall. The innate immune system of insects utilizes a group of peptidoglycan recognition proteins (PGRPs) for the recognition of specific peptidoglycans and activating immune signaling pathways. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are two important signaling molecules of the IMD pathway. Here we cloned and characterized PGRP-L1 and IMD from the domesticated silkworm Bombyx mori (BmPGRP-L1 and BmIMD). BmPGRP-L1 gene consists of five exons that encodes a polypeptide of 304 amino acids with a transmembrane region and an extracellular PGRP domain. The PGRP domain lacks key residues for the amidase activity. BmIMD cDNA encodes a polypeptide of 250 amino acids with a death domain. BmPGRP-L1 and BmIMD were expressed in various tissues and induced by bacterial challenges. In addition, in vivo blocking of the PGRP domain by the antiserum or purified antibody significantly reduced the expression of some antimicrobial peptide genes. The extracellular region of BmPGRP-L1 bound to diaminopimelic acid-type and lysine-type peptidoglycans. Overexpression of full-length BmIMD in Drosophila Schneider 2 cells significantly induced three antimicrobial peptide genes. These results suggest that BmPGRP-L1 and BmIMD may be players in the IMD pathway of B. mori. This study provides a foundation for further studies on the functions of silkworm IMD pathway.

Cloning and analysis of peptidoglycan recognition protein-LC and immune deficiency from the diamondback moth, Plutella xylostella.

Peptidoglycan (PGN) exists in both Gram-negative and Gram-positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP-LC and IMD (PxPGRP-LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP-LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP-LC and IMD homologs. PxPGRP-LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP-LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full-length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP-LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella.

Immune functions of insect ßGRPs and their potential application.

Insects rely completely on the innate immune system to sense the foreign bodies and to mount the immune responses. Germ-line encoded pattern recognition receptors play crucial roles in recognizing pathogen-associated molecular patterns. Among them, ß-1,3-glucan recognition proteins (ßGRPs) and gram-negative bacteria-binding proteins (GNBPs) belong to the same pattern recognition receptor family, which can recognize ß-1,3-glucans. Typical insect ßGRPs are comprised of a tandem carbohydrate-binding module in the N-terminal and a glucanase-like domain in the C-terminal. The former can recognize triple-helical ß-1,3-glucans, whereas the latter, which normally lacks the enzymatic activity, can recruit adapter proteins to initiate the protease cascade. According to studies, insect ßGRPs possess at least three types of functions. Firstly, some ßGRPs cooperate with peptidoglycan recognition proteins to recognize the lysine-type peptidoglycans upstream of the Toll pathway. Secondly, some directly recognize fungal ß-1,3-glucans to activate the Toll pathway and melanization. Thirdly, some form the 'attack complexes' with other immune effectors to promote the antifungal defenses. The current review will focus on the discovery of insect ßGRPs, functions of some well-characterized members, structure-function studies and their potential application.

Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori.

C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities.

A single-CRD C-type lectin is important for bacterial clearance in the silkworm.

C-type lectins (CTLs) depend on the carbohydrate-recognition domain (CRD) to recognize carbohydrates by a Ca(2+)-dependent mechanism. In animals, CTLs play critical roles in pathogen recognition, activation of the complement system and signaling pathways. Immulectins (Dual-CRD CTLs) in lepidopteran are involved in recognizing pathogens. However, little is known about the immune-related functions of insect single-CRD CTLs. Here, we reported the characterization of C-type lectin-S3 (CTL-S3), a single-CRD CTL from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S3 gene is 672 bp, which encodes a putative protein of 223 amino acids. CTL-S3 gene was expressed in a variety of tissues. Levels of CTL-S3 mRNA in fertilized eggs and whole larvae were elevated upon bacterial challenges. CTL-S3 was secreted to larval hemolymph. The recombinant protein (rCTL-S3) binds to bacterial cell wall components and bacteria. CTL-S3 inhibited the growth of Bacillus subtilis and caused agglutination of Staphylococcus aureus. More importantly, CTL-S3 facilitated the rapid clearance of Escherichia coli and Staphylococcus aureus from the body cavity of larvae. Taken together, our results suggested that CTL-S3 may function as an opsonin in larval hemolymph to enhance the clearance of pathogens.