The parasitoid wasp, Trichogramma dendrolimi, can enter diapause at the prepupal stage. Thus, diapause is an efficient preservation method during the mass production of T. dendrolimi. Previous studies on diapause have mainly focused on ecological characteristics, so the molecular basis of diapause in T. dendrolimi is unknown. We compared transcriptomes of diapause and non-diapause T. dendrolimi to identify key genes and pathways involved in diapause development. Transcriptome sequencing was performed on diapause prepupae, pupae after diapause, non-diapause prepupae, and pupae. Analysis yielded a total of 87,022 transcripts with an average length of 1604 bp. By removing redundant sequences and those without significant BLAST hits, a non-redundant dataset was generated, containing 7593 sequences with an average length of 3351 bp. Among them, 5702 genes were differentially expressed. The result of Gene Ontology (GO) enrichment analysis revealed that regulation of transcription, DNA-templated, oxidation-reduction process, and signal transduction were significantly affected. Ten genes were selected for validation using quantitative real-time PCR (qPCR). The changes showed the same trend as between the qPCR and RNA-Seq results. Several genes were identified as involved in diapause, including ribosomal proteins, zinc finger proteins, homeobox proteins, forkhead box proteins, UDP-glucuronosyltransferase, Glutathione-S-transferase, p53, and DNA damage-regulated gene 1 (pdrg1). Genes related to lipid metabolism were also included. We generated a large amount of transcriptome data from T. dendrolimi, providing a resource for future gene function research. The diapause-related genes identified help reveal the molecular mechanisms of diapause, in T. dendrolimi, and other insect species.

/pdf/high-throughput-profiling-of-diapause-regulated-genes-from-4xts2rj3hc.pdf

High-throughput profiling of diapause regulated genes from Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Diapause is a form of dormancy, and Bombyx mori silkworm embryos are ideal models for studying diapause in insects. However, molecular events in eggs during the onset of diapause remain unclear. In this study, transcriptome analyses were performed on silkworm diapause eggs via RNA sequencing at 20 and 48 h after oviposition. A total of 6402 differentially expressed genes (DEGs) were detected in diapause eggs at 48 h versus that at 20 h after oviposition. Gene ontology enrichment analysis showed that DEGs in diapause eggs at 48 h versus that at 20 h after oviposition were involved in ribosome-related metabolism and hydrogen transport. Kyoto Encyclopedia of Genes and Genomes analysis revealed several significantly enriched biological pathways, namely the oxidative phosphorylation, Forkhead box protein O3 (FoxO) signaling, ribosome, endoplasmic reticular protein processing, and autophagy pathways. Fifteen DEGs from the FoxO signaling pathway were selected, and their expression profiles were consistent with the transcriptome results from real-time quantitative reverse transcription polymerase chain reaction. Our results can improve understanding of the diapause mechanism in silkworm eggs and identified key pathways for future studies.

Early Molecular Events during Onset of Diapause in Silkworm Eggs Revealed by Transcriptome Analysis

Improved wound healing of burnt skin and skin lesions, as well as medical implants and replacement products, requires the support of synthetical matrices. Yet, producing synthetic biocompatible matrices that exhibit specialized flexibility, stability, and biodegradability is challenging. Synthetic chitin/chitosan matrices may provide the desired advantages for producing specialized grafts but must be modified to improve their properties. Synthetic chitin/chitosan hydrogel and aerogel techniques provide the advantages for improvement with a bioinspired view adapted from the natural molecular toolbox. To this end, animal genetics provide deep knowledge into which molecular key factors decisively influence the properties of natural chitin matrices. The genetically identified proteins and enzymes control chitin matrix assembly, architecture, and degradation. Combining synthetic chitin matrices with critical biological factors may point to the future direction with engineering materials of specific properties for biomedical applications such as burned skin or skin blistering and extensive lesions due to genetic diseases.

/pdf/improving-polysaccharide-based-chitin-chitosan-aerogel-2ltw1j29.pdf

Improving Polysaccharide-Based Chitin/Chitosan-Aerogel Materials by Learning from Genetics and Molecular Biology

Biochemical characterization of three midgut chitin deacetylases of the Lepidopteran insect Bombyx mori.

Chitin deacetylases (CDAs) are a group of enzymes involved in chitin metabolism in insects; they play a critical role in molting, pupation, and the modification of chitin. In this study, we identified several CDAs in the silkworm, Bombyx mori (BmCDA), and investigated the effect of various hormones on their expression in B. mori larvae and embryo cell lines (BmE). Eight genes encoding BmCDAs were identified in the silkworm genome. They showed different expression patterns in different tissues, and were classified into three types based on where they were expressed: the exoskeleton, digestive organs, and genital organs. Moreover, we found that some BmCDAs showed upregulated expression during the molting period, especially during the fourth molting period in larvae. We also verified that the expression of BmCDA1–6 was upregulated by treatment with 20-hydroxyecdysone not only in larvae, but also in BmE cells. Interestingly, juvenile hormone analog treatment also upregulated the expression of some BmCDAs. The overexpression of several transcription factors revealed that the POU transcription factor POUM2 may play a major role in the regulation of BmCDA expression. Finally, the silencing of BmCDA1 and BmCDA2 did not lead to abnormal phenotypes or death, but may have led to delays in silkworm pupation. These results provide important information about lepidopteran insects in terms of chitin deacetylases and the regulation of their expression.

https://www.mdpi.com/1422-0067/20/7/1679/pdf

Genome-Wide Analysis and Hormone Regulation of Chitin Deacetylases in Silkworm.

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/1744-7917.12552

Genome-wide identification of chitin-binding proteins and characterization of BmCBP1 in the silkworm, Bombyx mori.

[Analysis of molting fluid in silkworm (Bombyx mori) by two-dimensional electrophoresis and mass spectrometry].

There is inconsistent evidence regarding whether the botulinum toxin A (BTA) injection can relieve pain caused by bruxism. This study aimed to estimate the efficiency of BTA injection in relieving pain caused by bruxism at different follow-up periods. Five electronic databases were searched from 2005 to 2022 using search terms related to botulinum toxin and bruxism. Only controlled clinical trials were included. Two investigators reviewed each article and discussed any disagreements until a consensus was reached. Pain outcomes as evaluated by the visual analogue scale (VAS) were subjected to single-arm and Bayesian network meta-analyses. Pooling data were measured by a random-effects model. Eleven studies with a total of 365 bruxism patients were included. According to the single-arm analyses of the pooled data, the reduction in bruxism-related pain after BTA injection measured 4.06 points (95% CI = 3.37 to 4.75) on the VAS, and the pain relief was significant in the first 6 months after treatment (P < 0.01). According to the Bayesian analysis, BTA also resulted in significantly greater pain relief than oral splinting (mean difference (MD), −1.5; 95% credible interval (CrI) = −2.7 to −0.19) or saline injection (MD, −3.3; 95% CrI = −6.2 to −0.32). BTA significantly relieves the pain of bruxism for 6 months after injection, and its therapeutic efficacy was higher than that of oral splinting. Nevertheless, further long-term follow-up randomized controlled trials comparing BTA with other management or drugs are warranted. 

Can botulinum toxin injection alleviate the pain of bruxism? A Bayesian network analysis and a single-arm analysis

Diapause is a common biological phenomenon that occurs in many organisms, including fish, insects, and nematodes. In the silkworm (Bombyx mori), diapause generally occurs in the egg stage. Treatment with O₂, HCl, or other compounds can prevent egg diapause. Here, we characterized the transcriptomic responses of newly laid eggs treated with O₂ or HCl. Digital gene expression analysis showed that 610 genes in O₂-treated eggs and 656 in HCl-treated eggs were differentially expressed. Of these, 343 genes were differentially expressed in both treatments. In addition to trehalases, sorbic acid dehydrogenases, and some enzymes involved in the carbohydrate metabolism, we also identified heat shock proteins, cytochrome P450, and GADD45, which are related to stress tolerance. Gene ontology enrichment analysis showed differentially expressed genes in O₂-treated eggs were involved in oxidoreductase activity as well as in binding, catalytic, and metabolic processes. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the pathways for ribosome biogenesis, spliceosome, and circadian rhythm were significantly enriched in HCl-treated eggs. The reliability of the data was confirmed by qRT-PCR analysis. Our results improved the understanding of the mechanism of diapause blocking in silkworm eggs treated with O₂ or HCl and identified novel molecular targets for future studies.

https://www.mdpi.com/1422-0067/17/12/1838/pdf

Transcriptional Response of Silkworm (Bombyx mori) Eggs to O₂ or HCl Treatment.

Catalases (CATs) are important self-originating enzymes and are involved in many of the biological functions of plants. Multiple forms of CATs suggest their versatile role in lesion mimic mutants (LMMs), H2O2 homeostasis and abiotic and biotic stress tolerance. In the current study, we identified a large lesion mimic mutant9428 (llm9428) from Ethyl-methane-sulfonate (EMS) mutagenized population. The llm9428 showed a typical phenotype of LMMs including decreased agronomic yield traits. The histochemical assays showed decreased cell viability and increased reactive oxygen species (ROS) in the leaves of llm9428 compared to its wild type (WT). The llm9428 showed enhanced blast disease resistance and increased relative expression of pathogenesis-related (PR) genes. Studies of the sub-cellular structure of the leaf and quantification of starch contents revealed a significant decrease in starch granule formation in llm9428. Genetic analysis revealed a single nucleotide change (C > T) that altered an amino acid (Ala > Val) in the candidate gene (Os03g0131200) encoding a CATALASE C in llm9428. CRISPR-Cas9 targetted knockout lines of LLM9428/OsCATC showed the phenotype of LMMs and reduced starch metabolism. Taken together, the current study results revealed a novel role of OsCATC in starch metabolism in addition to validating previously studied functions of CATs.

/pdf/disruption-of-llm9428-oscatc-represses-starch-metabolism-and-78149t0g.pdf

Disruption of LLM9428/OsCATC Represses Starch Metabolism and Confers Enhanced Blast Resistance in Rice

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