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Background 20-Hydroxyecdysone (20E) is an important hormone that regulates insect development and metamorphosis. The fat body of insects plays a crucial role in nutrient storage and energy metabolism and is considered the exchange center for regulating insect development. The fat body undergoes remarkable transformation during insect metamorphosis and is primarily regulated by 20E. microRNAs (miRNAs) have been identified in different insects and have multiple functions in various physiological processes. However, the interaction of 20E and miRNAs in fat body regulation remains unclear. Results We constructed six small RNA libraries using Bombyx mori fat body treated with 20E. Expression and functional analyses were conducted to identify 20E-responsive miRNAs. In total, 431 miRNAs were identified, including 389 known and 42 novel miRNAs. Differential expression analysis revealed significant expression changes in the expression of 40, 9, and 18 miRNAs at 2 h, 6 h, and 12 h after 20E treatment, respectively. The expression of 10 miRNAs was validated using quantitative real-time PCR. miR-34-5p is a highly conserved miRNA among the 10 validated miRNAs, and autophagy-related gene 1 ( Atg1 ) was considered a target gene of miR-34-5p . The expression analysis of miR-34-5p and Atg1 exhibited an opposite expression pattern in the fat body after the 20E treatment. Dual-luciferase assay indicated that miR-34-5p could inhibit Atg1 expression by targeting a binding site in CDS region of Atg1 . In larval fat body, overexpressing miR-34-5p by injecting miR-34-5p agomir suppressed the expression of Atg1 and autophagy, whereas knocking down miR-34-5p by injecting miR-34-5p antagomir induced the expression of Atg1 and autophagy. Meanwhile, Atg1 silencing by RNAi also inhibited autophagy. These results indicate that miR-34-5p participates in 20E-induced autophagy in B. mori fat body by interacting with Atg1 . Conclusions We systematically identified and functionally characterized miRNAs associated with 20E regulation in the fat body of B. mori . miR-34-5p is involved in 20E-induced autophagy in B. mori by regulating its target gene Atg1 . These results provide insight into the role of sophisticated interactions between miRNAs, 20E regulation, and autophagy in fat body remodeling and insect metamorphosis.

The fall armyworm, Spodoptera frugiperda, a lepidopteran pest from the family Noctuidae, has become a major invasive pest since 2016. Using RNAi methods to control S. frugiperda is currently under investigation. This study is the first to target the V-ATPaseD gene of S. frugiperda using RNAi. Injection of dsRNA-V-ATPaseD into the hemolymph of 4th-instar larvae significantly suppressed gene expression at 24 and 48 h post-injection. Treated larvae showed delayed development and reduced pupation after 7 days. Subsequently, V-ATPaseD silencing was achieved through topical or oral administration of chitosan/dsRNA-V-ATPaseD nanoparticles. Larvae fed these nanoparticles exhibited significant reductions in V-ATPaseD mRNA at 72 h, persisting until 96 h before normalizing. Additionally, the treated larvae displayed disrupted molting and impaired pupation. Furthermore, larvae fed chitosan/dsRNA-V-ATPaseD were more susceptible to emamectin benzoate–lufenuron at LC30 concentrations, resulting in 68% mortality—27% higher than the pesticide alone—72 h post-exposure. Combining chitosan/dsRNA-V-ATPaseD nanoparticles with emamectin benzoate–lufenuron significantly enhanced pest control efficacy, providing new insights into pesticide reduction and sustainable pest control methods for this invasive species.

Active DNA demethylation is an important part of epigenetic regulation in plants and animals. How active DNA demethylation is regulated and its relationship with histone modification patterns are unclear. Here, we report the discovery of IDM1, a regulator of DNA demethylation in Arabidopsis. IDM1 is required for preventing DNA hypermethylation of highly homologous multicopy genes and other repetitive sequences that are normally targeted for active DNA demethylation by Repressor of Silencing 1 and related 5-methylcytosine DNA glycosylases. IDM1 binds methylated DNA at chromatin sites lacking histone H3K4 di- or trimethylation and acetylates H3 to create a chromatin environment permissible for 5-methylcytosine DNA glycosylases to function. Our study reveals how some genes are indicated by multiple epigenetic marks for active DNA demethylation and protection from silencing.

With the rapid development of China’s livestock industry, the demand for protein feed has concomitantly increased, underscoring the importance of developing and utilizing new types of feed. As a novel and unconventional protein source, fermented Broussonetia papyrifera feed has become a promising alternative to traditional feeds due to its high nutritional value, abundance of bioactive compounds, and wide range of applications. However, the efficient utilization of crude proteins in B. papyrifera has been hindered by the currently used microorganisms and fermentation processes. In the present study, nine proteolytic bacterial strains and eight yeast and fungal strains were isolated from the root zone soil of B. papyrifera and screened using qualitative and quantitative methods. The protease activities of strains Paenibacillus sp. AY and Rhodotorula mucilaginosa FG were determined to be 21.95 and 55.16 U/mL, respectively. Compared with the control group, Paenibacillus sp. AY significantly increased the acid-soluble protein and ammonia nitrogen contents in the fermented feed by 26.7% and 12.2% (p < 0.05), respectively, while R. mucilaginosa FG enhanced them by 43.3% and 24.5%; these isolates’ effects were comparable to those of the type strains. Finally, to increase the quality of fermented B. papyrifera feed, the cultivation conditions were further optimized using single-factor experiments and an orthogonal design. Under optimal conditions, the acid-soluble protein content reached 7.63%, which was 27.2% higher than that of the control. Our results provide a basis for developing a novel process to efficiently utilize the crude proteins in B. papyrifera feed and can accelerate the application of this feed in animal production.

Peritrophins are associated with structural and functional integrity of peritrophic membranes (PM), structures composed of chitin and proteins. PM lines the insect midgut and has roles in digestion and protection from toxins. We report the full-length cDNA cloning, molecular characterization and functional analysis of SfPER, a novel PM peritrophin A protein, in Spodoptera frugiperda . The predicted amino acid sequence indicated SfPER’s domain structure as a CMCMC-type, consisting of a signal peptide and three chitin-binding (C) domains with two intervening mucin-like (M) domains. Phylogenetic analysis determined a close relationship between SfPER and another S. frugiperda PM peritrophin partial sequence. SfPER transcripts were found in larvae and adults but were absent from eggs and pupae. Chitin affinity studies with a recombinant SfPER-C1 peritrophin A-type domain fused to SUMO/His-tag confirmed that SfPER binds to chitin. Western blots of S. frugiperda larval proteins detected different sized variants of SfPER along the PM, with larger variants found towards the posterior PM. In vivo suppression of SfPER expression did not affect susceptibility of larvae to Bacillus thuringiensis toxin, but significantly decreased pupal weight and adult emergence, possibly due to PM structural alterations impairing digestion. Our results suggest SfPER could be a novel target for insect control.

Gyrovirus galga1 (GyVg1), a member of the Anelloviridae family and Gyrovirus genus, has been detected in chicken and human tissue samples. In this study, the DNA of GyVg1-related gyroviruses in the sera of six dogs and three cats from Central and Eastern China was identified using PCR. Alignment analysis between the nine obtained and reference GyVg1 strains revealed that the genome identity ranged from 99.20% (DOG03 and DOG04 strains) to 96.17% (DOG01 and DOG06 strains). Six recombination events were predicted in multiple strains, including DOG01, DOG05, DOG06, CAT01, CAT02, and CAT03. The predicted major and minor parents of DOG05 came from Brazil. The DOG06 strain is potentially recombined from strains originating from humans and cats, whereas DOG01 is potentially recombined from G17 (ferret-originated) and Ave3 (chicken-originated), indicating that transmissions across species and regions may occur. Sixteen representative amino acid mutation sites were identified: nine in VP1 (12 R/H, 114S/N, 123I/M, 167 L/P, 231 P/S, 237 P/L, 243 R/W, 335 T/A, and 444S/N), four in VP2 (81 A/P, 103 R/H, 223 R/G, and 228 A/T), and three in VP3 (38 M/I, 61 A/T, and 65 V/A). These mutations were only harbored in strains identified in dogs and cats in this study. Whether this is related to host tropism needs further investigation. In this study, GyVg1 was identified in the sera of dogs and cats, and the molecular characteristics prompted the attention of public health.

Background 20-hydroxyecdysone (20E) plays important roles in insect molting and metamorphosis. 20E-induced autophagy has been detected during the larval–pupal transition in different insects. In Bombyx mori , autophagy is induced by 20E in the larval fat body. Long non-coding RNAs (lncRNAs) function in various biological processes in many organisms, including insects. Many lncRNAs have been reported to be potential for autophagy occurrence in mammals, but it has not been investigated in insects. Results RNA libraries from the fat body of B. mori dissected at 2 and 6 h post-injection with 20E were constructed and sequenced, and comprehensive analysis of lncRNAs and mRNAs was performed. A total of 1035 lncRNAs were identified, including 905 lincRNAs and 130 antisense lncRNAs. Compared with mRNAs, lncRNAs had longer transcript length and fewer exons. 132 lncRNAs were found differentially expressed at 2 h post injection, compared with 64 lncRNAs at 6 h post injection. Thirty differentially expressed lncRNAs were common at 2 and 6 h post-injection, and were hypothesized to be associated with the 20E response. Target gene analysis predicted 6493 lncRNA-mRNA cis pairs and 42,797 lncRNA-mRNA trans pairs. The expression profiles of LNC_000560 were highly consistent with its potential target genes, Atg4B , and RNAi of LNC_000560 significantly decreased the expression of LNC_000560 and Atg4B . These results indicated that LNC_000560 was potentially involved in the 20E-induced autophagy of the fat body by regulating Atg4B . Conclusions This study provides the genome-wide identification and functional characterization of lncRNAs associated with 20E-induced autophagy in the fat body of B. mori . LNC_000560 and its potential target gene were identified to be related to 20-regulated autophagy in B. mori . These results will be helpful for further studying the regulatory mechanisms of lncRNAs in autophagy and other biological processes in this insect model.

The necrotrophic pathogen Sclerotinia sclerotiorum is widely distributed and infects a broad range of hosts, making it one of the most economically damaging plant pathogens. This study demonstrated that pyrimethanil, an anilinopyrimidine fungicide, exhibited potent activity against S. sclerotiorum, with EC50 values ranging from 0.411 to 0.610 μg/mL. Four highly pyrimethanil-resistant mutants were obtained through chemical taming, with EC50 values of 7.247 to 24.718 μg/mL. These mutants exhibited significantly reduced mycelial growth, sclerotia production, and pathogenicity compared to their wild-type parental isolates, indicating that pyrimethanil resistance suffered a fitness penalty in S. sclerotiorum. Notably, three mutants (DDJH-Pyri-R1, DDJH-Pyri-R3, and DDJH-Pyri-R4), completely lose the capacity to infect detached tomato leaves. Point mutations that cause amino acid changes in the predicted sequence of cystathione-γ synthase (CGS) and cystathione-β lyase (CBL), encoded by SsCGS1 and SsCGS2, were identified in three mutants. However, one mutant (DDJH-Pyri-R2) showed no mutations in these genes, suggesting an alternative resistance mechanism. Molecular docking revealed that mutations in SsCGS1-R3, SsCGS1-R4, and SsCGS2-R1 reduced the binding affinity between pyrimethanil and SsCGSs. No cross-resistance was observed between pyrimethanil and other commonly used fungicides, including carbendazim, fludioxonil, prochloraz, tebuconazole, pyraclostrobin, boscalid, fluazinam, and cyprodinil. These findings provide valuable insights for designing resistance inhibitors and suggest that pyrimethanil has significant potential for controlling soybean sclerotinia stem rot (SSR) caused by S. sclerotiorum.

Background Enterobacter hormaechei is commonly considered a causative pathogen for nosocomial infections and it does not usually cause diseases in animals. However, researchers have recently dissociated the pathogenic Enterobacter hormaechei from foxes and piglets. Here, the Enterobacter hormaechei was first found to be associated with respiratory disease in unweaned calves in China. Case presentation A 2-month-old calf was severely sick and diagnosed with respiratory infection by a rural veterinarian, and it died 5 days after treatment with penicillin G. The lung sample was then run through histopathological analysis and pathogen isolation. The sequence analysis and biochemical tests results showed the isolated bacterium strain to be Enterobacter hormaechei , and drug sensitivity tests showed resistance to all β-lactam antimicrobials and sensitivity to quinolones. Thickened alveoli septum, inflammatory cell infiltration, and erythrocyte diapedesis around the pulmonary alveoli septum were visible in lung histopathological sections. One week later, at the same farm, another calf showed similar clinical signs, and the Enterobacter hormaechei strain was isolated from its nasal discharge; after a week of treatment with enrofloxacin, as suggested by the results of drug sensitivity tests, this calf fully recovered. Conclusions To the best of our knowledge, this is the first case report of calves with respiratory disease that was associated with E. hormaechei , and multi-drug resistance was observed in isolates.

The development and maturation of maize kernel involves meticulous and fine gene regulation at transcriptional and post-transcriptional levels, and miRNAs play important roles during this process. Although a number of miRNAs have been identified in maize seed, the ones involved in the early development of grains and in different lines of maize have not been well studied. Here, we profiled four small RNA libraries, each constructed from groups of immature grains of Zea mays inbred line Chang 7-2 collected 4-6, 7-9, 12-14, and 18-23 days after pollination (DAP). A total of 40 known (containing 111 unique miRNAs) and 162 novel (containing 196 unique miRNA candidates) miRNA families were identified. For conserved and novel miRNAs with over 100 total reads, 44% had higher accumulation before the 9th DAP, especially miR166 family members. 42% of miRNAs had highest accumulation during 12-14 DAP (which is the transition stage from embryogenesis to nutrient storage). Only 14% of miRNAs had higher expression 18-23 DAP. Prediction of potential targets of all miRNAs showed that 165 miRNA families had 377 target genes. For miR164 and miR166, we showed that the transcriptional levels of their target genes were significantly decreased when co-expressed with their cognate miRNA precursors in vivo. Further analysis shows miR159, miR164, miR166, miR171, miR390, miR399, and miR529 families have putative roles in the embryogenesis of maize grain development by participating in transcriptional regulation and morphogenesis, while miR167 and miR528 families participate in metabolism process and stress response during nutrient storage. Our study is the first to present an integrated dynamic expression pattern of miRNAs during maize kernel formation and maturation.