Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Comprehensive insights into host-pathogen interaction between brown planthopper and a fungal entomopathogen by dual RNA sequencing.

BACKGROUND: The brown planthopper (BPH) is one of the most destructive pests of rice, causing tremendous yield and economic losses every year. The fungal entomopathogen Metarhizium anisopliae was previously proved to have great potential for BPH biocontrol. Genome-wide insight into the insect-fungus interaction is crucial for genetic improvement of M. anisopliae to enhance its virulence to BPH but still has been poorly explored. RESULTS: Using dual RNA-seq approach, we present here a global view of host and fungal gene expressions in BPH adults during the fungal infection. The results revealed that BPH could initiate strong defense responses against the fungal attack by upregulating the expressions of a large number of genes, including genes involved in cuticle formation, immune response, cell detoxification and biomacromolecule metabolism. Correspondingly, the fungal entomopathogen could induce a series of genes to infect and modulate BPH, including genes involved in fungal penetration, invasive growth, stress resistance and virulence. Three host defense-related genes (NlPCE4, NlPOD1 and NlCYP4DE1) were chosen for further function analysis. RNAi-mediated knockdown of NlPCE4 caused a significant decrease in BPH survival, but no obvious effects on the survival rates were detected by the suppression of NlPOD1 and NlCYP4DE1. Combination of dsRNA injection and fungal infection could significantly enhance the BPH-killing speed, as synergistic mortalities were observed in co-treatments of RNAi and M. anisopliae infection. CONCLUSION: Our study provides a comprehensive insight into molecular mechanisms of host-pathogen interaction between BPH and M. anisopliae and contributes to future development of new efficient biocontrol strategies for BPH biocontrol.

Host-Plant Induced Shifts in Microbial Community Structure in Small Brown Planthopper, Laodelphax striatellus (Homoptera: Delphacidae).

Microbiome associated with insects play vital roles in host ecology and physiology. The small brown planthopper (SBPH), Laodelphax striatellus, is a polyphagous insect pest that caused enormous damage to a wide range of cereal crops. Previous studies have assessed the effects of environmental factors, such as antibiotics, insecticide, and geographical habitat on the bacterial composition of SBPH. However, the influence of host plants on the microbial community in SBPH still unclear. Here, we characterized and compared the microbial community in three SBPH populations feeding on rice, barley, and wheat, respectively, using high-throughput amplicon sequencing. Our observations revealed that the microbiome harbored by SBPH was abundant and diverse. Ten phyla comprising 141 genera of bacteria were annotated, and four fungal phyla consisting of 47 genera were assigned. The bacteria belonging to the phylum Proteobacteria were the most prevalent and the fungi with the highest abundance were from the order Hypocreales. Comparative analysis showed that host plants could significantly induce structural changes of SBPH microbiome. Significant differences in abundance were observed in two main bacterial orders (Rickettsiales and Rhodospirillales) and three fungal classes (Sordariomycetes, an unclassified class in Ascomycota and Eurotiomycetes) among three host-adapted SBPH populations. Our results could broaden our understanding of interactions among SBPH, its microbial associates and host plants, and also represented the basis of future SBPH biological management.

The gut bacterial flora associated with brown planthopper is affected by host rice varieties.

Gut microbiota plays vital roles in the development, evolution and environmental adaptation of the host insects. The brown planthopper (BPH) is one of the most destructive pests of rice, but little is known about its gut microbiota. In this study, we investigated the gut bacterial communities in two BPH populations feeding on susceptible and resistant rice varieties by high-throughput amplicon sequencing. Our results revealed that the gut bacterial communities in BPH were species diverse. A total of 29 phyla and 367 genera were captured, with Proteobacteria and Acinetobacter being the most prominent phylum and genus, respectively. Comparative analysis showed that significant differences in the profile of gut bacterial communities existed between the two BPH populations. The species richness detected in the population feeding on the resistant rice variety was significantly higher than that in the population rearing on the susceptible rice variety. Although the most dominant gut bacteria at all taxonomic levels showed no significant differences between the two BPH populations, the relative abundances of two subdominant phyla (Firmicutes and Bacteroidetes) and two subdominant classes (Bacteroidia and Clostridia) were significantly different. FAPROTAX analysis further indicated that host rice varieties might induce changes of the gut bacterial flora in BPH, as significant differences in five metabolism-related functional categories (fermentation, methylotrophy, xylanolysis, nitrate reduction and ureolysis) were detected between the two BPH populations. Our results are informative for studies which focused on the interactions between BPH and its symbiotic microbes and could also provide the basis of future BPH biological management.