Reoxygenation Mitigates Intermittent Hypoxia-Induced Systemic Inflammation and Gut Microbiota Dysbiosis in High-Fat Diet-Induced Obese Rats.

Background: Obstructive sleep apnea (OSA) is a prevalent sleep breathing disorder characterized by intermittent hypoxia (IH), with continuous positive airway pressure (CPAP) as its standard treatment. However, the effects of intermittent hypoxia/reoxygenation (IH/R) on weight regulation in obesity and its underlying mechanism remain unclear. Gut microbiota has gained attention for its strong association with various diseases. This study aims to explore the combined influence of IH and obesity on gut microbiota and to investigate the impact of reoxygenation on IH-induced alterations. Methods: Diet-induced obese (DIO) rats were created by 8-week high-fat diet (HFD) feeding and randomly assigned into three groups (n=15 per group): normoxia (NM), IH (6% O2, 30 cycles/h, 8 h/day, 4 weeks), or hypoxia/reoxygenation (HR, 2-week IH followed by 2-week reoxygenation) management. After modeling and exposure, body weight and biochemical indicators were measured, and fecal samples were collected for 16S rRNA sequencing. Results: DIO rats in the IH group showed increased weight gain (p=0.0016) and elevated systemic inflammation, including IL-6 (p=0.0070) and leptin (p=0.0004). Moreover, IH rats exhibited greater microbial diversity (p<0.0167), and significant alterations in the microbial structure (p=0.014), notably the order Clostridiales, accompanied by an upregulation of bile acid metabolism predicted pathway (p=0.0043). Reoxygenation not only improved IH-exacerbated obesity, systemic inflammation, leptin resistance, and sympathetic activation, but also showed the potential to restore IH-induced microbial alterations. Elevated leptin levels were associated with Ruminococcaceae (p=0.0008) and Clostridiales (p=0.0019), while body weight was linked to Blautia producta (p=0.0377). Additionally, the abundance of Lactobacillus was negatively correlated with leptin levels (p=0.0006) and weight (p=0.0339). Conclusion: IH leads to gut dysbiosis and metabolic disorders, while reoxygenation therapy demonstrates a potentially protective effect by restoring gut homeostasis and mitigating inflammation. It highlights the potential benefits of CPAP in reducing metabolic risk among obese patients with OSA.

Topological arrangement of coronal segments in human callosal fibers in vivo tractography.

The topography of human callosal fibers in the midsagittal corpus callosum (mid-CC), in terms of cortical termination, is inconsistent in the literature. Despite being a high-profile and controversial topic, heterotopic callosal bundles (HeCBs) have not been studied from a whole-brain perspective. Here, we used multi-modal magnetic resonance imaging data from Human Connectome Project Development to explore these two topographic aspects by combining whole-brain tractography based on multi-shell multi-tissue constrained spherical deconvolution, the post-tractography reducing-false-positive-streamline algorithm of Convex Optimization Modeling for Microstructure Informed Tractography 2, and the new cortex parcellation atlas of Human Connectome Project multi-modal parcellation, version 1.0. We proposed that the callosal streamlines would demonstrate a topological arrangement of coronal segments arranged from anterior to posterior, with each perpendicular to the long axis of the mid-CC following its natural curvature, and the adjacent segments overlapping one another owing to the existence of HeCBs. We found that the cortices connected by the coronal segments, from anterior to posterior, corresponded exactly to the cortices from anterior to posterior in the flattened cortical surfaces of this atlas, indicating the original relative positions of the neocortex before curling and flipping during brain evolution. For each cortical area defined by this atlas, the sum strength of the HeCBs was far greater than that of the homotopic callosal bundle. Our findings on the topography of the whole CC would help in further understanding the network between the bilateral hemispheres and preventing disconnection syndromes in clinical settings.

Probiotic potentials of the silkworm gut symbiont Enterococcus casseliflavus ECB140, a promising L-tryptophan producer living inside the host.

AIMS: L-tryptophan is an essential aromatic amino acid for the growth and development of animals. Studies about enteric L-tryptophan-producing bacteria are scarce. In this report, we characterized the probiotic potential of Enterococcus casseliflavus ECB140, focusing on its L-tryptophan production abilities. METHODS AND RESULTS: ECB140 strain was isolated from the silkworm gut and can survive under strong alkaline environmental conditions. Bacterial colonization traits (motility and biofilm) were examined and showed that only ECB140 produced flagellum and strong biofilms compared with other Enterococcus strains. Comparative genome sequence analyses showed that only ECB140 possessed a complete route for L-tryptophan synthesis among all 15 strains. High-performance liquid chromatography and qRT-PCR confirmed the capability of ECB140 to produce L-tryptophan. Besides, the genome also contains the biosynthesis pathways of several other essential amino acids, such as phenylalanine, threonine, valine, leucine, isoleucine and lysine. These results indicate that ECB140 has the ability to survive passage through the gut and could act as a candidate probiotic. CONCLUSIONS: The study describes a novel, natural silkworm gut symbiont capable of producing L-tryptophan. Enterococcus casseliflavus ECB140 physical and genomic attributes offer possibilities for its colonization and provide L-tryptophan for lepidopteran insects.

The gut commensal bacterium Enterococcus faecalis LX10 contributes to defending against Nosema bombycis infection in Bombyx mori.

BACKGROUND: Microsporidia, a group of obligate intracellular fungal-related parasites, have been used as efficient biocontrol agents for agriculture and forestry pests due to their host specificity and transovarial transmission. They mainly infect insect pests through the intestinal tract, but the interactions between microsporidia and the gut microbiota of the host have not been well demonstrated. RESULTS: Based on the microsporidia-Bombyx mori model, we report that the susceptibility of silkworms to exposure to the microsporidium Nosema bombycis was both dose and time dependent. Comparative analyses of the silkworm gut microbiome revealed substantially increased abundance of Enterococcus belonging to Firmicutes after N. bombycis infection. Furthermore, a bacterial strain (LX10) was obtained from the gut of B. mori and identified as Enterococcus faecalis based on 16S rRNA sequence analysis. E. faecalis LX10 reduced the N. bombycis spore germination rate and the infection efficiency in vitro and in vivo, as confirmed by bioassay tests and histopathological analyses. In addition, after simultaneous oral feeding with E. faecalis LX10 and N. bombycis, gene (Akirin, Cecropin A, Mesh, Ssk, DUOX and NOS) expression, hydrogen peroxide and nitric oxide levels, and glutathione S-transferase (GST) activity showed different degrees of recovery and correction compared with those under N. bombycis infection alone. Finally, the enterococcin LX protein was identified from sterile LX10 fermentation liquid based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. CONCLUSION: Altogether, the results revealed that E. faecalis LX10 with anti-N. bombycis activity might play an important role in protecting silkworms from microsporidia. Removal of these specific commensal bacteria with antibiotics and utilization of transgenic symbiotic systems may effectively improve the biocontrol value of microsporidia. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Comparative shotgun metagenomic data of the silkworm Bombyx mori gut microbiome.

Lepidoptera (butterflies and moths) is a major insect order including important pollinators and agricultural pests, however their microbiomes are little studied. Here, using next-generation sequencing (NGS)-based shotgun metagenomics, we characterize both the biodiversity and functional potential of gut microbiota of a lepidopteran model insect, the silkworm Bombyx mori. Two metagenomes, including the standard inbred strain Dazao (P50) and an improved hybrid strain Qiufeng × Baiyu (QB) widely used in commercial silk production, were generated, containing 45,505,084 and 69,127,002 raw reads, respectively. Taxonomic analysis revealed that a total of 663 bacterial species were identified in P50 silkworms, while 322 unique species in QB silkworms. Notably, Enterobacter, Acinetobacter and Enterococcus were dominated in both strains. The further functional annotation was performed by both BlastP and MG-RAST against various databases including Nr, COG, KEGG, CAZy and SignalP, which revealed >5 × 106 protein-coding genes. These datasets not only provide first insights into all bacterial genes in silkworm guts, but also help to generate hypotheses for subsequently testing functional traits of gut microbiota in an important insect group.

Gut bacterial and fungal communities of the domesticated silkworm (Bombyx mori) and wild mulberry-feeding relatives.

Bombyx mori, the domesticated silkworm, is of great importance as a silk producer and as a powerful experimental model for the basic and applied research. Similar to other animals, abundant microorganisms live inside the silkworm gut; however, surprisingly, the microbiota of this model insect has not been well characterized to date. Here, we comprehensively characterized the gut microbiota of the domesticated silkworm and its wild relatives. Comparative analyses with the mulberry-feeding moths Acronicta major and Diaphania pyloalis revealed a highly diverse but distinctive silkworm gut microbiota despite thousands of years of domestication, and stage-specific signatures in both total (DNA-based) and active (RNA-based) bacterial populations, dominated by the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Most fungal sequences were assigned to the phyla Ascomycota and Basidiomycota. Environmental factors, including diet and human manipulation (egg production), likely influence the silkworm gut composition. Despite a lack of spatial variation along the gut, microbial community shifts were apparent between early instars and late instars, in concert with host developmental changes. Our results demonstrate that the gut microbiota of silkworms assembles into increasingly identical community throughout development, which differs greatly from those of other mulberry-feeding lepidopterans from the same niche, highlighting host-specific effects on microbial associations and the potential roles these communities play in host biology.

Insect symbionts as valuable grist for the biotechnological mill: an alkaliphilic silkworm gut bacterium for efficient lactic acid production.

Insects constitute the most abundant and diverse animal class and act as hosts to an extraordinary variety of symbiotic microorganisms. These microbes living inside the insects play critical roles in host biology and are also valuable bioresources. Enterococcus mundtii EMB156, isolated from the larval gut (gut pH >10) of the model organism Bombyx mori (Lepidoptera: Bombycidae), efficiently produces lactic acid, an important metabolite for industrial production of bioplastic materials. E. mundtii EMB156 grows well under alkaline conditions and stably converts various carbon sources into lactic acid, offering advantages in downstream fermentative processes. High-yield lactic acid production can be achieved by the strain EMB156 from renewable biomass substrates under alkaline pretreatments. Single-molecule real-time (SMRT) sequencing technology revealed its 3.01 Mbp whole genome sequence. A total of 2956 protein-coding sequences, 65 tRNA genes, and 6 rRNA operons were predicted in the EMB156 chromosome. Remarkable genomic features responsible for lactic acid fermentation included key enzymes involved in the pentose phosphate (PP)/glycolytic pathway, and an alpha amylase and xylose isomerase were characterized in EMB156. This genomic information coincides with the phenotype of E. mundtii EMB156, reflecting its metabolic flexibility in efficient lactate fermentation, and established a foundation for future biotechnological application. Interestingly, enzyme activities of amylase were quite stable in high-pH broths, indicating a possible mechanism for strong EMB156 growth in an alkaline environment, thereby facilitating lactic acid production. Together, these findings implied that valuable lactic acid-producing bacteria can be discovered efficiently by screening under the extremely alkaline conditions, as exemplified by gut microbial symbionts of Lepidoptera insects.

Quantitative Proteomic Analysis of Germination of Nosema bombycis Spores under Extremely Alkaline Conditions.

The microsporidian Nosema bombycis is an obligate intracellular pathogen of the silkworm Bombyx mori, causing the epidemic disease Pebrine and extensive economic losses in sericulture. Although N. bombycis forms spores with rigid spore walls that protect against various environmental pressures, ingested spores germinate immediately under the extremely alkaline host gut condition (Lepidoptera gut pH > 10.5), which is a key developmental turning point from dormant state to infected state. However, to date this process remains poorly understood due to the complexity of the animal digestive tract and the lack of genetic tools for microsporidia. Here we show, using an in vitro spore germination model, how the proteome of N. bombycis changes during germination, analyse specific metabolic pathways employed in detail, and validate key functional proteins in vivo in silkworms. By a label-free quantitative proteomics approach that is directly based on high-resolution mass spectrometry (MS) data, a total of 1136 proteins were identified with high confidence, with 127 proteins being significantly changed in comparison to non-germinated spores. Among them, structural proteins including polar tube protein 1 and 3 and spore wall protein (SWP) 4 and 30 were found to be significantly down-regulated, but SWP9 significantly up-regulated. Some nucleases like polynucleotide kinase/phosphatase and flap endonucleases 1, together with a panel of hydrolases involved in protein degradation and RNA cleavage were overrepresented too upon germination, which implied that they might play important roles during spore germination. The differentially regulated trends of these genes were validated, respectively, by quantitative RT-PCR and 3 proteins of interest were confirmed by Western blotting analyses in vitro and in vivo. Furthermore, the pathway analysis showed that abundant up- and down-regulations appear involved in the glycolysis, pentose phosphate pathway, purine, and pyrimidine metabolism, suggesting preparations of energy generation and substance synthesis for the following invasion and proliferation inside the host. This report, to our knowledge, provides the first proteomic landscape of N. bombycis spores, and also a stepping stone on the way to further study of the unique infection mode of this economically important pathogen and other microsporidia in general.