Tissue ontogeny and chemical composition influence bacterial biodiversity in the wood and shoot tip of Populus nigra.

Plant-microbe interactions significantly influence plant growth dynamics and adaptability. This study explores the impact of metabolites on microbial biodiversity in shoot tips and wood of Populus nigra under greenhouse conditions, using high-throughput sequencing and metabolite profiling. Branches from P. nigra were harvested, rooted, and transplanted into pots for growth. After 3 months, tissue samples from shoot tips and wood were collected, and metabolites extracted and analysed using GC-MS and LC-MS. Genomic DNA was extracted and subjected to high-throughput sequencing for bacterial biodiversity profiling. Both datasets were analysed using bioinformatic and statistical pipelines. Metabolite profiling indicated that shoot tips had a higher relative abundance of primary and secondary metabolites, including sugars, fatty acids, organic acids, phenolic acid derivatives and salicinoids, while wood was enriched in flavonoids. Bacterial biodiversity also differed significantly between these tissues, with Clostridiales, Bacteroidales and Bacillales dominating in shoot tips, associated with rapid growth and anaerobic fermentation, while wood tissues were characterized by diazotrophs from Rhizobiales, Sphingomonadales and Frankiales. PCoA clustering confirmed tissue-specific microbial differences. Functional analysis revealed an enrichment of fundamental cellular processes in shoot tips, while wood exhibited pathways related to degradation and mortality. Metabolite profiling revealed significant variations in primary and secondary metabolites, highlighting their influence on microbial biodiversity across plant tissues. The dominance of specific bacterial orders and distinct functional pathways in each tissue suggests a tailored microbial response to the unique environments of shoot tips and wood.

Barcoding of Italian mosquitoes (BITMO): generation and validation of DNA barcoding reference libraries for native and alien species of Culicidae.

BACKGROUND: Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2). METHODS: A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed. RESULTS: Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI. CONCLUSIONS: This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches.

Characterization of Intestinal Flora in Osteoporosis Patients Based on 16S rDNA Sequencing.

Aim: This study investigated differences in gut flora between osteoporosis (OP) patients and healthy individuals using 16S rDNA sequencing. The correlation between differential flora abundance and bone mineral density (BMD) was analyzed, and key flora and potential mechanisms associated with OP were explored. Methods: Forty-three OP patients and twenty-four healthy volunteers were recruited. Gender, age, height, weight, and BMD data were collected. DNA from fecal samples was extracted for 16S rDNA sequencing. The Kruskal-Wallis test assessed differences in gut flora composition, while LEfSe analysis identified significant flora. Spearman correlation analysis examined the relationship between differential flora and BMD, and PICRUSt predicted pathways involved in OP. Results: Significant differences in microbial composition were found between the two groups. Klebsiella, Escherichia-Shigella, and Akkermansia were biomarkers in OP patients, with Faecalibacterium in the healthy group. Akkermansia abundance negatively correlated with lumbar BMD, while Klebsiella and Escherichia-Shigella negatively correlated with femoral neck and hip BMD. Faecalibacterium showed a positive correlation with BMD. Functional predictions indicated differences in metabolism-related pathways between the groups. Conclusion: Gut flora differed significantly between OP patients and healthy individuals. Akkermansia, Klebsiella, and Escherichia-Shigella could serve as diagnostic biomarkers for OP, highlighting the potential of gut flora in OP diagnosis and treatment.

Assessing Fecal Microbial Diversity and Hormone Levels as Indicators of Gastrointestinal Health in Reintroduced Przewalski's Horses (Equus ferus przewalskii).

Diarrhea serves as a vital health indicator for assessing wildlife populations post-reintroduction. Upon release into the wild, wild animals undergo adaptation to diverse habitats and dietary patterns. While such changes prompt adaptive responses in the fecal microbiota, they also render these animals susceptible to gastrointestinal diseases, particularly diarrhea. This study investigates variations in fecal microorganisms and hormone levels between diarrhea-afflicted and healthy Przewalski's horses. The results demonstrate a significant reduction in the alpha diversity of the fecal bacterial community among diarrheal Przewalski's horses, accompanied by notable alterations in taxonomic composition. Firmicutes, Proteobacteria, and Bacteroidetes emerge as dominant phyla across all fecal samples, irrespective of health status. However, discernible differences in fecal bacterial abundance are observed between healthy and diarrhea-stricken individuals at the genus level, specifically, a diminished relative abundance of Pseudobutyrivibrio is observed. The majority of the bacteria that facilitate the synthesis of short-chain fatty acids, Christensenellaceae_R_7_group (Christensenellaceae), NK4A214_group (Ruminococcus), Lachnospiraceae_XPB1014_group (Lachnospiraceae), [Eubacterium]_coprostanoligenes_group (Eubacterium), Rikenellaceae_RC9_gut_group, Lachnospiraceae_AC2044_group (Lachnospiraceae), and Prevotellaceae_UcG_001 (Prevotella) are noted in diarrhea-affected Przewalski's horses, while Erysipelotrichaceae, Phoenicibacter, Candidatus_Saccharimonas (Salmonella), and Mogibacterium are present in significantly increased amounts. Moreover, levels of immunoglobulin IgA and cortisol are significantly elevated in the diarrhea group compared with the non-diarrhea group. Overall, this study underscores substantial shifts in fecal bacterial diversity, abundance, and hormone levels in Przewalski's horses during episodes of diarrhea.

Wolbachia of phylogenetic supergroup K identified in oribatid mite Nothrus anauniensis (Acari: Oribatida: Nothridae).

Heritable endosymbionts widely occur in arthropod and nematode hosts. Among these endosymbionts, Wolbachia has been extensively detected in many arthropods, such as insects and crustaceans. Maternal inheritance is the most basic and dominant mode of transmission of Wolbachia, and it might regulate the reproductive system of the host in four ways: feminization, parthenogenesis, male killing, and cytoplasmic incompatibility. There is a relatively high percentage (10%) of thelytokous species in Oribatida, a suborder under the subclass Acari of arthropods, but the study of the endosymbionts in oribatid mites is almost negligible. In this paper, we detected endosymbiotic bacteria in two parthenogenetic oribatid species, Nothrus anauniensis Canestrini and Fanzago, 1877, which has never been tested for endosymbionts, and Oppiella nova, in which Wolbachia and Cardinium have been reported before. The results showed that Wolbachia was first found in N. anauniensis with an infection rate of 100% across three populations. Phylogenetic analysis showed that Wolbachia in N. anauniensis belonged to the supergroup K, marking the second supergroup of Wolbachia found in oribatid mites. Unlike previous studies, our study did not detect Wolbachia in O. nova, leading to the exclusion of Wolbachia's role in mediating thelytoky in this species.

Comprehensive analysis of microbiome biodiversity in popular date palm (Phoenix dactylifera L.) fruit varieties.

Due to its nutritional value and health benefits, the date palm (Phoenix dactylifera L.) is an essential dietary food crop throughout Middle Eastern and African countries. Consumers are concerned about the possible microbial contamination of dates, especially since most dates arriving in local markets are unprocessed. The absence of processing increases the possibility of microbial contamination, which raises the probability of microbial contamination. This study aims to analyze and evaluate the variability of fungal and bacterial microbiota identified in the most popular date palm fruits in Saudi Arabia. The study assessed ten date variety fruits from the most popular date palm varieties for consumption in Saudi Arabia and analyzed the microbial count. Morphological and molecular characterization and comparison of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences identified 78 fungi, including 36 distinct species across 15 fungal genera. Alternaria, Fusarium, Curvilaria, Aspergillus, and Penicillium were the most frequent genera among the ten fruit cultivars studied, according to ITS-rDNA sequence analysis. Furthermore, 36 bacterial isolates were obtained from ten date varieties studied, each with a unique colony morphology. These isolates were identified based on sequence alignment and comparison of their 16S rDNA internal spacer regions to those available in public databases. The results showed that the bacterial isolates included 15 species from five bacterial genera. The results suggested that Bacillus, Stenotrophomonas, and Brucella were the prevailing genera among the ten tested fruit varieties. Some bacterial genera, such as Brucella, Achromobacter, and Stenotrophomonas, are well-known potential human pathogens. Chaetomium globosum was also recognized as air pollution causing adverse health effects such as allergies and as the causal agent of human fungal infections among the tested date varieties; the Rashodiah type exhibited the highest fungal contamination, whereas the Sagai variety displayed the lowest fungal contamination. Conversely, the Sukkari, Barhi, and Mejdool varieties were the most contaminated with bacteria among the ten tested varieties, while the Khalas variety showed the least bacterial contamination. To the best of the authors' knowledge, this study provides the initial comprehensive account of the molecular and morphological identification of all fungal and bacterial genera associated with date palm (P. dactylifera) fruits.

Altered microbial diversity and composition of multiple mucosal organs in cervical cancer patients.

OBJECTIVES: The aim of this study was to characterize the microbiome of multiple mucosal organs in cervical cancer (CC) patients. METHODS: We collected oral, gut, urinary tract, and vaginal samples from enrolled study participants, as well as tumor tissue from CC patients. The microbiota of different mucosal organs was identified by 16S rDNA sequencing and correlated with clinical-pathological characteristics of cervical cancer cases. RESULTS: Compared with controls, CC patients had reduced α-diversity of oral and gut microbiota (pOral_Sob < 0.001, pOral_Shannon = 0.049, pOral_Simpson = 0.013 pFecal_Sob = 0.030), although there was an opposite trend in the vaginal microbiota (pVaginal_Pielou = 0.028, pVaginal_Simpson = 0.006). There were also significant differences in the ß-diversity of the microbiota at each site between cases and controls (pOral = 0.002, pFecal = 0.037, pUrine = 0.001, pVaginal = 0.001). The uniformity of urine microbiota was lower in patients with cervical squamous cell carcinoma (pUrine = 0.036) and lymph node metastasis (pUrine_Sob = 0.027, pUrine_Pielou = 0.028, pUrine_Simpson = 0.021, pUrine_Shannon = 0.047). The composition of bacteria in urine also varied among patients with different ages (p = 0.002), tumor stages (p = 0.001) and lymph node metastasis (p = 0.002). In CC cases, Pseudomonas were significantly enriched in the oral, gut, and urinary tract samples. In addition, Gardnerella, Anaerococcus, and Prevotella were biomarkers of urinary tract microbiota; Abiotrophia and Lautropia were obviously enriched in the oral microbiota. The microbiota of tumor tissue correlated with other mucosal organs (except the gut), with a shift in the microflora between mucosal organs and tumors. CONCLUSIONS: Our study not only revealed differences in the composition and diversity of the vaginal and gut microflora between CC cases and controls, but also showed dysbiosis of the oral cavity and urethra in cervical cancer cases.

Phyto-safe in vitro regeneration and harnessing antimicrobial-resistant endophytes as bioinoculants for enhanced growth and secondary metabolites yield in Nilgirianthus ciliatus.

Nilgirianthus ciliatus, extensively exploited for its pharmacological properties, is now classified as vulnerable. In vitro micropropagation offers a sustainable approach for ecological conservation and rational utilization of this biodiversity resource. This study aimed to reduce endophytes during in vitro propagation and isolating antimicrobial-resistant endophytes from N. ciliatus by employing various concentrations and exposure times of Plant Preservative Mixture (PPM). Optimal results were observed when nodal explants treated with 0.3% PPM for 8 h, followed by inoculation in Murashige and Skoog (MS) medium supplemented with 3 mg/L 6-benzylaminopurine (BAP) and 0.3% PPM. This protocol achieved 82% shoot regeneration with minimal endophytic contamination, suggesting that the duration of explant exposure to PPM significantly influences endophyte reduction. Two antimicrobial-resistant endophytes were isolated and identified as Bacillus cereus and Acinetobacter pittii through 16S rDNA sequencing. These endophytes exhibited plant growth-promoting characteristics, including amylolytic, proteolytic, lipolytic activities, indole-3-acetic acid production, phosphate solubilization, and stress tolerance. In vivo application of these endophytes as bioinoculants to N. ciliatus not only improved growth parameters but also significantly increased the levels of pharmacologically important compounds, squalene, and stigmasterol, as confirmed by High-performance thin-layer chromatography (HPTLC). This study demonstrates that PPM is a promising alternative for sustainable micropropagation of N. ciliatus. Furthermore, it highlights the potential of antimicrobial-resistant endophytes as bioinoculants to improve growth and medicinal value, offering a sustainable solution for conservation and large-scale cultivation of this species.

Relationship between deltamethrin resistance and gut symbiotic bacteria of Aedes albopictus by 16S rDNA sequencing.

BACKGROUND: Aedes albopictus is an important vector for pathogens such as dengue, Zika, and chikungunya viruses. While insecticides is the mainstay for mosquito control, their widespread and excessive use has led to the increased resistance in Ae. albopictus globally. Gut symbiotic bacteria are believed to play a potential role in insect physiology, potentially linking to mosquitoes' metabolic resistance against insecticides. METHODS: We investigated the role of symbiotic bacteria in the development of resistance in Ae. albopictus by comparing gut symbiotic bacteria between deltamethrin-sensitive and deltamethrin-resistant populations. Adults were reared from field-collected larvae. Sensitive and resistant mosquitoes were screened using 0.03% and 0.09% deltamethrin, respectively, on the basis of the World Health Organization (WHO) tube bioassay. Sensitive and resistant field-collected larvae were screened using 5 × LC50 (lethal concentration at 50% mortality) and 20 × LC50 concentration of deltamethrin, respectively. Laboratory strain deltamethrin-sensitive adults and larvae were used as controls. The DNA of gut samples from these mosquitoes were extracted using the magnetic bead method. Bacterial 16S rDNA was sequenced using BGISEQ method. We isolated and cultured gut microorganisms from adult and larvae mosquitoes using four different media: Luria Bertani (LB), brain heart infusion (BHI), nutrient agar (NA), and salmonella shigella (SS). RESULTS: Sequencing revealed significantly higher gut microbial diversity in field-resistant larvae compared with field-sensitive and laboratory-sensitive larvae (P < 0.01). Conversely, gut microorganism diversity in field-resistant and field-sensitive adults was significantly lower compared with laboratory-sensitive adults (P < 0.01). At the species level, 25 and 12 bacterial species were isolated from the gut of field resistant larvae and adults, respectively. The abundance of Flavobacterium spp., Gemmobacter spp., and Dysgonomonas spp. was significantly higher in the gut of field-resistant larvae compared with sensitive larvae (all P < 0.05). Furthermore, the abundance of Flavobacterium spp., Pantoea spp., and Aeromonas spp. was significantly higher in the gut of field-resistant adults compared with sensitive adults (all P < 0.05). The dominant and differentially occurring microorganisms were also different between resistant larval and adult mosquitoes. These findings suggest that the gut commensal bacteria of Ae. albopictus adults and larvae may play distinct roles in their deltamethrin resistance. CONCLUSIONS: This study provides an empirical basis for further exploration of the mechanisms underlying the role of gut microbial in insecticide resistance, potentially opening a new prospect for mosquito control strategies.

Effect of High Dietary Iron on Fat Deposition and Gut Microbiota in Chickens.

To meet the demand of consumers for chicken products, poultry breeders have made improvements to chickens. However, this has led to a new problem in the modern poultry industry, namely excessive fat deposition. This study aims to understand the effects of dietary iron supplementation on fat deposition and gut microbiota in chickens. In this study, we investigated the effects of iron on the growth performance, fat deposition, and gut microbiota of silky fowl black-bone chickens. A total of 75 7-week-old silky fowl black-bone chickens were randomly divided into three groups (five replicates per group, five chickens per replicate) and fed them for 28 days using a growing diet (control group), a growing diet + 10% tallow (high-fat diet group, HFD group), and a growing diet + 10% tallow + 500 mg/kg iron (HFDFe500 group), respectively. We detected the effects of iron on the growth performance, fat deposition, and gut microbiota of silky fowl black-bone chickens using the growth performance index test, oil red O staining, and HE staining, and found that the high-fat diet significantly increased liver and serum fat deposition and liver injury, while the addition of iron to the diet could reduce the fat deposition caused by the high-fat diet and alleviate liver injury. In addition, 16S rDNA sequencing was used to compare the relative abundance of gut microbiota in the cecal contents in different feeding groups. The results showed that the high-fat diet could induce gut microbiota imbalance in chickens, while the high-iron diet reversed the gut microbiota imbalance. PICRUSt functional prediction analysis showed that dietary iron supplementation affected amino acid metabolism, energy metabolism, cofactors, and vitamin metabolism pathways. In addition, correlation analysis showed that TG was significantly associated with Firmicutes and Actinobacteriota (p < 0.05). Overall, these results revealed high dietary iron (500 mg/kg) could reduce fat deposition and affect the gut microbiota of silky fowl black-bone chickens, suggesting that iron may regulate fat deposition by influencing the gut microbiota of chickens and provides a potential avenue that prevents excessive fat deposition in chickens by adding iron to the diet.