Ribosomal RNA Research Articles

Mitochondrial Genomes of Mammals from the Brazilian Cerrado and Phylogenetic Considerations for the Orders Artiodactyla, Carnivora, and Chiroptera (Chordata: Mammalia)

We assembled and annotated the complete mitochondrial genomes of Lycalopex vetulus (hoary fox), Cerdocyon thous (bush dog), Tayassu pecari (white-lipped peccary), and Tadarida brasiliensis (Brazilian free-tailed bat). The mitogenomes exhibited typical vertebrate structures, containing 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and a D-loop region. Phylogenetic reconstruction using the 13 protein-coding genes revealed robust relationships among species within Carnivora, Chiroptera, and Artiodactyla, corroborating previous studies. Secondary structure analysis of tRNAs and ribosomal genes showed slight variations among species of the same order. This research highlights the importance of mitochondrial genomics in understanding the evolutionary relationships and genetic diversity of Cerrado mammals, contributing to conservation efforts for this unique ecosystem.

Rubus tingzhouensis (Rosaceae), a new species from Fujian, China

Rubus tingzhouensis C.An & G.C.Lin, a newly-defined species within the family Rosaceae from Fujian Province, China, is formally described and illustrated here. R. tingzhouensis is morphologically similar to R. swinhoei, but can be distinguished by several key characteristics, such as long, reddish-purple stipitate glands, soft bristles, light yellow short trichomes (vs. shortly grey tomentose at the early stage, glabrescent) and scattered epidermal prickles (vs. few to many curved prickles). Furthermore, the stipules are pinnately deeply laciniate, measuring 1–1.5 cm (vs. ovate-oblong to ovate-lanceolate, 0.5–0.8 cm). Phylogenetic analyses, based on partial sequences and the complete plastome data, provide robust support for a close relationship between R. tingzhouensis and R. swinhoei, while also highlighting distinct genetic differentiation between these two species. The chloroplast genome of R. tingzhouensis is 156,311 bp in length and comprises 132 unique genes, including 86 protein-coding genes, 37 transfer RNAs, eight ribosomal RNAs and one pseudogene.

Rethreading the needle: A novel molecular index of soil health (MISH) using microbial functional genes to predict soil health management

Soil health relies on the actions and interactions of an abundant and diverse biological community. Current soil health assessments rely heavily on a suite of soil biological, chemical, and physical indicators, often excluding molecular information. Soil health is critical for sustainable agricultural production, and a comprehensive understanding of how microbial communities provide ecosystem services can help guide management practices. To explore the role of microbial function in soil health, 536 soil samples were collected from 26 U.S. states, representing 52 different crops and grazing lands, and analyzed for various soil health indicators. The bacterial functional profile was characterized using 16S ribosomal RNA gene sequencing paired with PICRUSt2 to predict metagenome functions. Functional data were used as predictors in eXtreme Gradient Boosting (XGBoost), a powerful machine learning algorithm, and enzymes important to soil health indicators were compiled into a Molecular Index of Soil Health (MISH). The overall MISH score significantly correlated with non-molecular measures of soil health and management practice adoption. Additionally, several new enzymes were identified as potential targets to better understand microbial mediation of soil health. This low-cost, DNA-based approach to measuring soil health is robust and generalizable across climates.

Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action.

Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted-repeat lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form 2 or 3 intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +8 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea, causal agent of gray mold disease in fruits, vegetables and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides a structural framework for design of novel, potent antifungal peptides for agricultural use.

Characterising healthy Australian oral microbiomes for ‘super donor’ selection

ObjectivesAmong healthy people, we understand very little about the sociodemographic, lifestyle, and dental hygiene behaviours that shape their oral microbiota. This study investigates how sociodemographic, lifestyle and dental hygiene behaviours shape oral microbiota diversity and composition in an Australian population to better inform healthy oral microbiota donors for Oral Microbiota Transplantation (OMT). MethodsThe study comprised 93 healthy people who underwent comprehensive oral examinations and questionnaires to assess their health status. Participants were excluded if they had any active systemic or oral disease. All completed a questionnaire containing information on socio-economic, lifestyle, behavioural, and oral health factors. Supragingival plaque was collected, and 16S ribosomal RNA (rRNA) amplicon sequencing was used to analyse microbial composition. Associations between the core microbiome, alpha- (within-sample), beta-diversity (between-sample) and an individual's co-variates were tested for statistical significance. A redundancy analysis (RDA), multivariate adonis, differential abundance and correlation analysis were performed to characterise which factors drive the variation in the healthy oral microbiome. ResultsStreptococcus and Corynebacterium were the most prevalent and abundant genera among healthy Australians. The alpha and beta diversity were higher among unemployed non-Australian-born students who consumed low carbohydrates, fat, and sugar and had not visited the dentist for over 12 months. Additionally, beta diversity was significantly higher among daily flossers who abstained from fluoride treatment and had high salivary pH, although no single factor explained more than 4% of the total variation (R2= 0.042). Alloprevotella, Lachnosporacea, and Parvimonas were significantly abundant among non-Australians who did not visit the dentist within a year. The RDA analysis revealed associations between microbiome composition and factors such as high carbohydrate, sugar, and fat consumption, low fibre intake, and regular dental checks among Australian-born individuals. ConclusionThese findings indicate that alpha and beta diversity of the oral microbiome varied significantly with sociodemographic, lifestyle, and dietary factors, including non-Australian birthplaces, unemployment, diet, and infrequent dental visits. Clinical SignificanceThese findings underscore the importance of considering diverse sociodemographic, lifestyle, and dietary factors in oral health management. Before microbiome transplantations, clinicians should account for individual characteristics that may be beneficial for shaping and maintaining optimal oral microbiome diversity and health.

The complete chloroplast genome sequence of Melothria scabra (Cucurbitaceae)

Melothria scabra has gradually become an economically important plant worldwide. The complete chloroplast genome of M. scabra has a length of 156,744 bp, contains a large single-copy (LSC) region (86,387 bp), a small single-copy (SSC) region (18,055 bp), and two inverted repeats (IRs) with the same length of 26,151 bp. In total, 126 genes were detected, including 83 protein-encoding genes, 35 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. For phylogenetic analysis, M. scabra has a closer genetic relationship with Cucumis sativus and Citrullus lanatus. The complete chloroplast genome sequence of M. scabra would promote the germplasm exploration, phylogenetic relationships, and molecular biology researches in Melothria.

Combined analysis of cross-population healthy adult human microbiome reveals consistent differences in gut microbial characteristics between Western and non-Western countries

Despite extensive research on the gut microbiome of healthy individuals from a single country, there are still a limited number of population-level comparative studies. Moreover, the sequencing approach used in most related studies involves 16S ribosomal RNA (rRNA) sequencing with a limited resolution, which cannot provide detailed functional profiles. In the present study, we applied a combined analysis approach to analyze whole metagenomic shotgun sequencing data from 2035 healthy adult samples from six countries across four continents. Analysis of core species revealed that 13 species were present in more than 90% of all investigated individuals, the majority of which produced short-chain fatty acids (SCFA)-producing bacteria. Our analysis revealed consistently significant differences in gut microbial species and pathways between Western and non-Western countries, such as Escherichia coli and the relation of MetaCyc pathways to the TCA cycle. Specific changes in microbial species and pathways are potentially related to lifestyle and diet. Furthermore, we identified several noteworthy microbial species and pathways that exhibit distinct characteristics specific to China. Interestingly, we observed that China (CHN) was more similar to the United States (USA) and United Kingdom (GBR) in terms of the taxonomic and functional composition of the gut microbiome than India (IND) and Madagascar (MDG), which were more similar to the China (CHN) diet. The current study identified consistent microbial features associated with population and geography, which will inspire further clinical translations that consider paying attention to differences in microbiota backgrounds and confounding factors.

Engineered FnCas9 mediated mutation profiling for clarithromycin resistance in Helicobacter pylori strains isolated from Indian patients with gastrointestinal disorders

Helicobacter pylori is a highly prevalent gut pathogen with reported implications in a wide range of gastrointestinal disorders. Antibiotic based therapy, especially with clarithromycin is one of most effective treatment strategies against H. pylori. However, rising global prevalence of clarithromycin resistance in certain H. pylori strains, primarily attributed to point mutations in the 23 s ribosomal RNA coding gene, pose a major challenge in the effective eradication of this pathogen. There are a number of established methodologies devised for H. pylori mutation detection, so as to provide a tailored treatment plan to the patients and resist further transmissions of antibiotic resistant strains. However, there is no ‘gold standard’ method available to detect mutation status in clinical isolates of H. pylori from infected patients. CRISPR-Cas9 based technologies have revolutionized the field of mutation detection in biological samples, particularly during the recent COVID-19 pandemic. Although multiple assays have been reported for detection of H. pylori in clinical samples including CRISPR diagnostics (CRISPRDx) platforms, there is no such assay reported till date to detect specific mutations that confer antibiotic resistance to this pathogen. In this study, we have developed an assay using engineered FnCas9 (en31-FnCas9) protein to effectively detect the A2142G and A2143G mutations in the 23 s rDNA of H. pylori strains isolated from gastric biopsy samples. The data from in vitro cleavage assays and strip-based lateral flow tests using en31-FnCas9 and guide RNAs targeting the conserved and mutated loci of H. pylori 23 s rDNA are in perfect congruence with the data from Sanger sequencing and restriction fragment length polymorphism (RFLP) analysis. Our results indicate that en31-FnCas9 based mutation analysis can be deployed as an efficient diagnostic methodology to detect clarithromycin susceptibility in patients with suspected H. pylori infections.

Taxonomic Revision of Ningshan Odd-Scaled Snake, Achalinus ningshanensis (Serpentes, Xenodermidae), with Description of a New Subspecies from Western China

Achalinu ningshanensis (Yang, Huang, Jiang, Burbrink, and Huang, 2022) was first described in Ningshan County, Shaanxi Province, China in 2022, based on seven female specimens. In this study, based on phylogenetic analyses using mitochondrial 12S ribosomal RNA (12S), 16S ribosomal RNA (16S), cytochrome c oxidase subunit 1 (CO1), cytochrome b (cyt b) gene fragments, and morphological examinations of specimens, we revise the taxonomic status of A. ningshanensis, and provide additional data on this species. The molecular phylogeny indicated that A. ningshanensis is nested in a highly supported monophyletic group, forming a sister taxon to A. spinalis, and is divided into two well-supported lineages, A and B, with an uncorrected p-distance between lineages from 3.6 to 4.3% for CO1. Therefore, we proposed that Lineage B from western Sichuan and southwestern Shaanxi is a new subspecies, Achalinus ningshanensis occidentalis ssp. nov., and Lineage A from southern Shaanxi and northeastern Sichuan is allocated as Achalinus ningshanensis ningshanensis. Morphologically, the new subspecies can be distinguished from its congeners, especially from Achalinus ningshanensis ningshanensis, by the following characteristics: (1) the tail is relatively short, with a TAL/TL ratio of 0.202–0.226 in males, and 0.155–0.178 in females; (2) there are two pairs of chin-shields; (3) there are 21–22 maxillary teeth; (4) the length of the suture between internasals is significantly shorter than that between prefrontals, with an LSBI/LSBP ratio of 0.502–0.773; (5) there are six supralabials, with the fourth and fifth in contact with the eye; (6) there are five to six infralabials, and the first to third or fourth touches the first pair of chin-shields; (7) there is one hexagonal loreal, with an LorH/LorL ratio of 0.612–1.040; (8) the two anterior temporals are in contact with the eye; (9) there are 155–160 ventrals in males, and 165–174 in females; (10) there are 60–65 subcaudals in males, and 49–53 in females, which are not paired; and (11) the dorsum is iridescent and uniformly charcoal black, lacks a longitudinal vertebral line, and has a dark brown or dark gray ventral area.

Therapeutic effects and mechanisms of berberine on enteritis caused by Salmonella in poultry

The present study aimed to investigate the therapeutic effects of berberine (BBR) on Salmonella enteritis in broiler chickens and to elucidate its mechanisms of action preliminarily. Blood samples were collected from 21- to 35-day-old Sanhuang male chicks to measure immune and biochemical indicators and to calculate the organ coefficients for the liver, spleen, bursa of Fabricius, and thymus. The caecal microbiota was analysed through 16S ribosomal RNA (rRNA) gene sequencing, and transcriptome sequencing was conducted. Compared with the positive control group (S), the berberine-treated group (BS) presented increased serum immunoglobulin M (IgM) levels, serum IgG levels, and total antioxidant capacity; berberine ameliorated the increase in the thymus index caused by Salmonella administration. The addition of berberine to the diet increased the abundance of beneficial bacterial genera, including Bacteroides and Lactobacillus. It also decreased the abundance of harmful bacterial genera, including Faecalibacterium and Streptococcus. Transcriptome analysis revealed that gene expression in the S and BS groups was associated with T cell selection and B cell receptor signalling pathways, which are enriched primarily in multiple immune-related signalling pathways, including the B cell receptor signalling pathway, NF-κ B signalling pathway, intestinal immune network for IgA production, asthma, and African trypanosomiasis. The significantly expressed genes included ATAD5, ERP29, MGST2, PIK3CA, and HSP90AA1. The present study demonstrated that berberine has a good therapeutic effect on Salmonella infection in chicks, as it inhibits the occurrence and development of Salmonella-induced intestinal inflammation by regulating the balance of the gut microbiota and the expression of related genes, including ATAD5, ERP29, MGST2, PIK3CA, and HSP90AA1.

DEAD-box RNA helicase 10 is required for 18S rRNA maturation by controlling the release of U3 snoRNA from pre-rRNA in embryonic stem cells

Ribosome biogenesis plays a pivotal role in maintaining stem cell homeostasis, yet the precise regulatory mechanisms governing this process in mouse embryonic stem cells (mESCs) remain largely unknown. In this investigation, we ascertain that DEAD-box RNA helicase 10 (DDX10) is indispensable for upholding cellular homeostasis and the viability of mESCs. Positioned predominantly at the nucleolar dense fibrillar component (DFC) and granular component (GC), DDX10 predominantly binds to 45S ribosomal RNA (rRNA) and orchestrates ribosome biogenesis. Degradation of DDX10 prevents the release of U3 snoRNA from pre-rRNA, leading to perturbed pre-rRNA processing and compromised maturation of the 18S rRNA, thereby disrupting the biogenesis of the small ribosomal subunit. Moreover, DDX10 participates in the process of liquid-liquid phase separation (LLPS), which is necessary for efficient ribosome biogenesis. Notably, the NUP98-DDX10 fusion associated with acute myelocytic leukemia (AML) alters the cellular localization of DDX10 and results in loss of ability to regulate pre-rRNA processing. Collectively, this study reveals the critical role of DDX10 as a pivotal regulator of ribosome biogenesis in mESCs.

CO2 protects cells from iron-Fenton oxidative DNA damage in Escherichia coli and humans

While hydroxyl radical is commonly named as the Fenton product responsible for DNA and RNA damage in cells, here we demonstrate that the cellular reaction generates carbonate radical anion due to physiological bicarbonate levels. In human and Escherichia coli models, their transcriptomes were analyzed by RNA direct nanopore sequencing of ribosomal RNA and chromatography coupled to electrochemical detection to quantify oxidation products in order to follow the bicarbonate dependency in H2O2-induced oxidation. These transcriptomic studies identified physiologically relevant levels of bicarbonate focused oxidation on the guanine base favorably yielding 8-oxo-7,8-dihydroguanine (OG). In human cells, the bicarbonate-dependent oxidation was further analyzed in the metabolome by mass spectrometry, and a glycosylase-dependent qPCR assay to quantify oxidation sites in telomeres. These analyses further identify guanine as the site of oxidation when bicarbonate is present upon H2O2 exposure. Labile iron as the catalyst for forming carbonate radical anion was demonstrated by repeating the bicarbonate-dependent oxidations in cells experiencing ferroptosis, which had a >fivefold increase in redox-active iron, to find enhanced overall guanine-specific oxidation when bicarbonate was present. The complete profiling of nucleic acid oxidation in the genome, transcriptome, and metabolome supports the conclusion that a cellular Fe(II)-carbonate complex redirects the Fenton reaction to yield carbonate radical anion. Focusing H2O2-induced oxidative modification on one pathway is consistent with the highly evolved base excision repair suite of enzymes to locate G-oxidation sites for repair and gene regulation in response to oxidative stress.

The transcription factor FgSfp1 orchestrates mycotoxin deoxynivalenol biosynthesis in Fusarium graminearum

Fusarium graminearum (F. graminearum) and its derivative mycotoxin deoxynivalenol (DON) are highly concerned with food safety and sustainability worldwide. Although several transcription factors (TFs) had been elucidated, molecular mechanism participates in DON biosynthesis regulation remains largely unrevealed. Here, we first characterized a zinc finger-contained TF in F. graminearum, FgSfp1, which is indispensable for DON production since its depletion resulting in a 95.4% DON yielding reduction. Interestingly, contrast to previous knowledge, all TRI-cluster genes were abnormally upregulated in ΔFgSfp1 while Tri proteins abundance rationally decreased simultaneously. Further evidence show FgSfp1 could coordinate genetic translation pace by manipulating ribosomal biogenesis process. Specifically, FgSfp1-depletion leads to ribosome biogenesis assembly factor (RiBi) expression attenuation along with DON precursor acetyl-CoA synthase reduction since FgSfp1 actively interacts with RNA 2’-O-methylation enzyme FgNop1 revealed by Bi-FC. It subsequently influences mRNA translation pace. In conclusion, we elucidated that the FgSfp1 orchestrates DON biosynthesis via participating RNA posttranscriptional modification for ribosomal RNA maturation, offering insights into the DON biosynthesis regulation. Ultimately, this TF might be a key regulator for DON contamination control in the whole food chain.

DNA barcode reference database and gap analysis for biomonitoring Hong Kong’s marine animals

DNA-based taxonomic identification relies on comprehensive DNA reference libraries, but uneven barcode coverage poses a global challenge. Here we determined the gaps in BOLD and GenBank for marine animals in Hong Kong’s subtropical waters. We compiled 170,529 sequences from four marker genes [cytochrome c oxidase subunit I (COI), 12S ribosomal RNA (rRNA), 16S rRNA, and 18S rRNA] representing 4074 marine animal species spanning 17 phyla. Gap analysis highlighted variations in COI barcode coverage across phyla, ranging from 0 % in Ctenophora, Gnathostomulida, Hemichordata, Phoronida and Platyhelminthes to 90.44 % in Chordata. The average coverage of COI (53.24 % in BOLD and 58.47 % in GenBank) was substantially higher than that of the other three rRNA genes (19.46–32.25 %). In total, 4392 Barcode Index Numbers (BINs) were assigned to 2169 species, of which 41.13 % were taxonomically concordant and 50.71 % were displayed multiple BINs, indicating the presence of cryptic species and the potential for undiscovered diversity. Overall, this study provided a valuable DNA barcode library for marine animals in Hong Kong, revealed that many marine animals lack a barcode, and many species may be undescribed, highlighting the need for enhanced reference libraries. We also outlined a strategy for filling the gaps in the database. Utilizing the local reference database can significantly enhance molecular-based biodiversity assessment and improve the identification of cryptic (morphologically similar) and invasive species, thereby providing more informed support for coastal management and conservation efforts in Hong Kong waters and the surrounding areas of China’s Hong Kong-Zhuhai-Macau Greater Bay Area.

Detection and Characterization of Visceral Anisakid Nematodes in Blue Whiting from Portuguese Waters

This study employs molecular detection techniques, including conventional PCR and Sanger sequencing, to investigate the prevalence, species composition and public health implications of Anisakid nematodes in blue whiting (Micromesistius poutassou) caught off the Portuguese coast. With Portugal’s high fish consumption rates and increasing preference for raw or undercooked seafood, the risk of parasitic infections, particularly anisakidosis, is rising. Fifty blue whiting fish were examined, showing a 100% infection rate with Anisakid larvae. Molecular analysis identified 68.1% of the larvae as Anisakis simplex, 18.1% as Anisakis pegreffii, and 13.8% as Hysterothylacium aduncum, marking the first report of H. aduncum in blue whiting in Portugal. Phylogenetic analysis based on the internal transcribed spacer (ITS) 1, 5.8S ribosomal RNA and ITS-2 confirmed the species classification. Notably, 42.9% of the fish were infected with multiple Anisakid species, increasing the risk of allergenic sensitization. Statistical analysis showed no significant correlation between fish width and parasitic load, and a weak negative correlation was found between fish length and parasitic load. The study contributes to food safety by integrating molecular tools that enable rapid and accurate species identification, offering new insights into the detection of biological contaminants in seafood. These findings are significant considering the rising trend in raw seafood consumption, underscoring the urgent need for enhanced detection strategies and broader parasite monitoring programs to mitigate public health risks. The high prevalence of parasitized fish highlights the necessity for the implementation of safe cooking practices to reduce the risk of anisakidosis. Further research into the allergenic potential of Hysterothylacium spp. and the ecological factors influencing this nematode distribution is recommended.

Propionimicrobium lymphophilum in urine of children with monosymptomatic nocturnal enuresis

BackgroundDespite a unique microbiome in urine, the relationship between nocturnal enuresis and the urobiome remains unclear. This study aimed to compare the presence of specific bacterial species in the urine of children with and without nocturnal enuresis.MethodsWe used 16S ribosomal RNA gene sequencing to analyze the urobiome in urine samples obtained from the two groups of children. The presence of Propionimicrobium lymphophilum was examined using real-time PCR in the urine of 25 children diagnosed with monosymptomatic nocturnal enuresis (MNE), and 17 children without this condition.ResultsChildren with MNE exhibited a significantly higher prevalence of P. lymphophilum: 16 out of 25 (64.0%) compared to 4 out of 17 (23.5%) in the control group. Among children with frequent bedwetting, there was a significantly higher prevalence of P. lymphophilum;15 out of 16 (93.8%) compared to 2 out of 9 (22.2%) in those with infrequent bedwetting. Bacterial culture tests confirmed the anaerobic growth of P. lymphophilum isolates from urine samples of two PCR-positive patients with MNE. These isolates were found to be susceptible to ampicillin.ConclusionThese findings suggest that P. lymphophilum may be associated with chronic urinary tract infections and potentially contribute to the development of MNE in children.

Long-term co-circulation of multiple arboviruses in southeast Australia revealed by xeno-monitoring and viral whole genome sequencing

Abstract Arbovirus surveillance of wild-caught mosquitoes is an affordable and sensitive means of monitoring virus transmission dynamics at various spatial-temporal scales, and emergence and re-emergence during epidemic and interepidemic periods. A variety of molecular diagnostics for arbovirus screening of mosquitoes (known as xeno-monitoring) are available, but most provide limited information about virus diversity. PCR-based screening coupled with RNA sequencing is an increasingly affordable and sensitive pipeline for integrating complete viral genome sequencing into surveillance programs. This enables large-scale, high-throughput arbovirus screening from diverse samples. We collected mosquitoes in CO2-baited light traps from five urban parks in Brisbane from March 2021 to May 2022. Mosquito pools of ≤200 specimens were screened for alphaviruses and flaviviruses using virus genus-specific primers and reverse transcription quantitative PCR (qRT-PCR). A subset of virus-positive samples was then processed using a mosquito-specific ribosomal RNA depletion method and then sequenced on the Illumina NextSeq. Overall, 54,670 mosquitoes, representing 26 species were screened in 382 pools. Thirty detections of arboviruses were made in 28 pools. Twenty of these positive pools were further characterised using RNA sequencing generating 18 full-length genomes. These full-length sequences belonged to four medically relevant arboviruses: Barmah Forest, Ross River, Sindbis-like and Stratford viruses. Phylogenetic and evolutionary analyses revealed the evolutionary progression of arbovirus lineages over the last 100 years, demonstrating that different epidemiological, immunological, and evolutionary processes may actively shape the evolution of Australian arboviruses. These results underscore the need for more genomic surveillance data to explore the complex evolutionary pressures acting on arboviruses. Overall, our findings highlight the effectiveness of our methodology, which can be applied broadly to enhance arbovirus surveillance in various ecological contexts and improve understanding of transmission dynamics.

Influence of different diet categories on gut bacterial diversity in Frankliniella occidentalis.

The microbial composition of insect guts is typically influenced by the type of food consumed, and conversely, these microbes influence the food habits of insects. Western flower thrips (WFT; Frankliniella occidentalis) is an invasive pest with a wide range of hosts, including vegetables and horticultural crops. To elucidate variations in gut bacteria among WFT feeding on rose (Rosa rugosa) flowers (FF), kidney bean (Phaseolus vulgaris) pods (PF), and kidney bean leaves (LF), we collected adult guts and extracted DNA for 16S ribosomal RNA gene sequencing of microbial communities. The results revealed that the FF population had the highest number of annotations. Alpha diversity analysis revealed that the Chao and Ace indexes were the greatest in the PF population, indicating a higher abundance of gut bacteria. Moreover, the Simpson index was the highest in the FF population, indicating that gut bacterial diversity was the highest in the FF population. Comparison of species composition demonstrated that Proteobacteria dominated all 3 populations at the phylum level, with Actinobacteria being the subdominant phylum. At the genus level, Stenotrophomonas was the dominant bacteria in the PF and LF populations, whereas Rosenbergiella was dominant in the FF population. KEGG pathway annotation predicted that the gut bacteria of adult WFT were mainly involved in carbohydrate and amino acid metabolism. Our results revealed that the diversity and composition of WFT gut microbiota are influenced by diet, offering evidence for future studies on the ecological adaptability of WFT and the mechanisms underlying the interaction between gut microbiota and host.

Fast and accurate multi-bacterial identification using cleavable and FRET-based peptide nucleic acid probes

Fast and accurate identification of pathogenic microbes in patient samples is crucial for the timely treatment of acute infectious diseases such as sepsis. The fluorescence in situ hybridization (FISH) technique allows the rapid detection and identification of microbes based on their variation in genomic sequence without time-consuming culturing or sequencing. However, the recent explosion of microbial genomic data has made it challenging to design an appropriate set of probes for microbial mixtures. We developed a novel set of peptide nucleic acid (PNA)-based FISH probes with optimal target specificity by analyzing the variations in 16S ribosomal RNA sequence across all bacterial species. Owing to their superior penetration into bacteria and higher mismatch sensitivity, the PNA probes distinguished seven bacterial species commonly observed in bacteremia with 96–99.9% accuracy using our optimized FISH procedure. Detection based on Förster resonance energy transfer (FRET) between pairs of adjacent binding PNA probes eliminated crosstalk between species. Rapid sequential species identification was implemented, using chemically cleavable fluorophores, without compromising detection accuracy. Owing to their outstanding accuracy and enhanced speed, this set of techniques shows great potential for clinical use.

Morphological and biological characters of the entomophilic stage of Schistonchus pumilae (Aphelenchoididae) with an update of its molecular profiles

Summary Schistonchus pumilae was first reported from the syconi of a creeper fig, Ficus pumila, from Miyazaki, Japan, and the species was described based on propagative (plant-parasitic) females and males. Thus, the premature female (entomophilic form) of the species was not described. In the present study, male syconi of F. pumila were collected from the type locality of the nematode (Miyazaki, Japan), and the premature females of S. pumilae were collected from inside the syconi and also from the emerging pollinator fig wasps (Wiebesia pumilae). Although only 26 female wasps were recovered for dissections, seven of them harboured 1-7 nematodes each. Nematodes associated with the wasp were all premature females, and their typological and morphometric characters were similar with those isolated from inside the syconi. In addition, the premature females were slightly smaller than propagative females and had a less developed gonad, suggesting that the nematode is a phoretic associate, not a parasite. Regardless of its phylogenetic status, the carrier insect usage of S. pumilae is somewhat similar to two Ficophagus species, and clearly different from S. caprifici, which propagates in the host wasp haemocoel as a parasite. Additionally, the molecular profiles of S. pumilae (SSU and D1-D3 LSU of ribosomal RNA genes) were re-sequenced using a cloning technique, and the information was updated for phylogenetic analysis in the future.