Microbial communities play key roles both for humans and the environment. They are involved in ecosystem functions, maintaining their stability, and provide important services, such as carbon cycle and nitrogen cycle. Acting both as symbionts and as pathogens, description of the structure and composition of these communities is important. Metabarcoding uses ribosomal DNA (rDNA) (eukaryotic) or rRNA gene (prokaryotic) sequences for identification of species present in a site and measuring their abundance. This procedure requires several technical steps that could be source of bias producing a distorted view of the real community composition. In this work, we took advantage of an innovative 'long-read' next-generation sequencing (NGS) technology (MinION) amplifying the DNA spanning from the internal transcribed spacer (ITS) to large subunit (LSU) that can be read simultaneously in this platform, providing more information than 'short-read' systems. The experimental system consisted of six fungal mock communities composed of species present at various relative amounts to mimic natural situations characterized by predominant and low-frequency species. The influence of the sequencing platform (MinION and Illumina MiSeq) and the effect of different reference databases and marker sequences on metagenomic identification of species were evaluated. The results showed that the ITS-based database provided more accurate species identification than LSU. Furthermore, a procedure based on a preliminary identification with standard reference databases followed by the production of custom databases, including only the best outputs of the first step, is proposed. This additional step improved the estimate of species proportion of the mock communities and reduced the number of ghost species not really present in the simulated communities.IMPORTANCE Metagenomic analyses are fundamental in many research areas; therefore, improvement of methods and protocols for the description of microbial communities becomes more and more necessary. Long-read sequencing could be used for reducing biases due to the multicopy nature of rDNA sequences and short-read limitations. However, these novel technologies need to be assessed and standardized with controlled experiments, such as mock communities. The interest behind this work was to evaluate how long reads performed identification and quantification of species mixed in precise proportions and how the choice of database affects such analyses. Development of a pipeline that mitigates the effect of the barcoding sequences and the impact of the reference database on metagenomic analyses can help microbiome studies go one step further.Metagenomic analyses are fundamental in many research areas; therefore, improvement of methods and protocols for the description of microbial communities becomes more and more necessary. Long-read sequencing could be used for reducing biases due to the multicopy nature of rDNA sequences and short-read limitations.

Potentially toxic dinoflagellates of the genus Ostreopsis are expanding their distribution globally, with a marked increase in the frequency of Benthic Harmful Algal Blooms (BHABs). Six of the twelve species of Ostreopsis produce a highly potent non-proteinaceous toxin known as palytoxin (PLTX) and its analogs, posing a significant threat to human and environmental health. The study of BHAB species, particularly the genus Ostreopsis, is a recent development in Mexico. With a comprehensive approach, this study aimed to taxonomically and toxicologically characterize Ostreopsis strains from three locations in the central and southeastern Gulf of California. Morphometric and tabular analyses suggested that Ostreopsis cf. ovata and O. fattorussoi are the most probable morphospecies. However, phylogenetic analyses inferred from LSU rDNA (D1-D3) and ITS rDNA sequences further narrowed our results to the Ostreopsis ovata species complex, which also suggests its dominance in the region. In a composite sample (three strains from each of the three locations) chemically analyzed with LC-MS/MS, no intact palytoxins were found; however, amine aldehyde fragment derivatives common to PLTX, OST-D, OVTX, 42-OH PLTX, deoxy PLTX, and isobaric PLTX, were found at low intracellular concentrations. These findings were consistent with an Artemia franciscana bioassay, as the mortality rate after 48 h of exposure was below 10 % and showed no significant differences (p > 0.05) between the control and experimental conditions. Therefore, it is concluded that the tested strains of O. cf. ovata do not currently represent a risk to human or ecosystem health.

Alexandrium species are among the most frequently reported in harmful algal blooms (HABs), posing risks to both the environment and human health. During a study of a bloom event in Paracas Bay in the austral autumn of 2018, a dinoflagellate of the genus Alexandrium was detected in phytoplankton samples, and a clonal culture was established. Morphological analyses identified A. ostenfeldii, which was confirmed by phylogenetic analyses based on the LSU rDNA D1/D2 regions, confirming that strain IMP-BG-507 clustered with strains isolated from El Callao and Chilean Patagonia belonging to sub-clade 6. LC-MS analyses revealed an atypical Paralytic Shellfish Toxin profile dominated by dcSTX, dcGTX3, and dcGTX2, followed by doSTX, doGTX2, and doGTX3. Additionally, LC-HRMS revealed the presence of 13-desmethyl spirolide C, which is the first report of this toxin in an A. ostenfeldii strain isolated from the Pacific coast of South America. The unique toxin profile of strain IMP-BG-507 reveals the need to continue studies on different strains and populations of A. ostenfeldii and to deepen the search for new species of the Alexandrium genus that could affect aquaculture development on the coasts of Peru.

A molecular hypothesis of the Infraorder Rhigonematomorpha is presented. The phylogeny recovered using combined SSU and LSU markers suggests that the Rhigonematomorpha is nested within a larger clade that includes Ascaridomorpha, Spiruromorpha and Oxyuridomorpha and is strongly supported by maximum likelihood (ML) bootstrap support values (BS) and Bayesian posterior probabilities (BPP). SSU and LSU ML and Bayesian analyses recovered Rhigonematomorpha as a paraphyletic clade. In the ML and Bayesian analyses of a combined matrix of complete to partial sequences of SSU and LSU, respectively, Rhigonematomorpha is recovered as a monophyletic clade with moderate BPP but low BS. Highly supported BS and BPP of a combined SSU and LSU matrix support a hypothesis of a monophyletic Superfamily Ransomnematoidea that includes the families Carnoyidae, Hethidae, Ransomnematidae plus a Brumptaemilius, Cattiena, Insulanema clade, and a monophyletic Superfamily Rhigonematoidea that probably includes the paraphyletic families Rhigonematidae and Ichthyocephalidae. It is suggested that the future inclusion of more families and genera might help resolve the monophyly of the Infraorder Rhigonematomorpha as advanced here. [ABSTRACT FROM AUTHOR]

Squash (Cucurbita spp.) is an economically important vegetable crop in Pakistan, yet its productivity is severely impacted by powdery mildew. Despite frequent outbreaks, limited data exist on its causal agents and host range in Pakistan. This study aimed to assess the prevalence, incidence, characterization and host range of associated squash powdery mildew (SPM) pathogens. Surveys conducted during 2020 and 2021 in the Pothwar region revealed 100% disease prevalence with mean incidence of 50.47% and 59.78%, respectively. Disease incidence was positively correlated with humidity and rainfall. Healthy plant assays for culture maintenance and preservation of inoculum at −20 °C were found the most efficient for maintaining the pathogen virulence even after a year whereas, 500 mg powder/L (3.4 × 104 spores) was standardized as optimum inoculum load for the onset of utmost disease severity. Microscopic observation of each sample identified two coexisting SPM causal agents i.e. Podosphaera xanthii (Px.) and Golovinomyces cichoracearum (Gc.). Nucleotide sequences of highly virulent isolates from pathogenicity tests confirmed the species identity and phylogenetic analyses revealed genetic similarity with global isolates. All cucurbit crops were susceptible to SPM whereas, the weeds were infected by other powdery mildew species. Interestingly, sunflower (both wild and cultivated) (Helianthus annuus) was found infected with both SPM species which confirms the sunflower as potential alternate host for SPM.

8065 LSU Eye Center Clinical Retina Fellowships To offers complete vitreoretinal capabilities with digital imaging, ICG angiography, OCT, ultrasound, and PDT. Ophthalmology. Applicants must be graduates of ACGME-accredited ophthalmology residency [...]