Publications by authors named "D Minardi"

90 Publications

Improved high throughput protocol for targeting eukaryotic symbionts in metazoan and eDNA samples.

Mol Ecol Resour 2022 Feb 1;22(2):664-678. Epub 2021 Oct 1.

Centre for Environment, Fisheries and Aquaculture Research, Weymouth, Dorset, UK.

Eukaryote symbionts of animals are major drivers of ecosystems not only because of their diversity and host interactions from variable pathogenicity but also through different key roles such as commensalism and to different types of interdependence. However, molecular investigations of metazoan eukaryomes require minimising coamplification of homologous host genes. In this study we (1) identified a previously published "antimetazoan" reverse primer to theoretically enable amplification of a wider range of microeukaryotic symbionts, including more evolutionarily divergent sequence types, (2) evaluated in silico several antimetazoan primer combinations, and (3) optimised the application of the best performing primer pair for high throughput sequencing (HTS) by comparing one-step and two-step PCR amplification approaches, testing different annealing temperatures and evaluating the taxonomic profiles produced by HTS and data analysis. The primer combination 574*F - UNonMet_DB tested in silico showed the largest diversity of nonmetazoan sequence types in the SILVA database and was also the shortest available primer combination for broadly-targeting antimetazoan amplification across the 18S rRNA gene V4 region. We demonstrate that the one-step PCR approach used for library preparation produces significantly lower proportions of metazoan reads, and a more comprehensive coverage of host-associated microeukaryote reads than the two-step approach. Using higher PCR annealing temperatures further increased the proportion of nonmetazoan reads in all sample types tested. The resulting V4 region amplicons were taxonomically informative even when only the forward read is analysed. This region also revealed a diversity of known and putatively parasitic lineages and a wider diversity of host-associated eukaryotes.
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http://dx.doi.org/10.1111/1755-0998.13509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9292944PMC
February 2022

Global mRNA and miRNA Analysis Reveal Key Processes in the Initial Response to Infection with WSSV in the Pacific Whiteleg Shrimp.

Viruses 2021 06 13;13(6). Epub 2021 Jun 13.

Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, UK.

White Spot Disease (WSD) presents a major barrier to penaeid shrimp production. Mechanisms underlying White Spot Syndrome Virus (WSSV) susceptibility in penaeids are poorly understood due to limited information related to early infection. We investigated mRNA and miRNA transcription in over 36 h following infection. Over this time course, 6192 transcripts and 27 miRNAs were differentially expressed-with limited differential expression from 3-12 h post injection (hpi) and a more significant transcriptional response associated with the onset of disease symptoms (24 hpi). During early infection, regulated processes included cytoskeletal remodelling and alterations in phagocytic activity that may assist WSSV entry and translocation, novel miRNA-induced metabolic shifts, and the downregulation of ATP-dependent proton transporter subunits that may impair cellular recycling. During later infection, uncoupling of the electron transport chain may drive cellular dysfunction and lead to high mortalities in infected penaeids. We propose that post-transcriptional silencing of the immune priming gene Dscam (downregulated following infections) by a novel shrimp miRNA (Pva-pmiR-78; upregulated) as a potential mechanism preventing future recognition of WSSV that may be suppressed in surviving shrimp. Our findings improve our understanding of WSD pathogenesis in and provide potential avenues for future development of prophylactics and treatments.
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http://dx.doi.org/10.3390/v13061140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231841PMC
June 2021

Normal human macula densa morphology and cell turnover: A histological, ultrastructural, and immunohistochemical investigation.

Anat Rec (Hoboken) 2020 11 28;303(11):2904-2916. Epub 2020 Jun 28.

Department of Experimental and Clinical Medicine, Section of Neuroscience and Cell Biology, School of Medicine, Università Politecnica delle Marche, Ancona, Italy.

The aim was to analyze the morphology of normal human macula densa (MD), evaluate the cells that may be responsible for its turnover, and collect quantitative data. Of four samples of normal human renal tissue, two were embedded in resin to measure the longitudinal extension and examine the ultrastructure of the MD, the other two were embedded in paraffin to study apoptosis and cell proliferation. The MD is composed of a monolayer tissue about 40 μm long, which includes 35-40 cells arranged in overlapping rows. Ultrastructurally, MD cells show two polarized portions: an apical end, with sensory features, and a basolateral aspect, with paracrine function. MD cells are connected apically by tight junctions, with/without adherens junctions, which form a barrier between the distal tubule lumen and the interstitium. Cells in degeneration, often associated with macrophages, and undifferentiated cells were found in the MD and adjacent distal tubule. A filamentous mat previously described in proximal tubule scattered tubular cells (STCs) was detected in the basal cytoplasm in undifferentiated cells. The tissue was consistently negative for the proliferation marker Ki67 and for the apoptotic markers caspase-3 and caspase-9. This work confirms our earlier morphological findings and provides new data: (a) MD cells display both apical adherens and tight junctions, the latter forming a tubulo-mesangial barrier; (b) the MD is a monolayer made up of about 40 cells arranged in rows; (c) the simultaneous presence of degenerating (8-13%) and undifferentiated (4-13%) cells reminiscent of STCs suggests a non-negligible cell turnover.
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http://dx.doi.org/10.1002/ar.24465DOI Listing
November 2020

De novo transcriptome assembly of the Qatari pearl oyster Pinctada imbricata radiata.

Mar Genomics 2020 Jun 7;51:100734. Epub 2019 Dec 7.

Environmental Science Center (ESC), Qatar University, P. O. Box: 2713, Doha, Qatar.

The pearl oyster Pinctada imbricata radiata is an iconic species in Qatar, representing an integral part of the nation's cultural heritage and one of the main economic foundations upon which the nation developed. During the early part of the 20th century, nearly half the Qatar population was involved in the pearl oyster industry. However, the fishery has undergone steady decline since the 1930s, and the species is now under threat due to multiple confounding pressures. This manuscript presents the first de novo transcriptome of the Qatari pearl oyster assembled into 30,739 non-redundant coding sequences and with a BUSCO completeness score of 98.4%. Analysis of the transcriptome reveals the close evolutionary distance to the conspecific animal Pinctada imbricata fucata but also highlights differences in immune genes and the presence of distinctive transposon families, suggesting recent adaptive divergence. This data is made available for all to utilise in future studies on the species.
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http://dx.doi.org/10.1016/j.margen.2019.100734DOI Listing
June 2020

Improved method for genotyping the causative agent of crayfish plague (Aphanomyces astaci) based on mitochondrial DNA.

Parasitology 2019 07 12;146(8):1022-1029. Epub 2019 Apr 12.

Biosciences, University of Exeter,Stocker Road, EX4 4QD, Exeter,UK.

Aphanomyces astaci causes crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction of A. astaci into Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carry A. astaci in their cuticle as a benign infection. Aphanomyces astaci rapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis of A. astaci pure cultures characterized five genotype groups (A, B, C, D and E). Current A. astaci genotyping techniques (microsatellites and genotype-specific regions, both targeting nuclear DNA) can be applied directly to DNA extracted from infected cuticles but require high infection levels. Therefore, they are not suitable for genotyping benign infections in North American crayfish (carriers). In the present study, we combine bioinformatics and molecular biology techniques to develop A. astaci genotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts of A. astaci pure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence of A. astaci genotype groups A and B in UK waters.
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http://dx.doi.org/10.1017/S0031182019000283DOI Listing
July 2019
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