Biology subjects

Visscher, P. T.

Publications and source records attributed to Visscher, P. T..

5 recordsLinked to original sources

bioRxiv2024

High Throughput Viral Enumeration of Aquatic Ecosystems via Flow Cytometry

For the past 25 years, flow cytometry has been a gold standard for the direct measurement of viral-like particles (VLP) in aquatic ecosystems. Flow cytometry allows for higher throughput and costs less than alternative enumeration methods, leading to its broad usage in aquatic viral ecology. A major challenge associated with flow cytometry is the degradation of VLPs over time, making the use of high throughput plates not possible, thus lowering overall throughput. It has also been difficult to maintain a method with low contamination, high signal-to-noise ratios, and observations of real VLPs vs. fake particles. For these reasons, the use of flow cytometry has rapidly declined over the years due to the advent of massively parallel sequencing. Here, we describe a high throughput method in a 96 well plate format that provides a hands-free approach to viral enumeration. Our approach limits fake particles, noise levels, and cross-sample contamination. In a standard run, 60 samples can be measured for VLPs within 2.25 hours, which is [~]1 hour faster than the standard single tube approach and [~]1.5 hours faster than epifluorescence microscopy (EFM). Direct measurement of VLPs still provides a window into the viral-host interactions within aquatic ecosystems, which can be rapidly measured and resolved in a high throughput manner.

microbiology↗

bioRxiv2024

Resolving and Quantifying Viral-Like Particles via Blind Deconvolution

Viruses represent the most numerous biological entities on Earth; but the direct quantification of viruses within ecosystems reminds an ongoing challenge. The classical method of epifluorescence microscopy (EFM) reminds the gold standard measurement of viral-like particles (VLPs) within ecosystems. Quantifying VLPs in epifluorescence microscopy is burdened by ongoing challenges that include manual human counting, an absence of accurate morphological sizing, and the a range of viral sizes (20-300 nm) falling below the diffraction limit of light microscopy. Here, a proof-of-concept computer vision framework for the automated enumeration and sizing of viral-like particles is presented, known as EpiVirQuant. A novel tunable pointspread function is introduced which allows for a dynamic blind deconvolution. Final enumeration by EpiVirQuant was directly compared to manual human counting which yielded 18% more VLPs identified. EpiVirQuant quantified average VLP size of 179.5 nm, which is consistent with median size of VLPs in nature of of _160 nm. Runtime ranged from 60-80 seconds-perimage depending on parameter selection. This provides a viable proof-of-concept cost-effective solution for the enumeration and large-scale morphological analysis of VLPs.

bioinformatics↗

bioRxiv2023

The eukaryome of modern microbialites reveals distinct colonization across aquatic ecosystems

Microbial diversity includes bacteria, archaea, eukaryotes, and viruses; however, protists are less studied for their impact and diversity within ecosystems. Protists have been suggested to shape the emergence and decline of ancient stromatolites. Modern microbialites offer a unique proxy to study the deposition of carbonate by microbial communities due to analog status for ancient ecosystems and their cosmopolitan abundance. We examined protists across aquatic ecosystems between freshwater (Kelly and Pavilion Lake in British Columbia, Canada) and marine microbialites (Shark Bay, Australia and Highborne Cay, Bahamas) to decipher the transition with respect to diversity and composition. While factors such as sequencing technology and primer-bias might influence our conclusions, we found that at the taxonomic compositional-level, the freshwater microbialite communities were clearly distinct from the marine microbialite communities. Chlorophytes were significantly more abundant in the freshwater microbialites, while saltwater microbialites communities were primarily composed of pennate diatoms. Despite the differences in taxonomic make-up, we can infer the convergent important role of these protists to microbialite community health and function. These results highlight not only the consistency and potential role of microbialite eukaryotic communities across geographic locations, but also that other factors such as salinity seem to be the main drivers of community composition.

microbiology↗

bioRxiv2023

Viral enumeration using cost-effective wet-mount epifluorescence microscopy for aquatic ecosystems and modern microbialites

Enumeration is a fundamental measure of community ecology in which viruses represent the most numerous biological identities. Epifluorescence microscopy (EFM) has been the gold standard method for environmental viral enumeration for over 25 years. Currently, standard EFM methods using the Anodisc filters are no longer cost-effective (>$15 per slide) and have yet to be applied to modern microbialites. Microbialites are microbially driven benthic organosedimentary deposits that have been present for most of Earths history. We present a cost-effective method for environmental viral enumeration from aquatic samples, microbial mats, and exopolymeric substances (EPS) within modern microbialites using EFM. Our integrated approach, which includes filtration, differential centrifugation, chloroform treatment, glutaraldehyde fixation, benzonase nuclease treatment, probe sonication (EPS and mat only), SYBR Gold staining, wet-mounting, and imaging, provides a robust method for modern microbialites and aquatic samples. Viral abundances of modern microbialites and aquatic samples collected from Fayetteville Green Lake (FGL) and Great Salt Lake (GSL) did not differ across ecosystems by sample type. EPS and microbial mat samples had an order of magnitude higher viral-like particle (VLP) abundance when compared to water regardless of the ecosystem (107 vs. 106). Viral enumeration allows for estimates of total viral numbers and weights. The entire weight of all the viruses in FGL and GSL are [~]598 g and [~]2.2 kg, respectively; this is equivalent to a loaf of bread for FGL and standard brick for GSL. Further development of EFM methods and software is needed for viral enumeration. Our method provides a robust and cost-effective ([~]$0.75 per sample) viral enumeration within modern microbialites and aquatic ecosystems.

microbiology↗

bioRxiv2020

Microbial dark matter filling the niche in hypersaline microbial mats

Shark Bay, Australia, harbours one of the most extensive and diverse systems of living microbial mats, that are proposed to be analogs of some of the earliest ecosystems on Earth. These ecosystems have been shown to possess a substantial abundance of uncultivable microorganisms. These enigmatic groups - microbial dark matter (MDM) - are hypothesised to play key roles in microbial mats. We reconstructed 115 metagenome-assembled genomes (MAGs) affiliated to MDM, spanning 42 phyla within the bacterial and archaeal domains. We classified bacterial MDM from the PVC group, FCB group, Microgenomates, Parcubacteria, and Peregrinibacteria, as well as a high proportion of archaeal MDM under the TACK, DPANN, Altiarchaeales, and Asgard archaea. The latter includes the first putative Heimdallarchaeota MAG obtained from any microbial mat system. This study reports novel microorganisms (Zixibacterial order GN15) putatively taking part in dissimilatory sulfate reduction in surface hypersaline settings, as well as novel eukaryote signature proteins in the Asgard archaea. Despite possessing reduced-size genomes, the MDM MAGs are capable of fermenting and degrading organic carbon, suggesting a role in recycling organic carbon. Several forms of RuBisCo were identified, allowing putative CO2 incorporation into nucleotide salvaging pathways, which may act as an alternative carbon and phosphorus source. High capacity of hydrogen production was found among Shark Bay MDM. Putative schizorhodopsins were also identified in Parcubacteria, Asgard archaea, DPANN archaea, and Bathyarchaeota, allowing these members to potentially capture light energy. Diversity-generating retroelements were prominent in DPANN archaea that likely facilitate the adaptation to a dynamic, host-dependent lifestyle. In light of our findings, we propose H2, ribose and CO/CO2 as the main energy currencies of the MDM community in these mat systems.

microbiology↗