Identifying early life stages of Great Lakes fishes using a metabarcoding approach

Kavishka S. Gallage, Alexander Van Nynatten, Nathan K. Lujan, Nathan R. Lovejoy, and Nicholas E. Mandrak

Canadian Journal of Fisheries and Aquatic Sciences. 6 September 2023. doi: 10.1139/cjfas-2023-0061


Detection of early life stages of fishes is important for understanding life history patterns and critical spawning habitats. When feasible, identifying early life stages of fishes using morphology requires taxonomic expertise and can be challenging, time consuming, and imprecise. In this study, we used DNA metabarcoding to identify egg and larval batch samples from two sites in the species-rich East Sydenham River, Ontario, Canada. We used a two-step PCR metabarcoding approach to amplify a highly variable region of the mitochondrial COI gene from 1075 mixed species batch samples. Amplicon libraries were sequenced with Illumina Mi-seq and the sequencing reads were filtered and assembled using the software package mothur. Barcodes were then classified using a reference library comprised of Great Lakes fishes and potential invaders. In total, 34 species, including three at-risk species and three invasive species, were detected at the two sampling sites. This study shows the potential utility of metabarcoding for detection and identification of early life stage Great Lake fishes.

Ichthyoplankton metabarcoding: An efficient tool for early detection of invasive species establishment

Alexander Van Nynatten, Kavishka S. Gallage, Nathan K. Lujan, Nicholas E. Mandrak, Nathan R. Lovejoy

Mol Ecol Resour 2023 Aug;23(6):1319-1333 doi: 10.1111/1755-0998

Pubmed ID: 37101312


Detection of invasive species is critical for management but is often limited by challenges associated with capture, processing and identification of early life stages. DNA metabarcoding facilitates large-scale monitoring projects to detect establishment early. Here, we test the use of DNA metabarcoding to monitor invasive species by sequencing over 5000 fishes in bulk ichthyoplankton samples (larvae and eggs) from four rivers of ecological and cultural importance in southern Canada. We were successful in detecting species known from each river and three invasive species in two of the four rivers. This includes the first detection of early life-stage rudd in the Credit River. We evaluated whether sampling gear affected the detection of invasive species and estimates of species richness, and found that light traps outperform bongo nets in both cases. We also found that the primers used for the amplification of target sequences and the number of sequencing reads generated per sample affect the consistency of species detections. However, these factors have less impact on detections and species richness estimates than the number of samples collected and analysed. Our analyses also show that incomplete reference databases can result in incorrectly attributing DNA sequences to invasive species. Overall, we conclude that DNA metabarcoding is an efficient tool for monitoring the early establishment of invasive species by detecting evidence of reproduction but requires careful consideration of sampling design and the primers used to amplify, sequence and classify the diversity of native and potentially invasive species.

Habitats Within the Plant Root Differ in Bacterial Network Topology and Taxonomic Assortativity

Connor R. Fitzpatrick, Julia Copeland, Pauline W. Wang, David S. Guttman, Peter M. Kotanen, Marc T. J. Johnson

Mol Plant Microbe Interact 2023 Mar;36(3):165-175. doi: 10.1094/MPMI-09-22-0188-R.

PMID: 36463399


The root microbiome is composed of distinct epiphytic (rhizosphere) and endophytic (endosphere) habitats. Differences in abiotic and biotic factors drive differences in microbial community diversity and composition between these habitats, though how they shape the interactions among community members is unknown. Here, we coupled a large-scale characterization of the rhizosphere and endosphere bacterial communities of 30 plant species across two watering treatments with co-occurrence network analysis to understand how root habitats and soil moisture shape root bacterial network properties. We used a novel bootstrapping procedure and null network modeling to overcome some of the limitations associated with microbial co-occurrence network construction and analysis. Endosphere networks had elevated node betweenness centrality versus the rhizosphere, indicating greater overall connectivity among core bacterial members of the root endosphere. Taxonomic assortativity was higher in the endosphere, whereby positive co-occurrence was more likely between bacteria within the same phylum while negative co-occurrence was more likely between bacterial taxa from different phyla. This taxonomic assortativity could be driven by positive and negative interactions among members of the same or different phylum, respectively, or by similar niche preferences associated with phylum rank among root inhabiting bacteria across plant host species. In contrast to the large differences between root habitats, drought had limited effects on network properties but did result in a higher proportion of shared co-occurrences between rhizosphere and endosphere networks. Our study points to fundamentally different ecological processes shaping bacterial co-occurrence across root habitats.

Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Detection of Covid-19 Outbreaks Using Built Environment Testing for SARS-CoV-2

Michael Fralick, Caroline Nott, Jason Moggridge, Lucas Castellani, Ashley Raudanskis, David S. Guttman, Aaron Hinz, Nisha Thampi, Alex Wong, Doug Manuel, Allison McGeer, Evgueni Doukhanine, Hebah Mejbel, Veronica Zanichelli, Madison Burella, Sylva L. Donaldson, Pauline W. Wang, Rees Kassen, Derek R. MacFadden

NEJM Evid 2023;2(3) doi: 10.1056/EVIDoa2200203

Pubmed: 38320044



Environmental surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through wastewater has become a useful tool for population-level surveillance. Built environment sampling may provide a more spatially refined approach for surveillance in congregate living settings.


We conducted a prospective study in 10 long-term care homes (LTCHs) between September 2021 and November 2022. Floor surfaces were sampled weekly at multiple locations within each building and analyzed for the presence of SARS-CoV-2 using quantitative reverse transcriptase polymerase chain reaction. The primary outcome was the presence of a coronavirus disease 2019 (Covid-19) outbreak in the week that floor sampling was performed.


Over the 14-month study period, we collected 4895 swabs at 10 LTCHs. During the study period, 23 Covid-19 outbreaks occurred with 119 cumulative weeks under outbreak. During outbreak periods, the proportion of floor swabs that were positive for SARS-CoV-2 was 54.3% (95% confidence interval [CI], 52 to 56.6), and during non-outbreak periods it was 22.3% (95% CI, 20.9 to 23.8). Using the proportion of floor swabs positive for SARS-CoV-2 to predict Covid-19 outbreak status in a given week, the area under the receiver-operating characteristic curve was 0.84 (95% CI, 0.78 to 0.9). Among 10 LTCHs with an outbreak and swabs performed in the prior week, eight had positive floor swabs exceeding 10% at least 5 days before outbreak identification. For seven of these eight LTCHs, positivity of floor swabs exceeded 10% more than 10 days before the outbreak was identified.


Detection of SARS-CoV-2 on floors is strongly associated with Covid-19 outbreaks in LTCHs. These data suggest a potential role for floor sampling in improving early outbreak identification.

A defect in cell wall recycling confers antibiotic resistance and sensitivity in Staphylococcus aureus

Stephanie Tan, Kelvin Cho, Justin R. Nodwell

J Biol Chem 2022 Oct;298(10):102473. doi: 10.1016/j.jbc.2022.102473.

PMID: 36089064


WalKR is a two-component system that is essential for viability in Gram-positive bacteria that regulates the all-important autolysins in cell wall homeostasis. Further investigation of this essential system is important for identifying ways to address antibiotic resistance. Here, we show that a T101M mutation in walR confers a defect in autolysis, a thickened cell wall, and decreased susceptibility to antibiotics that target lipid II cycle, a phenotype that is reminiscent of the clinical resistance form known as vancomycin intermediate-resistant Staphylococcus aureus. Importantly, this is accompanied by dramatic sensitization to tunicamycin. We demonstrate that this phenotype is due to partial collapse of a pathway consisting of autolysins, AtlA and Sle1, a transmembrane sugar permease, MurP, and GlcNAc recycling enzymes, MupG and MurQ. We suggest that this causes a shortage of substrate for the peptidoglycan biosynthesis enzyme MraY, causing it to be hypersensitive to competitive inhibition by tunicamycin. In conclusion, our results constitute a new molecular model for antibiotic sensitivity in S. aureus and a promising new route for antibiotic discovery.

A comparative study across the parasitic plants of Cuscuta subgenus Grammica (Convolvulaceae) reveals a possible loss of the plastid genome in its section Subulatae

Arjan Banerjee, Sasa Stefanovic

Planta 2023 Feb 24;257(4):66. doi: 10.1007/s00425-023-04099-y.

PMID: 36826697


Most species in Cuscuta subgenus Grammica retain many photosynthesis-related plastid genes, generally under purifying selection. A group of holoparasitic species in section Subulatae may have lost their plastid genomes entirely. The c. 153 species of plants belonging to Cuscuta subgenus Grammica are all obligate stem parasites. However, some have completely lost the ability to conduct photosynthesis while others retain photosynthetic machinery and genes. The plastid genome that primarily encodes key photosynthesis genes functions as a bellwether for how reliant plants are on primary production. This research assembles and analyses 17 plastomes across Cuscuta subgenus Grammica with the aim of characterizing the state of the plastome in each of its sections. By comparing the structure and content of plastid genomes across the subgenus, as well as by quantifying the selection acting upon each gene, we reconstructed the patterns of plastome change within the phylogenetic context for this group. We found that species in 13 of the 15 sections that comprise Grammica retain the bulk of plastid photosynthesis genes and are thus hemiparasitic. The complete loss of photosynthesis can be traced to two clades: the entire section Subulatae and a complex of three species within section Ceratophorae. We were unable to recover any significant plastome sequences from section Subulatae, suggesting that plastomes in these species are either drastically reduced or lost entirely.

The Evolution of C4 Photosynthesis in Flaveria (Asteraceae): Insights from the Flaveria linearis Complex

 Shunsuke Adachi, Matt Stata, Duncan G Martin, Shifeng Cheng, Hongbing Liu, Xin-Guang Zhu, Rowan F Sage

Plant Physiol 2023 Jan 2;191(1):233-251. doi: 10.1093/plphys/kiac467.

PMID: 36200882


Flaveria is a leading model for C4 plant evolution due to the presence of a dozen C3-C4 intermediate species, many of which are associated with a phylogenetic complex centered around Flaveria linearis. To investigate C4 evolution in Flaveria, we updated the Flaveria phylogeny and evaluated gas exchange, starch δ13C, and activity of C4 cycle enzymes in 19 Flaveria species and 28 populations within the F. linearis complex. A principal component analysis identified six functional clusters: (1) C3, (2) sub-C2, (3) full C2, (4) enriched C2, (5) sub-C4, and (6) fully C4 species. The sub-C2 species lacked a functional C4 cycle, while a gradient was present in the C2 clusters from little to modest C4 cycle activity as indicated by δ13C and enzyme activities. Three Yucatan populations of F. linearis had photosynthetic CO2 compensation points equivalent to C4 plants but showed little evidence for an enhanced C4 cycle, indicating they have an optimized C2 pathway that recaptures all photorespired CO2 in the bundle sheath (BS) tissue. All C2 species had enhanced aspartate aminotransferase activity relative to C3 species and most had enhanced alanine aminotransferase activity. These aminotransferases form aspartate and alanine from glutamate and in doing so could help return photorespiratory nitrogen (N) from BS to mesophyll cells, preventing glutamate feedback onto photorespiratory N assimilation. Their use requires upregulation of parts of the C4 metabolic cycle to generate carbon skeletons to sustain N return to the mesophyll, and thus could facilitate the evolution of the full C4 photosynthetic pathway.

Lung Allograft Microbiome Association with Gastroesophageal Reflux, Inflammation, and Allograft Dysfunction

Pierre H. H. Schneeberger, Chen Yang Kevin Zhang, Jessica Santilli, Bo Chen, Wei Xu, Youngho Lee, Zonelle Wijesinha Elaine Reguera-Nuñez, Noelle Yee, Musawir Ahmed, Kristen Boonstra, Rayoun Ramendra, Courtney W. Frankel, Scott M. Palmer  Jamie L. Todd , Tereza Martinu, Bryan Coburn

Am J Respir Crit Care Med 2022 Dec 15;206(12):1495-1507. doi: 10.1164/rccm.202110-2413OC.

PMID: 35876129


Rationale: It remains unclear how gastroesophageal reflux disease (GERD) affects allograft microbial community composition in lung transplant recipients and its impact on lung allograft inflammation and function.

Objectives: Our objective was to compare the allograft microbiota in lung transplant recipients with or without clinically diagnosed GERD in the first year after transplant and assess associations between GERD, allograft microbiota, inflammation, and acute and chronic lung allograft dysfunction (ALAD and CLAD).

Methods: A total of 268 BAL samples were collected from 75 lung transplant recipients at a single transplant center every 3 months after transplant for 1 year. Ten transplant recipients from a separate transplant center provided samples before and after antireflux Nissen fundoplication surgery. Microbial community composition and density were measured using 16S ribosomal RNA gene sequencing and quantitative polymerase chain reaction, respectively, and inflammatory markers and bile acids were quantified.

Measurements and Main Results: We observed a range of allograft community composition with three discernible types (labeled community state types [CSTs] 1-3). Transplant recipients with GERD were more likely to have CST1, characterized by high bacterial density and relative abundance of the oropharyngeal colonizing genera Prevotella and Veillonella. GERD was associated with more frequent transitions to CST1. CST1 was associated with lower inflammatory cytokine concentrations than pathogen-dominated CST3 across the range of microbial densities observed. Cox proportional hazard models revealed associations between CST3 and the development of ALAD/CLAD. Nissen fundoplication decreased bacterial load and proinflammatory cytokines.

Conclusions: GERD was associated with a high bacterial density, Prevotella- and Veillonella-dominated CST1. CST3, but not CST1 or GERD, was associated with inflammation and early development of ALAD and CLAD. Nissen fundoplication was associated with a reduction in microbial density in BAL fluid samples, especially the CST1-specific genus, Prevotella.

Effect of faecal microbial transplant via colonoscopy in patients with severe obesity and insulin resistance: A randomized double-blind, placebo-controlled Phase 2 trial

Yasaman Ghorbani BSc, Katherine J. P. Schwenger PhD, Divya Sharma PhD, Hyejung Jung MSc, Jitender Yadav PhD, Wei Xu PhD, Wendy Lou PhD, Susan Poutanen MD, Susy S. Hota MD, Elena M. Comelli

Diabetes Obes Metab 2023 Feb;25(2):479-490. doi: 10.1111/dom.14891.

PMID: 36239189


Aim: To assess the effects of faecal microbial transplant (FMT) from lean people to subjects with obesity via colonoscopy.

Material and methods: In a double-blind, randomized controlled trial, subjects with a body mass index ≥ 35 kg/m2 and insulin resistance were randomized, in a 1:1 ratio in blocks of four, to either allogenic (from healthy lean donor; n = 15) or autologous FMT (their own stool; n = 13) delivered in the caecum and were followed for 3 months. The main outcome was homeostatic model assessment of insulin resistance (HOMA-IR) and secondary outcomes were glycated haemoglobin levels, lipid profile, weight, gut hormones, endotoxin, appetite measures, intestinal microbiome (IM), metagenome, serum/faecal metabolites, quality of life, anxiety and depression scores.

Results: In the allogenic versus autologous groups, HOMA-IR and clinical variables did not change significantly, but IM and metabolites changed favourably (P < 0.05): at 1 month, Coprococcus, Bifidobacterium, Bacteroides and Roseburia increased, and Streptococcus decreased; at 3 months, Bacteroides and Blautia increased. Several species also changed significantly. For metabolites, at 1 month, serum kynurenine decreased and faecal indole acetic acid and butenylcarnitine increased, while at 3 months, serum isoleucine, leucine, decenoylcarnitine and faecal phenylacetic acid decreased. Metagenomic pathway representations and network analyses assessing relationships with clinical variables, metabolites and IM were significantly enhanced in the allogenic versus autologous groups. LDL and appetite measures improved in the allogenic (P < 0.05) but not in the autologous group.

Conclusions: Overall, in those with obeisty, allogenic FMT via colonoscopy induced favourable changes in IM, metabolites, pathway representations and networks even though other metabolic variables did not change. LDL and appetite variables may also benefit.

Elevated tyrosine results in the cytosolic retention of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in Arabidopsis thaliana

Michael Kanaris, Jimmy Poulin, Dea Shahinas, Daniel Johnson, Valerie M. Crowley, Geoffrey Fucile, Nicholas Provart, Dinesh Christendat

Plant J 2022 Feb;109(4):789-803. doi: 10.1111/tpj.15590.

PMID: 34797933


The shikimate pathway plays a central role in the biosynthesis of aromatic amino acids and specialized metabolites in plants. The first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) serves as a key regulatory point for the pathway in various organisms. These enzymes are important in regulating the shikimate pathway in multiple microbial systems. The mechanism of regulation of DAHPS is poorly understood in plants, and the role of tyrosine (Tyr) with respect to the three DAHPS isozymes from Arabidopsis thaliana was investigated. In vitro enzymatic analyses established that Tyr does not function as an allosteric regulator for the A. thaliana DAHPS isozymes. In contrast, Arabidopsis T-DNA insertional mutants for the DAHPS1 locus, dahps1, are hypersensitive to elevated Tyr. Tyr hypersensitivity can be reversed with tryptophan and phenylalanine supplementation, indicating that Tyr is affecting the shikimate pathway flux in the dahps1 mutant. Tyr treatment of Arabidopsis seedlings showed reduced accumulation of overexpressed DAHPS2 in the chloroplast. Further, bimolecular fluorescence complementation studies revealed that DAHPS2 interacts with a 14-3-3 protein in the cytosol, and this interaction is enhanced with Tyr treatment. This interaction with 14-3-3 may retain DAHPS2 in the cytosol, which prevents its ability to function in the chloroplast with elevated Tyr.