Targeted metagenomics reveals pangenomic diversity of the nitroplast (UCYN-A) and its algal host plastid.

UCYN-A (Cand. Atelocyanobacterium thalassa) has recently been recognized as a globally-distributed, early stage, nitrogen-fixing organelle (the 'nitroplast') of cyanobacterial origin present in select species of haptophyte algae (e.g., Braarudosphaera bigelowii). Although the nitroplast was recognized as the UCYN-A2 sublineage, it is yet to be confirmed in other sublineages of the algal/UCYN-A complex. We used water samples collected from Halifax Harbour (Bedford Basin, Nova Scotia, Canada) and the offshore Scotian Shelf to further our understanding of B. bigelowii and UCYN-A in the coastal Northwest Atlantic. Sequencing data revealed UCYN-A-associated haptophyte signatures and yielded near-complete metagenome-assembled genomes (MAGs) for UCYN-A1, UCYN-A4, and the plastid of the A4-associated haptophyte. Comparative genomics provided new insights into the pangenome of UCYN-A. The UCYN-A4 MAG is the first genome sequenced from this sublineage and shares ~85% identity with the UCYN-A2 nitroplast. Genes missing in the reduced genome of the nitroplast were also missing in the A4 MAG supporting its likely classification as a nitroplast as well. The UCYN-A1 MAG was found to be nearly 100% identical to the reference genome despite coming from different ocean basins. Time-series data paired with the recurrence of specific microbes in enrichment cultures gave insight into the microbes that frequently co-occur with the algal/UCYN-A complex (e.g., Pelagibacter ubique). Overall, our study expands knowledge of UCYN-A and its host across major ocean basins and investigates their co-occurring microbes in the coastal Northwest Atlantic (NWA), thereby facilitating future studies on the underpinnings of haptophyte-associated diazotrophy in the sea.

Determinants of influenza non-vaccination among Canadian children: insights from a nationwide survey.

Background: To identify determinants influencing Canadian parents' decision not to vaccinate their children aged 6 months to 17 years against seasonal influenza. Methods: Data from the 2022 Childhood COVID-19 Immunization Coverage Survey, a national survey of approximately 10,500 Canadian parents/guardians and their children, was analyzed. The survey examined influenza vaccine coverage, parental perspectives on vaccines, reasons for hesitancy, and factors influencing immunization. Socio-demographic characteristics, including ethnicity, household income, working sector, educational attainment, and prevalence of chronic medical conditions among children were considered. Historical vaccine uptake and the impact of the COVID-19 pandemic on immunization decisions were also reviewed. Key determinants of non-vaccination in the 2021-2022 influenza season were analyzed using multivariable logistic regression, with a statistical significance level set at p-value <0·05. Results: 70% of children aged 6 months to 17 years did not receive the seasonal influenza vaccine. Key predictors for non-vaccination included: residing in rural settings (aOR 1·35, 95% CI 1·13-1·60), parental education attainment of less than high school (aOR 2·48, 95% CI 1·24-4·97), and the absence of chronic medical conditions in children (aOR 1.60, 95% CI 1.34-1.91)· Other strong predictors included lower household income; deterrence due to the COVID-19 pandemic; and parental hesitancy stemming from concerns about the vaccine's safety, effectiveness, and by beliefs that their child was not at risk of contracting the influenza or severe consequences from the infection. Conclusion: This research underscores pivotal determinants of parental decisions not to vaccinate their children against seasonal influenza and sheds light on the impact of the COVID-19 pandemic. The results highlight the importance of addressing safety concerns and providing clear information to alleviate hesitancy.

Factors associated with childhood non-vaccination against COVID-19 in Canada: A national survey analysis.

Background: COVID-19 vaccination efforts are critical in mitigating the impact of the virus, but despite proven safety and efficacy, vaccination rates among children in Canada are lower than in adults, prompting a need to explore determinants of childhood COVID-19 non-vaccination to improve uptake. Method: This study analyzed data from the Canadian COVID-19 Immunization Coverage Survey 2022. Using multivariable logistic regression, it examined the association between COVID-19 non-vaccination among children aged 5-17 and factors such as parental sociodemographic characteristics, vaccine knowledge, attitudes, and beliefs (KAB), and vaccination history. Results: The analysis revealed that negative KAB towards vaccines, reflected in higher KAB composite scores, significantly increased the likelihood of non-vaccination. Additionally, factors such as lower household incomes, rural residence, employment in sectors not at risk for vaccine-preventable diseases, and younger parental age were associated with higher non-vaccination. The study also highlighted ethnic disparities in vaccination odds and found that children with incomplete routine vaccinations or inconsistent flu vaccination histories were more likely to be unvaccinated against COVID-19. Surprisingly, children of parents who consistently received flu vaccinations were more likely to be unvaccinated against COVID-19. Furthermore, parental education levels showed a complex relationship with children's COVID-19 vaccination status, indicating nuanced influences on vaccination decisions. Conclusion: The findings offer vital insights into the factors influencing COVID-19 vaccination uptake among children in Canada, suggesting avenues for targeted strategies to improve vaccine coverage.

Mock microbial community meta-analysis using different trimming of amplicon read lengths.

Trimming of sequencing reads is a pre-processing step that aims to discard sequence segments such as primers, adapters and low quality nucleotides that will interfere with clustering and classification steps. We evaluated the impact of trimming length of paired-end 16S and 18S rRNA amplicon reads on the ability to reconstruct the taxonomic composition and relative abundances of communities with a known composition in both even and uneven proportions. We found that maximizing read retention maximizes recall but reduces precision by increasing false positives. The presence of expected taxa was accurately predicted across broad trim length ranges but recovering original relative proportions remains a difficult challenge. We show that parameters that maximize taxonomic recovery do not simultaneously maximize relative abundance accuracy. Trim length represents one of several experimental parameters that have non-uniform impact across microbial clades, making it a difficult parameter to optimize. This study offers insights, guidelines, and helps researchers assess the significance of their decisions when trimming raw reads in a microbiome analysis based on overlapping or non-overlapping paired-end amplicons.

Marine nitrogen-fixers in the Canadian Arctic Gateway are dominated by biogeographically distinct noncyanobacterial communities.

We describe diazotrophs present during a 2015 GEOTRACES expedition through the Canadian Arctic Gateway (CAG) using nifH metabarcoding. In the less studied Labrador Sea, Bradyrhizobium sp. and Vitreoscilla sp. nifH variants were dominant, while in Baffin Bay, a Stutzerimonas stutzeri variant was dominant. In comparison, the Canadian Arctic Archipelago (CAA) was characterized by a broader set of dominant variants belonging to Desulfobulbaceae, Desulfuromonadales, Arcobacter sp., Vibrio spp., and Sulfuriferula sp. Although dominant diazotrophs fell within known nifH clusters I and III, only a few of these variants were frequently recovered in a 5-year weekly nifH times series in the coastal NW Atlantic presented herein, notably S. stutzeri and variants belonging to Desulfobacterales and Desulfuromonadales. In addition, the majority of dominant Arctic nifH variants shared low similarity (< 92% nucleotide identities) to sequences in a global noncyanobacterial diazotroph catalog recently compiled by others. We further detected UCYN-A throughout the CAG at low-levels using quantitative-PCR assays. Temperature, depth, salinity, oxygen, and nitrate were most strongly correlated to the Arctic diazotroph diversity observed, and we found a stark division between diazotroph communities of the Labrador Sea versus Baffin Bay and the CAA, hence establishing that a previously unknown biogeographic community division can occur for diazotrophs in the CAG.

Microevolutionary patterns in ecotypes of the symbiotic cyanobacterium UCYN-A revealed from a Northwest Atlantic coastal time series.

UCYN-A is a globally important nitrogen-fixing symbiotic microbe often found in colder regions and coastal areas where nitrogen fixation has been overlooked. We present a 3-year coastal Northwest Atlantic time series of UCYN-A by integrating oceanographic data with weekly nifH and16S rRNA gene sequencing and quantitative PCR assays for UCYN-A ecotypes. High UCYN-A relative abundances dominated by A1 to A4 ecotypes reoccurred annually in the coastal Northwest Atlantic. Although UCYN-A was detected every summer/fall, the ability to observe separate ecotypes may be highly dependent on sampling time given intense interannual and weekly variability of ecotype-specific occurrences. Additionally, much of UCYN-A's rarer diversity was populated by short-lived neutral mutational variants, therefore providing insight into UCYN-A's microevolutionary patterns. For instance, rare ASVs exhibited community composition restructuring annually, while also sharing a common connection to a dominant ASV within each ecotype. Our study provides additional perspectives for interpreting UCYN-A intraspecific diversity and underscores the need for high-resolution datasets when deciphering spatiotemporal ecologies within UCYN-A.

Compact and automated eDNA sampler for in situ monitoring of marine environments.

Using environmental DNA (eDNA) to monitor biodiversity in aquatic environments is becoming an efficient and cost-effective alternative to other methods such as visual and acoustic identification. Until recently, eDNA sampling was accomplished primarily through manual sampling methods; however, with technological advances, automated samplers are being developed to make sampling easier and more accessible. This paper describes a new eDNA sampler capable of self-cleaning and multi-sample capture and preservation, all within a single unit capable of being deployed by a single person. The first in-field test of this sampler took place in the Bedford Basin, Nova Scotia, Canada alongside parallel samples taken using the typical Niskin bottle collection and post-collection filtration method. Both methods were able to capture the same aquatic microbial community and counts of representative DNA sequences were well correlated between methods with R[Formula: see text] values ranging from 0.71-0.93. The two collection methods returned the same top 10 families in near identical relative abundance, demonstrating that the sampler was able to capture the same community composition of common microbes as the Niskin. The presented eDNA sampler provides a robust alternative to manual sampling methods, is amenable to autonomous vehicle payload constraints, and will facilitate persistent monitoring of remote and inaccessible sites.

Cobalamin producers and prokaryotic consumers in the Northwest Atlantic.

Cobalamin availability can influence primary productivity and ecological interactions in marine microbial communities. The characterization of cobalamin sources and sinks is a first step in investigating cobalamin dynamics and its impact on productivity. Here, we identify potential cobalamin sources and sinks on the Scotian Shelf and Slope in the Northwest Atlantic Ocean. Functional and taxonomic annotation of bulk metagenomic reads, combined with analysis of genome bins, were used to identify potential cobalamin sources and sinks. Cobalamin synthesis potential was mainly attributed to Rhodobacteraceae, Thaumarchaeota, and cyanobacteria (Synechococcus and Prochlorococcus). Cobalamin remodelling potential was mainly attributed to Alteromonadales, Pseudomonadales, Rhizobiales, Oceanospirilalles, Rhodobacteraceae, and Verrucomicrobia, while potential cobalamin consumers include Flavobacteriaceae, Actinobacteria, Porticoccaceae, Methylophiliaceae, and Thermoplasmatota. These complementary approaches identified taxa with the potential to be involved in cobalamin cycling on the Scotian Shelf and revealed genomic information required for further characterization. The Cob operon of Rhodobacterales bacterium HTCC2255, a strain with known importance in cobalamin cycling, was similar to a major cobalamin producer bin, suggesting that a related strain may represent a critical cobalamin source in this region. These results enable future inquiries that will enhance our understanding of how cobalamin shapes microbial interdependencies and productivity in this region.

Short-term response to iron resupply in an iron-limited open ocean diatom reveals rapid decay of iron-responsive transcripts.

In large areas of the ocean, iron concentrations are insufficient to promote phytoplankton growth. Numerous studies have been conducted to characterize the effect of iron on algae and how algae cope with fluctuating iron concentrations. Fertilization experiments in low-iron areas resulted primarily in diatom-dominated algal blooms, leading to laboratory studies on diatoms comparing low- and high-iron conditions. Here, we focus on the short-term temporal response following iron addition to an iron-starved open ocean diatom, Thalassiosira oceanica. We employed the NanoString platform and analyzed a high-resolution time series on 54 transcripts encoding proteins involved in photosynthesis, N-linked glycosylation, iron transport, as well as transcription factors. Nine transcripts were iron-responsive, with an immediate response to the addition of iron. The fastest response observed was the decrease in transcript levels of proteins involved in iron uptake, followed by an increase in transcript levels of iron-containing enzymes and a simultaneous decrease in the transcript levels of their iron-free replacement enzymes. The transcription inhibitor actinomycin D was used to understand the underlying mechanisms of the decrease of the iron-responsive transcripts and to determine their half-lives. Here, Mn-superoxide dismutase (Mn-SOD), plastocyanin (PETE), ferredoxin (PETF) and cellular repressor of EA1-stimulated genes (CREGx2) revealed longer than average half-lives. Four iron-responsive transcripts showed statistically significant differences in their decay rates between the iron-recovery samples and the actD treatment. These differences suggest regulatory mechanisms influencing gene transcription and mRNA stability. Overall, our study contributes towards a detailed understanding of diatom cell biology in the context of iron fertilization response and provides important observations to assess oceanic diatom responses following sudden changes in iron concentrations.

Seasonal bacterial niche structures and chemolithoautotrophic ecotypes in a North Atlantic fjord.

Quantifying the temporal change of bacterial communities is essential to understanding how both natural and anthropogenic pressures impact the functions of coastal marine ecosystems. Here we use weekly microbial DNA sampling across four years to show that bacterial phyla have distinct seasonal niches, with a richness peak in winter (i.e., an inverse relationship with daylength). Our results suggest that seasonal fluctuations, rather than the kinetic energy or resource hypotheses, dominated the pattern of bacterial diversity. These findings supplement those from global analyses which lack temporal replication and present few data from winter months in polar and temperate regions. Centered log-ratio transformed data provided new insights into the seasonal niche partitioning of conditionally rare phyla, such as Modulibacteria, Verrucomicrobiota, Synergistota, Deinococcota, and Fermentibacterota. These patterns could not be identified using the standard practice of ASV generation followed by rarefaction. Our study provides evidence that five globally relevant ecotypes of chemolithoautotrophic bacteria from the SUP05 lineage comprise a significant functional group with varying seasonal dominance patterns in the Bedford Basin.

Combining multi-marker metabarcoding and digital holography to describe eukaryotic plankton across the Newfoundland Shelf.

The planktonic diversity throughout the oceans is vital to ecosystem functioning and linked to environmental change. Plankton monitoring tools have advanced considerably with high-throughput in-situ digital cameras and genomic sequencing, opening new challenges for high-frequency observations of community composition, structure, and species discovery. Here, we combine multi-marker metabarcoding based on nuclear 18S (V4) and plastidial 16S (V4-V5) rRNA gene amplicons with a digital in-line holographic microscope to provide a synoptic diversity survey of eukaryotic plankton along the Newfoundland Shelf (Canada) during the winter transition phase of the North Atlantic bloom phenomenon. Metabarcoding revealed a rich eukaryotic diversity unidentifiable in the imaging samples, confirming the presence of ecologically important saprophytic protists which were unclassifiable in matching images, and detecting important groups unobserved or taxonomically unresolved during similar sequencing campaigns in the Northwest Atlantic Ocean. In turn, imaging analysis provided quantitative observations of widely prevalent plankton from every trophic level. Despite contrasting plankton compositions portrayed by each sampling method, both capture broad spatial differences between the northern and southern sectors of the Newfoundland Shelf and suggest complementary estimations of important features in eukaryotic assemblages. Future tasks will involve standardizing digital imaging and metabarcoding for wider use and consistent, comparable ocean observations.

Highly-resolved interannual phytoplankton community dynamics of the coastal Northwest Atlantic.

Microbial observatories can track phytoplankton at frequencies that resolve monthly, seasonal, and multiyear trends in environmental change from short-lived events. Using 4-years of weekly flow cytometry along with chloroplast and cyanobacterial 16S rRNA gene sequence data from a time-series station in the coastal Northwest Atlantic (Bedford Basin, Nova Scotia, Canada), we analyzed temporal observations for globally-relevant genera (e.g., Bolidomonas, Teleaulax, Minidiscus, Chaetoceros, Synechococcus, and Phaeocystis) in an oceanic region that has been recognized as a likely hotspot for phytoplankton diversity. Contemporaneous Scotian Shelf data also collected during our study established that the major phytoplankton within the Bedford Basin were important in the Scotian Shelf during spring and fall, therefore pointing to their broader significance within the coastal Northwest Atlantic (NWA). Temporal trends revealed a subset of indicator taxa along with their DNA signatures (e.g., Eutreptiella and Synechococcus), whose distribution patterns make them essential for timely detection of environmentally-driven shifts in the NWA. High-resolution sampling was key to identifying important community shifts towards smaller phytoplankton under anomalous environmental conditions, while further providing a detailed molecular view of community compositions underpinning general phytoplankton succession within the coastal NWA. Our study demonstrates the importance of accessible coastal time-series sites where high-frequency DNA sampling allows for the detection of shifting baselines in phytoplankton communities.

Late reactivation of cherubism in an adult further to local inflammation.

Cherubism is a rare pediatric disease affecting the jaw. It appears among children between 2 and 5 years old. Maximum growth is observed at 7-8 years old, then lesions remain unchanged or increase slowly until puberty. Only 2 cases of later growth have been reported. We describe a case of cherubism reactivation in a 46-year-old woman. Appearance of a new lesion occurs in a context of local inflammation due to repeated friction of the dental prosthesis on the mandible. No article in the literature describes a similar case. This case shows the determining role of inflammation (local or general) in the pathophysiology of cherubism.

Niche partitioning by photosynthetic plankton as a driver of CO2-fixation across the oligotrophic South Pacific Subtropical Ocean.

Oligotrophic ocean gyre ecosystems may be expanding due to rising global temperatures [1-5]. Models predicting carbon flow through these changing ecosystems require accurate descriptions of phytoplankton communities and their metabolic activities [6]. We therefore measured distributions and activities of cyanobacteria and small photosynthetic eukaryotes throughout the euphotic zone on a zonal transect through the South Pacific Ocean, focusing on the ultraoligotrophic waters of the South Pacific Gyre (SPG). Bulk rates of CO2 fixation were low (0.1 µmol C l-1 d-1) but pervasive throughout both the surface mixed-layer (upper 150 m), as well as the deep chlorophyll a maximum of the core SPG. Chloroplast 16S rRNA metabarcoding, and single-cell 13CO2 uptake experiments demonstrated niche differentiation among the small eukaryotes and picocyanobacteria. Prochlorococcus abundances, activity, and growth were more closely associated with the rims of the gyre. Small, fast-growing, photosynthetic eukaryotes, likely related to the Pelagophyceae, characterized the deep chlorophyll a maximum. In contrast, a slower growing population of photosynthetic eukaryotes, likely comprised of Dictyochophyceae and Chrysophyceae, dominated the mixed layer that contributed 65-88% of the areal CO2 fixation within the core SPG. Small photosynthetic eukaryotes may thus play an underappreciated role in CO2 fixation in the surface mixed-layer waters of ultraoligotrophic ecosystems.

Heterogeneous viral contribution to dissolved organic matter processing in a long-term macrocosm experiment.

Viruses saturate environments throughout the world and play key roles in microbial food webs, yet how viral activities affect dissolved organic matter (DOM) processing in natural environments remains elusive. We established a large-scale long-term macrocosm experiment to explore viral dynamics and their potential impacts on microbial mortality and DOM quantity and quality in starved and stratified ecosystems. High viral infection dynamics and the virus-induced cell lysis (6.23-64.68% d-1) was found in the starved seawater macrocosm, which contributed to a significant transformation of microbial biomass into DOM (0.72-5.32 µg L-1 d-1). In the stratified macrocosm, a substantial amount of viral lysate DOM (2.43-17.87 µg L-1 d-1) was released into the upper riverine water, and viral lysis and DOM release (0.35-5.75 µg L-1 d-1) were reduced in the mixed water layer between riverine water and seawater. Viral lysis was stimulated at the bottom of stratified macrocosm, potentially fueled by the sinking of particulate organic carbon. Significant positive and negative associations between lytic viral production and different fluorescent DOM components were found in the starved and stratified macrocosm, indicating the potentially complex viral impacts on the production and utilization of DOM. Results also revealed the significant viral contribution to pools of both relatively higher molecular weight labile DOM and lower molecular weight recalcitrant DOM. Our study suggests that viruses have heterogeneous impact on the cycling and fate of DOM in aquatic environments.

Plankton classification with high-throughput submersible holographic microscopy and transfer learning.

BACKGROUND: Plankton are foundational to marine food webs and an important feature for characterizing ocean health. Recent developments in quantitative imaging devices provide in-flow high-throughput sampling from bulk volumes-opening new ecological challenges exploring microbial eukaryotic variation and diversity, alongside technical hurdles to automate classification from large datasets. However, a limited number of deployable imaging instruments have been coupled with the most prominent classification algorithms-effectively limiting the extraction of curated observations from field deployments. Holography offers relatively simple coherent microscopy designs with non-intrusive 3-D image information, and rapid frame rates that support data-driven plankton imaging tasks. Classification benchmarks across different domains have been set with transfer learning approaches, focused on repurposing pre-trained, state-of-the-art deep learning models as classifiers to learn new image features without protracted model training times. Combining the data production of holography, digital image processing, and computer vision could improve in-situ monitoring of plankton communities and contribute to sampling the diversity of microbial eukaryotes. RESULTS: Here we use a light and portable digital in-line holographic microscope (The HoloSea) with maximum optical resolution of 1.5 µm, intensity-based object detection through a volume, and four different pre-trained convolutional neural networks to classify > 3800 micro-mesoplankton (> 20 µm) images across 19 classes. The maximum classifier performance was quickly achieved for each convolutional neural network during training and reached F1-scores > 89%. Taking classification further, we show that off-the-shelf classifiers perform strongly across every decision threshold for ranking a majority of the plankton classes. CONCLUSION: These results show compelling baselines for classifying holographic plankton images, both rare and plentiful, including several dinoflagellate and diatom groups. These results also support a broader potential for deployable holographic microscopes to sample diverse microbial eukaryotic communities, and its use for high-throughput plankton monitoring.

Harnessing the Potential of Native Microbial Communities for Bioremediation of Oil Spills in the Iberian Peninsula NW Coast.

Oil spills are among the most catastrophic events to marine ecosystems and current remediation techniques are not suitable for ecological restoration. Bioremediation approaches can take advantage of the activity of microorganisms with biodegradation capacity thus helping to accelerate the recovery of contaminated environments. The use of native microorganisms can increase the bioremediation efficiency since they have higher potential to survive in the natural environment while preventing unpredictable ecological impacts associated with the introduction of non-native organisms. In order to know the geographical scale to which a native bioremediation consortium can be applied, we need to understand the spatial heterogeneity of the natural microbial communities with potential for hydrocarbon degradation. In the present study, we aim to describe the genetic diversity and the potential of native microbial communities to degrade petroleum hydrocarbons, at an early stage of bioremediation, along the NW Iberian Peninsula coast, an area particularly susceptible to oil spills. Seawater samples collected in 47 sites were exposed to crude oil for 2 weeks, in enrichment experiments. Seawater samples collected in situ, and samples collected after the enrichment with crude oil, were characterized for prokaryotic communities by using 16S rRNA gene amplicon sequencing and predictive functional profiling. Results showed a drastic decrease in richness and diversity of microbial communities after the enrichment with crude oil. Enriched microbial communities were mainly dominated by genera known to degrade hydrocarbons, namely Alcanivorax, Pseudomonas, Acinetobacter, Rhodococcus, Flavobacterium, Oleibacter, Marinobacter, and Thalassospira, without significant differences between geographic areas and locations. Predictive functional profiling of the enriched microbial consortia showed a high potential to degrade the aromatic compounds aminobenzoate, benzoate, chlorocyclohexane, chlorobenzene, ethylbenzene, naphthalene, polycyclic aromatic compounds, styrene, toluene, and xylene. Only a few genera contributed for more than 50% of this genetic potential for aromatic compounds degradation in the enriched communities, namely Alcanivorax, Thalassospira, and Pseudomonas spp. This work is a starting point for the future development of prototype consortia of hydrocarbon-degrading bacteria to mitigate oil spills in the Iberian NW coast.

Correcting a major error in assessing organic carbon pollution in natural waters.

Microbial degradation of dissolved organic carbon (DOC) in aquatic environments can cause oxygen depletion, water acidification, and CO2 emissions. These problems are caused by labile DOC (LDOC) and not refractory DOC (RDOC) that resists degradation and is thus a carbon sink. For nearly a century, chemical oxygen demand (COD) has been widely used for assessment of organic pollution in aquatic systems. Here, we show through a multicountry survey and experimental studies that COD is not an appropriate proxy of microbial degradability of organic matter because it oxidizes both LDOC and RDOC, and the latter contributes up to 90% of DOC in high-latitude forested areas. Hence, COD measurements do not provide appropriate scientific information on organic pollution in natural waters and can mislead environmental policies. We propose the replacement of the COD method with an optode-based biological oxygen demand method to accurately and efficiently assess organic pollution in natural aquatic environments.