Proliferation and anatoxin production of benthic cyanobacteria associated with canine mortalities along a stream-lake continuum.

Proliferations of benthic cyanobacteria are increasingly in the public eye, with rising animal deaths associated with benthic rather than planktonic blooms. In early June 2021, two dogs died after consuming material on the shore of Shubenacadie Grand Lake, Nova Scotia. Preliminary investigations indicated anatoxins produced by benthic cyanobacterial mats were responsible for the deaths. In this study, we monitored the growth of a toxic benthic cyanobacterial species (Microcoleus sp.) along a stream-lake continuum where the canine poisonings occurred. We found that the species was able to proliferate in both lentic and lotic environments, but temporal growth dynamics and the predominant sub-species were influenced by habitat type, and differed with hydrodynamic setting, nutrient and sunlight availability. Toxin concentration was greatest in cyanobacterial mats growing in the oligotrophic lakeshore environment (maximum measured total anatoxins (ATXs) >20 mg·kg-1 wet weight). This corresponded with a shift in the profile of ATX analogues, which also indicated changing sub-species dominance along the stream-lake transition.

Novel virulent and temperate cyanophages predicted to infect Microcoleus associated with anatoxin-producing benthic mats.

Cyanophages are crucial for regulating cyanobacterial populations, but their influence on anatoxin-producing Microcoleus mat dynamics remains unexplored. Here, we use metagenomics to explore phage presence in benthic mats from the Wolastoq|Saint John River (New Brunswick, Canada) and the Eel River (California, USA). We recovered multiple viral-like sequences associated with different putative bacterial hosts, including two cyanophage genomes with apparently different replication strategies. A temperate cyanophage was found integrated in the genomes of Microcoleus sp. 3 recovered from the Eel River and is phylogenetically related to Phormidium phages. We also recovered novel virulent cyanophage genomes from Wolastoq and Eel River mats that were dominated by anatoxin-producing Microcoleus species predicted to be the host. Despite the geographical distance, these genomes have similar sizes (circa 239 kbp) and share numerous orthologous genes with high sequence identity. A considerable reduction of the anatoxin-producing Microcoleus species in Wolastoq mats following the emergence of the virulent phage suggests that phage infections have an important role in limiting the abundance of this toxigenic cyanobacterium and releasing anatoxins into the surrounding water. Our results constitute the first report of cyanophages predicted to infect mat-forming Microcoleus species associated with anatoxin production.

Genomic characterization of coexisting anatoxin-producing and non-toxigenic Microcoleus subspecies in benthic mats from the Wolastoq, New Brunswick, Canada.

The presence of toxigenic benthic cyanobacteria in riverine ecosystems is an increasing concern around the world. In 2018, the death of three dogs along the Wolastoq (also known as the Saint John River) in New Brunswick, Canada, was attributed to anatoxin exposure after they ingested benthic microbial mats found along the shore. Here, we shotgun sequenced the DNA of 15 non-axenic cyanobacterial isolates derived from four anatoxin-containing benthic mat samples associated with the dog deaths. Anatoxins were produced by some of the isolates, but not all. We retrieved near-complete Microcoleus metagenome-assembled genomes (MAGs) from the isolates that are closely related to anatoxin-producing Microcoleus from the Cardrona River (New Zealand), although the Microcoleus MAGs from the Wolastoq varied in the presence/absence of the anatoxin-a biosynthesis cluster. Sequence similarity at the genomic level suggests that toxigenic and non-toxigenic Microcoleus MAGs from the Wolastoq belong to the same species but are separate subspecies. The toxigenic and nontoxic Wolastoq Microcoleus subspecies coexisted in the mat samples in similar relative abundance. Overall genomic comparisons revealed that toxigenic Microcoleus MAGs are longer and code for more accessory genes than their non-toxigenic relatives, suggesting a differential responsiveness to changing environments, stress conditions and nutrient availability.

Anatoxins from benthic cyanobacteria responsible for dog mortalities in New Brunswick, Canada.

In July 2018 three dogs died after visiting the Wolastoq (Saint John River) near Fredericton, New Brunswick, in Atlantic Canada. All showed signs of toxicosis, and necropsies revealed non-specific pulmonary edema and multiple microscopic brain hemorrhages. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of vomitus and stomach contents as well as water and biota from the mortality sites confirmed the presence of anatoxins (ATXs), a class of potent neurotoxic alkaloids. The highest levels were measured in a dried benthic cyanobacterial mat that two of the dogs had been eating before falling ill and in a vomitus sample collected from one of the dogs. Concentrations of 357 and 785 mg/kg for anatoxin-a and dihydroanatoxin-a, respectively, were measured in the vomitus. Known anatoxin-producing species of Microcoleus were tentatively identified using microscopy and confirmed by 16S rRNA gene sequencing. The ATX synthetase gene, anaC, was detected in the samples and isolates. The pathology and experimental results confirmed the role of ATXs in these dog mortalities. Further research is required to understand drivers for toxic cyanobacteria in the Wolastoq and to develop methodology for assessing occurrence.

Rapid Quantitation of Anatoxins in Benthic Cyanobacterial Mats Using Direct Analysis in Real-Time-High-Resolution Tandem Mass Spectrometry.

Toxic benthic cyanobacterial mats are increasingly reported worldwide as being responsible for animal mortalities due to their production of the potent neurotoxin anatoxin-a (ATX) and its analogues. Improved analytical methods for anatoxins are needed to address public health and watershed management challenges arising from extremely high spatial and temporal variability within impacted systems. We present the development, validation, and application of a direct analysis in real-time-high-resolution tandem mass spectrometry (DART-HRMS/MS) method for analysis of anatoxins in cyanobacterial field samples, including a simplified sample preparation approach. The method showed excellent sensitivity and selectivity for ATX, homoanatoxin-a, and dihydroanatoxin-a. Isotopically labeled ATX was used as an internal standard for all three analogues and successfully corrected for the matrix effects observed (86 ± 16% suppression). The limit of detection and recovery for ATX was estimated as 5 ng/g and 88%, respectively, using spiked samples. The total analysis time was ∼2 min, and excellent agreement was observed with results from a liquid chromatography-HRMS reference method. Finally, the DART-HRMS/MS method was applied to a set of 45 Microcoleus-dominated benthic cyanobacterial mat samples from the Wolastoq near Fredericton, Canada, demonstrating its power and applicability in enabling broad-scale field studies of ATX distribution.

Adaptive evolution of viruses infecting marine microalgae (haptophytes), from acute infections to stable coexistence.

Collectively known as phytoplankton, photosynthetic microbes form the base of the marine food web, and account for up to half of the primary production on Earth. Haptophytes are key components of this phytoplankton community, playing important roles both as primary producers and as mixotrophs that graze on bacteria and protists. Viruses influence the ecology and diversity of phytoplankton in the ocean, with the majority of microalgae-virus interactions described as 'boom and bust' dynamics, which are characteristic of acute virus-host systems. Most haptophytes are, however, part of highly diverse communities and occur at low densities, decreasing their chance of being infected by viruses with high host specificity. Viruses infecting these microalgae have been isolated in the laboratory, and there are several characteristics that distinguish them from acute viruses infecting bloom-forming haptophytes. Herein we synthesise what is known of viruses infecting haptophyte hosts in the ocean, discuss the adaptive evolution of haptophyte-infecting viruses -from those that cause acute infections to those that stably coexist with their host - and identify traits of importance for successful survival in the ocean.

Seasonality and distribution of cyanobacteria and microcystin toxin genes in an oligotrophic lake of Atlantic Canada.

Cyanotoxins are an emerging threat to freshwater resources worldwide. The most frequently reported cyanotoxins are the microcystins, which threaten the health of humans, wildlife, and ecosystems. Determining the potential for microcystin production is hindered by a lack of morphological features that correlate with microcystin production. However, amplicon-based methods permit the detection of microcystin biosynthesis genes and were employed to assess the toxin potential in Lake Utopia, NB, Canada, an oligotrophic lake that occasionally experiences cyanobacteria blooms. Samples collected at 2 week intervals from June 27th to September 27th, 2016, were screened by polymerase chain reaction (PCR) for the microcystin synthetase E gene (mcyE). The mcyE gene was present in some samples every sampling day, despite microcystin not being detected via ELISA, and was most frequently associated with the larger pore size fractions of the serially filtered samples. Further PCR surveys using primer sets to amplify genus-specific (e.g., Microcystis, Anabaena/Dolichospermum, and Planktothrix) mcyE fragments identified Microcystis as the only taxa in Lake Utopia with toxigenic potential. Sequencing of the 16S rRNA V3-V4 region revealed a community dominated by members of the order Synechococcales (from 38 to 96% relative abundance), but with significant presence of taxa from Cyanobacteriales including Microcystaceae and Nostocaceae. A persistent Microcystis population was detected in samples both testing positive and negative for the mcyE gene, highlighting the importance of identifying cyanotoxin production potential by gene presence and not species identity. To our knowledge, this study represents the first application of amplicon-based approaches to studying toxic cyanobacteria in an understudied region-Atlantic Canada.

Coleus blumei viroid 7: a novel viroid resulting from genome recombination between Coleus blumei viroids 1 and 5.

The genus Coleviroid, family Pospiviroidae, comprises six known viroids, all infecting Plectranthus scutellarioides (Coleus blumei; coleus). In 2017, a novel viroid-like RNA sequence that shares ca. 65% identity with Coleus blumei viroid 1 (CbVd-1) was identified in a coleus cultivar infected by multiple coleviroids. Further sequence and secondary structure analyses are consistent with the discovery of a seventh viroid in the genus Coleviroid: tentatively named "Coleus blumei viroid 7" (CbVd-7). The viroid appears to be the product of a natural recombination event between CbVd-1 and Coleus blumei viroid 5. We prove CbVd-7 to be infectious and in turn demonstrate the ability of all known coleviroid left- and right-arm segments to recombine. With a length of 234 nucleotides, this is the smallest viroid described to date.

Strawberry vein banding virus isolates in eastern Canada are molecularly divergent from other isolates.

The complete sequence of a strawberry vein banding virus (SVBV) isolate collected in Nova Scotia, Canada, and designated NS8, was determined. The 7,856-nucleotide circular double-stranded DNA genome contains seven open-reading frames (ORFs), which is consistent with other SVBV isolates and other members of the genus Caulimovirus. Comparison of NS8 with other whole-genome sequences retrieved from databases revealed that NS8 shares the highest sequence similarity (96.5% identity) with isolate China (accession number HE681085) and the lowest (88.3% identity) with clone pSVBV-E3 (accession number X97304). Despite the overall high sequence similarity between NS8 and China, the coat protein encoding ORF IV of NS8 shares only 90.9% sequence identity with the China isolate. Phylogenetic analysis at the complete-genome level placed NS8 and all Chinese isolates in one clade and clone pSVBV-E3 in a separate clade. Interestingly, phylogenetic analysis of all available ORF IV sequences, including those retrieved from databases and newly sequenced samples in this study from Canada, revealed three distinct clades. All Canadian isolates grouped together as one clade, pSVBV-E3 and several others from Europe, Egypt and the USA grouped as a second clade, and isolates from China formed a third clade. These results demonstrate that SVBV is more divergent than previously reported.

Effect of mid-oceanic ballast water exchange on virus-like particle abundance during two trans-Pacific voyages.

Ballast water is a potential source of invasive species, including viruses that target a variety of hosts. We sampled ballast during two trans-Pacific voyages and analyzed the efficacy of mid-oceanic exchange in reducing virus-like particle (VLP) abundance. Exchange did not significantly reduce virus abundance during the first voyage (P=0.874), whereas it reduced viral abundance 3.9-fold from 1.8 × 10(7) to 0.47 × 10(7) VLP mL(-1) during the second voyage (P<0.0001). Despite the impact of exchange during the second voyage, virus abundances were not significantly different between exchanged and unexchanged tanks upon arrival in Canada (P=0.363) and Canadian port water samples (P=0.502). Regressions between environmental parameters and VLP abundance uncovered negative correlations between salinity and viral abundance during one, and dissolved oxygen and viral abundance during the second voyage. In summary, ballast tanks are highly variable with respect to total virus abundance, and the efficacy of exchange requires investigation into the dynamics of specific viruses.

Resource dynamics during infection of Micromonas pusilla by virus MpV-Sp1.

Viruses infecting marine phytoplankton drive phytoplankton diversity, terminate blooms and shuttle genetic material. Assessments of the scale of viral impacts on trophic networks are, however, speculative. We investigated fluxes of DNA between host and virus during infection of the prasinophyte alga Micromonas pusilla by phycodnavirus MpV SP1. Under a light-dark regimen, viral genomes accumulated to a transient peak within 24 h, at the expense of both host DNA synthesis and nuclear DNA. Viral genome abundance then declined soon after host lysis. This release of a phosphate-rich nucleotide pool during viral infection of phytoplankton should be considered in trophic models. Lysis required light and was stalled in darkness, meanwhile viral genome replication proceeded slowly in the dark. Viral exploitation of this host is therefore only partially light-dependent and infected phytoplankton are poised to lyse at dawn or if mixed to the photic zone. The chloroplast genome remained intact until lysis, indicating that either this DNA pool is inaccessible or the virus spares the chloroplast for its energy and reductant generation. The photochemical turnover of residual Photosystem II complexes accelerated during lysis, indicating that events in late infection heighten demands on the remaining host photosynthetic systems, consistent with the light dependency of lysis.

Virus-specific responses of Heterosigma akashiwo to infection.

We used flow cytometry to examine the process of cell death in the bloom-forming alga Heterosigma akashiwo during infection by a double-stranded DNA virus (OIs1) and a single-stranded RNA virus (H. akashiwo RNA virus [HaRNAV]). These viruses were isolated from the same geographic area and infect the same strain of H. akashiwo. By use of the live/dead stains fluorescein diacetate and SYTOX green as indicators of cellular physiology, cells infected with OIs1 showed signs of infection earlier than HaRNAV-infected cultures (6 to 17 h versus 23 to 29 h). Intracellular esterase activity was lost prior to increased membrane permeability during infection with OIs1, while the opposite was seen with HaRNAV-infected cultures. In addition, OIs1-infected cells accumulated in the cultures while HaRNAV-infected cells rapidly disintegrated. Progeny OIs1 viruses consisted of large and small morphotypes with estimated latent periods of 11 and 17 h, respectively, and about 1,100 and 16,000 viruses produced per cell, respectively. In contrast, HaRNAV produced about 21,000 viruses per cell and had a latent period of 29 h. This study reveals that the characteristics of viral infection in algae are virus dependent and therefore are variable among viruses infecting the same species. This is an important consideration for ecosystem modeling exercises; calculations based on in situ measurements of algal physiology must be sensitive to the diverse responses of algae to viral infection.