Recovering sedimentary ancient DNA of harmful dinoflagellates accumulated over the last 9000 years off Eastern Tasmania, Australia.

Harmful algal blooms (HABs) have had significant adverse impacts on the seafood industry along the Tasmanian east coast over the past 4 decades. To investigate the history of regional HABs, we performed analyses of sedimentary ancient DNA (sedaDNA) in coastal sediments up to ~9000 years old collected inshore and offshore of Maria Island, Tasmania. We used metagenomic shotgun sequencing and a hybridisation capture array ("HABbaits1") to target three harmful dinoflagellate genera, Alexandrium, Gymnodinium, and Noctiluca. Bioinformatic and DNA damage analyses verified the authenticity of the sedaDNA sequences. Our results show that dinoflagellates of Alexandrium genera have been present off eastern Tasmania during the last ~8300 years, and we sporadically detected and unambiguously verified sequences of Gymnodinium catenatum that were present offshore up to ~7600 years ago. We also recovered sedaDNA of the fragile, soft-bodied Noctiluca scintillans with increased relative abundance since 2010, consistent with plankton surveys. This study enabled us to identify challenges of sedaDNA sequence validation (in particular for G. catenatum, a microreticulate gymnodinoid species) and provided guidance for the development of tools to monitor past and present HAB species and improvement of future HAB event predictions.

An On-Farm Workflow for Predictive Management of Paralytic Shellfish Toxin-Producing Harmful Algal Blooms for the Aquaculture Industry.

Paralytic shellfish toxins (PSTs) produced by marine dinoflagellates significantly impact shellfish industries worldwide. Early detection on-farm and with minimal training would allow additional time for management decisions to minimize economic losses. Here, we describe and test a standardized workflow based on the detection of sxtA4, an initial gene in the biosynthesis of PSTs. The workflow is simple and inexpensive and does not require a specialized laboratory. It consists of (1) water collection and filtration using a custom gravity sampler, (2) buffer selection for sample preservation and cell lysis for DNA, and (3) an assay based on a region of sxtA, DinoDtec lyophilized quantitative polymerase chain reaction (qPCR) assay. Water samples spiked with Alexandrium catenella showed a cell recovery of >90% when compared to light microscopy counts. The performance of the lysis method (90.3% efficient), Longmire's buffer, and the DinoDtec qPCR assay (tested across a range of Alexandrium species (90.7-106.9% efficiency; r2 > 0.99)) was found to be specific, sensitive, and efficient. We tested the application of this workflow weekly from May 2016 to 30th October 2017 to compare the relationship between sxtA4 copies L-1 in seawater and PSTs in mussel tissue (Mytilus galloprovincialis) on-farm and spatially (across multiple sites), effectively demonstrating an ∼2 week early warning of two A. catenella HABs (r = 0.95). Our tool provides an early, accurate, and efficient method for the identification of PST risk in shellfish aquaculture.

Genomic copy number variability at the genus, species and population levels impacts in situ ecological analyses of dinoflagellates and harmful algal blooms.

The application of meta-barcoding, qPCR, and metagenomics to aquatic eukaryotic microbial communities requires knowledge of genomic copy number variability (CNV). CNV may be particularly relevant to functional genes, impacting dosage and expression, yet little is known of the scale and role of CNV in microbial eukaryotes. Here, we quantify CNV of rRNA and a gene involved in Paralytic Shellfish Toxin (PST) synthesis (sxtA4), in 51 strains of 4 Alexandrium (Dinophyceae) species. Genomes varied up to threefold within species and ~7-fold amongst species, with the largest (A. pacificum, 130 ± 1.3 pg cell-1 /~127 Gbp) in the largest size category of any eukaryote. Genomic copy numbers (GCN) of rRNA varied by 6 orders of magnitude amongst Alexandrium (102- 108 copies cell-1) and were significantly related to genome size. Within the population CNV of rRNA was 2 orders of magnitude (105 - 107 cell-1) in 15 isolates from one population, demonstrating that quantitative data based on rRNA genes needs considerable caution in interpretation, even if validated against locally isolated strains. Despite up to 30 years in laboratory culture, rRNA CNV and genome size variability were not correlated with time in culture. Cell volume was only weakly associated with rRNA GCN (20-22% variance explained across dinoflagellates, 4% in Gonyaulacales). GCN of sxtA4 varied from 0-102 copies cell-1, was significantly related to PSTs (ng cell-1), displaying a gene dosage effect modulating PST production. Our data indicate that in dinoflagellates, a major marine eukaryotic group, low-copy functional genes are more reliable and informative targets for quantification of ecological processes than unstable rRNA genes.

Screening and activity of potential gastrointestinal probiotic lactic acid bacteria against Yersinia ruckeri O1b.

Yersiniosis of cultured Atlantic salmon is a recurrent fish health management challenge in many continents. The causative organism, Yersinia ruckeri, can reside latently in the gut and lead to acute infection and disease during hatchery and sea-transfer stages. One potential prevention approach is the administration of probiotic bacteria to suppress gut colonization of Y. ruckeri. Our study aimed to isolate and identify anti-Yersinia activity among lactic acid bacteria (LAB) isolated from the gastrointestinal tract (GIT) of aquatic animals. Of the 186 aquatic GIT isolates examined, three strains showed diffusible antimicrobial activity towards Y. ruckeri O1b. Analysis of 16 s rRNA gene sequences indicated the three bacterial strains were Enterococci, related to Enterococcus sp. (99%), Enterococcus thailandicus (99%), and Enterococcus durans (99%). Anti-Yersinia activity was maintained at neutral pH (~6.5-7.0), and in-vitro environmental tolerance assays showed the three strains could withstand simulated salmonids gastrointestinal tract conditions of: low pH (3.4) and 3% bile salt content. All three Enterococci strains showed higher adhesion to the intestinal mucus of Atlantic salmon than Y. ruckeri O1b (E. durans 24%, E. enterococcus sp. 25% and E. thailandicus 98%, compared to Y. ruckeri O1b 5%). However, only Enterococcus sp. and E. thailandicus were able to grow in the salmon intestinal mucus broth while E. durans showed no growth. Anti-Yersinia activity was completely inactivated by proteinase-K treatment, suggesting that the active compound/s are proteinaceous and may be bacteriocin-like inhibitory substances (BLIS). Our data indicate that Enterococcus sp. MA176 and E. thailandicus MA122 are potential probionts for the prevention of yersiniosis in salmonids. Further in-vivo studies are required to determine whether these bacteria reduce the incidence of yersiniosis in Atlantic salmon.

Morphological and phylogenetic data do not support the split of Alexandrium into four genera.

A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: "The polyphyly [sic] of Alexandrium is solved with the split into four genera". However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.

An optimized method for the extraction of ancient eukaryote DNA from marine sediments.

Marine sedimentary ancient DNA (sedaDNA) provides a powerful means to reconstruct marine palaeo-communities across the food web. However, currently there are few optimized sedaDNA extraction protocols available to maximize the yield of small DNA fragments typical of ancient DNA (aDNA) across a broad diversity of eukaryotes. We compared seven combinations of sedaDNA extraction treatments and sequencing library preparations using marine sediments collected at a water depth of 104 m off Maria Island, Tasmania, in 2018. These seven methods contrasted frozen versus refrigerated sediment, bead-beating induced cell lysis versus ethylenediaminetetraacetic acid (EDTA) incubation, DNA binding in silica spin columns versus in silica-solution, diluted versus undiluted DNA in shotgun library preparations to test potential inhibition issues during amplification steps, and size-selection of low molecular-weight (LMW) DNA to increase the extraction efficiency of sedaDNA. Maximum efficiency was obtained from frozen sediments subjected to a combination of EDTA incubation and bead-beating, DNA binding in silica-solution, and undiluted DNA in shotgun libraries, across 45 marine eukaryotic taxa. We present an optimized extraction protocol integrating these steps, with an optional post-library LMW size-selection step to retain DNA fragments of ≤500 base pairs. We also describe a stringent bioinformatic filtering approach for metagenomic data and provide a comprehensive list of contaminants as a reference for future sedaDNA studies. The new extraction and data-processing protocol should improve quantitative paleo-monitoring of eukaryotes from marine sediments, as well as other studies relying on the detection of highly fragmented and degraded eukaryote DNA in sediments.

Evaluation of spoilage potential and volatile metabolites production by Shewanella baltica isolated from modified atmosphere packaged live mussels.

Under the current commercial practice, live mussels only have 10days' shelf-life. Observed spoilage indices reduce consumers' acceptance, palatability and shelf-life of modified atmosphere packaged (MAP) live mussels. The aims of this study are to isolate specific spoilage bacteria from modified atmosphere packaged live mussels, evaluate isolates for microbial spoilage indices using qualitative methods and volatile metabolites production. Forty-six hydrogen sulphide producing bacteria were isolated and evaluated for trimethylamine n-oxide (TMAO) reduction, proteolytic and lipolytic activities and hydrogen sulphide production. Twenty-eight isolates were obtained from pouch water and 18 from mussel meat. All the isolates could produce H2S on Iron agar at 25°C while 30/46 produced H2S at 4°C and tolerate 0-6% NaCl. Four (4/46) isolates could not hydrolyse mussel protein. Over 80% isolates reduced TMAO to TMA in 3days with the production of H2S. Results of this study shows hydrogen sulphide producing bacteria isolated from MAP live mussels produce microbial spoilage indices. Isolate with highest enzymatic activities and hydrogen sulphide production was identified as Shewanella baltica using 16S rRNA gene. Axenic culture of the isolate was inoculated into sterile mussel broth. Inoculated sample was further stored at 4°C for 10days for spoilage study. Volatile metabolites produced during storage were evaluated using headspace solid phase micro-extraction gas chromatography mass spectrometry (HS-SPME GC/MS). A total of 44 compounds were identified in the sample after 10days while 27 compounds were identified in inoculated mussel broth. Group of compounds identified are alcohols, aldehydes, phenol, furans, ketone, esters, organic acid, aromatic hydrocarbons, alkanes, nitrogen and sulphur containing compounds. Dimethyl trisulphide, methyl-phenol, 3,5-octadiene and thiohexene were unique to inoculated mussel broth. Understanding spoilage mechanism and attendant spoilage indices will help in designing effective mussel quality protocols and shelf-life extension.

qPCR Assays for the Detection and Quantification of Multiple Paralytic Shellfish Toxin-Producing Species of Alexandrium.

Paralytic shellfish toxin producing dinoflagellates have negatively impacted the shellfish aquaculture industry worldwide, including in Australia and New Zealand. Morphologically identical cryptic species of dinoflagellates that may differ in toxicity, in particular, species of the former Alexandrium tamarense species complex, co-occur in Australia, as they do in multiple regions in Asia and Europe. To understand the dynamics and the ecological drivers of the growth of each species in the field, accurate quantification at the species level is crucial. We have developed the first quantitative polymerase chain reaction (qPCR) primers for A. australiense, and new primers targeting A. ostenfeldii, A. catenella, and A. pacificum. We showed that our new primers for A. pacificum are more specific than previously published primer pairs. These assays can be used to quantify planktonic cells and cysts in the water column and in sediment samples with limits of detection of 2 cells/L for the A. catenella and A. australiense assays, 2 cells/L and 1 cyst/mg sediment for the A. pacificum assay, and 1 cells/L for the A. ostenfeldii assay, and efficiencies of >90%. We utilized these assays to discriminate and quantify co-occurring A. catenella, A. pacificum, and A. australiense in samples from the east coast of Tasmania, Australia.

Historical demography and colonization pathways of the widespread intertidal seaweed Hormosira banksii (Phaeophyceae) in southeastern Australia.

The palaeoceanography of southern Australia has been characterized by fluctuating sea levels during glacial periods, changing temperature regimes and modified boundary currents. Previous studies on genetic structuring of species in southeastern Australia have focused mainly on the differentiation of eastern and western populations while the potential role of Bass Strait as a region of overlap for three biogeographic provinces (Peronia, Maugea, and Flindersia) has been largely ignored. This study aimed to explore the likely roles of historic and contemporary factors in determining divergence patterns in the habitat-forming intertidal seaweed Hormosira banksii in southeastern Australia with a special focus on postglacial dispersal into Bass Strait. We examined the genetic diversity of 475 Hormosira specimens collected from 19 sites around southern Australia using DNA sequence analysis of cytochrome oxidase 1. Three major haplotype groups were identified (western, centre and eastern) corresponding with the three existing biogeographical provinces in this region. Historic break points appeared to be retained and reinforced by modern day dispersal barriers. Phylogeographic grouping of Hormosira reflected a combination of historic and contemporary oceanography. As western and eastern group haplotypes were largely absent within Bass Strait, re-colonization after the last glacial maximum appeared to have originated from refuges within or near present day Bass Strait. Patterns of genetic structure for Hormosira are consistent with other marine species in this region and highlight the importance of biogeographical barriers in contributing to modern genetic structure.

Bacterial Associates Modify Growth Dynamics of the Dinoflagellate Gymnodinium catenatum.

Marine phytoplankton cells grow in close association with a complex microbial associate community known to affect the growth, behavior, and physiology of the algal host. The relative scale and importance these effects compared to other major factors governing algal cell growth remain unclear. Using algal-bacteria co-culture models based on the toxic dinoflagellate Gymnodinium catenatum, we tested the hypothesis that associate bacteria exert an independent effect on host algal cell growth. Batch co-cultures of G. catenatum were grown under identical environmental conditions with simplified bacterial communities composed of one-, two-, or three-bacterial associates. Modification of the associate community membership and complexity induced up to four-fold changes in dinoflagellate growth rate, equivalent to the effect of a 5°C change in temperature or an almost six-fold change in light intensity (20-115 moles photons PAR m-2 s-1). Almost three-fold changes in both stationary phase cell concentration and death rate were also observed. Co-culture with Roseobacter sp. DG874 reduced dinoflagellate exponential growth rate and led to a more rapid death rate compared with mixed associate community controls or co-culture with either Marinobacter sp. DG879, Alcanivorax sp. DG881. In contrast, associate bacteria concentration was positively correlated with dinoflagellate cell concentration during the exponential growth phase, indicating growth was limited by supply of dinoflagellate-derived carbon. Bacterial growth increased rapidly at the onset of declining and stationary phases due to either increasing availability of algal-derived carbon induced by nutrient stress and autolysis, or at mid-log phase in Roseobacter co-cultures potentially due to the onset of bacterial-mediated cell lysis. Co-cultures with the three bacterial associates resulted in dinoflagellate and bacterial growth dynamics very similar to more complex mixed bacterial community controls, suggesting that three-way co-cultures are sufficient to model interaction and growth dynamics of more complex communities. This study demonstrates that algal associate bacteria independently modify the growth of the host cell under non-limiting growth conditions and supports the concept that algal-bacterial interactions are an important structuring mechanism in phytoplankton communities.