Fatty acid production and associated gene pathways are altered by increased salinity and dimethyl sulfoxide treatments during cryopreservation of Symbiodinium pilosum (Symbiodiniaceae).

The Symbiodinium genus is ancestral among other Symbiodiniaceae lineages with species that are both symbiotic and free living. Changes in marine ecosystems threaten their existence and crucial ecological roles. Cryopreservation offers an avenue for their long-term storage for future habitat restoration after coral bleaching. In our previous study we demonstrated that high salinity treatments of Symbiodiniaceae isolates led to changes in their fatty acid (FA) profiles and higher cell viabilities after cryopreservation. In this study, we investigated the role of increased salinity on FA production and the genes involved in FA biosynthesis and degradation pathways during the cryopreservation of Symbiodinium pilosum. Overall, there was a twofold increase in mass of FAs produced by S. pilosum after being cultured in medium with increased salinity (54 parts per thousand; ppt). Dimethyl sulfoxide (Me2SO) led to a ninefold increase of FAs in standard salinity (SS) treatment, compared to a fivefold increase in increased salinity (IS) treatments. The mass of the FA classes returned to baseline during recovery. Transcriptomic analyses showed an acyl carrier protein gene was significantly upregulated after Me2SO treatment in the SS cultures. Cytochrome P450 reductase genes were significantly down regulated after Me2SO addition in SS treatment preventing FA degradation. These changes in the expression of FA biosynthesis and degradation genes contributed to more FAs in SS treated isolates. Understanding how increased salinity changes FA production and the roles of specific genes in regulating FA pathways will help improve current freezing protocols for Symbiodiniaceae and other marine microalgae.

Sedimentary ancient DNA reveals the impact of anthropogenic land use disturbance and ecological shifts on fish community structure in small lowland lake.

Freshwater fish biodiversity and abundance are decreasing globally. The drivers of decline are primarily anthropogenic; however, the causative links between disturbances and fish community change are complex and challenging to investigate. We used a suite of sedimentary DNA methods (droplet digital PCR and metabarcoding) and traditional paleolimnological approaches, including pollen and trace metal analysis, ITRAX X-ray fluorescence and hyperspectral core scanning to explore changes in fish abundance and drivers over 1390 years in a small lake. This period captured a disturbance trajectory from pre-human settlement through subsistence living to intensive agriculture. Generalized additive mixed models explored the relationships between catchment inputs, internal drivers, and fish community structure. Fish community composition distinctly shifted around 1350 CE, with the decline of a sensitive Galaxias species concomitant with early land use changes. Total fish abundance significantly declined around 1950 CE related to increases in ruminant bacterial DNA (a proxy for ruminant abundance) and cadmium flux (a proxy for phosphate fertilizers), implicating land use intensification as a key driver. Concurrent shifts in phytoplankton and zooplankton suggested that fish communities were likely impacted by food web dynamics. This study highlights the potential of sedDNA to elucidate the long-term disturbance impacts on biological communities in lakes.

A paleoecological investigation of recent cyanobacterial blooms and their drivers in two contrasting lakes.

Cyanobacterial blooms are one of the most significant threats to global water security and freshwater biodiversity. Interactions among multiple stressors, including habitat degradation, species invasions, increased nutrient runoff, and climate change, are key drivers. However, assessing the role of anthropogenic activity on the onset of cyanobacterial blooms and exploring response variation amongst lakes of varying size and depth is usually limited by lack of historical records. In the present study we applied molecular, paleolimnological (trace metal, Itrax-µ-XRF and hyperspectral scanning, chronology), paleobotanical (pollen) and historical data to reconstruct cyanobacterial abundance and community composition and anthropogenic impacts in two dune lakes over a period of up to 1200 years. Metabarcoding and droplet digital PCR results showed very low levels of picocyanobacteria present in the lakes prior to about CE 1854 (1839-1870 CE) in the smaller shallow Lake Alice and CE 1970 (1963-1875 CE) in the larger deeper Lake Wiritoa. Hereafter bloom-forming cyanobacteria were detected and increased notably in abundance post CE 1984 (1982-1985 CE) in Lake Alice and CE 1997 (1990-2007 CE) in Lake Wiritoa. Currently, the magnitude of blooms is more pronounced in Lake Wiritoa, potentially attributable to hypoxia-induced release of phosphorus from sediment, introducing an additional source of nutrients. Generalized linear modelling was used to investigate the contribution of nutrients (proxy = bacterial functions), temperature, redox conditions (Mn:Fe), and erosion (Ti:Inc) in driving the abundance of cyanobacteria (ddPCR). In Lake Alice nutrients and erosion had a statistically significant effect, while in Lake Wiritoa nutrients and redox conditions were significant.

An integrative approach to assess the impact of disturbance on native fish in lakes.

Freshwater fish are in a perilous state with more than 30% of species considered critically endangered. Yet significant ecological and methodological complexities constrain our ability to determine how disturbances are impacting native fish communities. We review current methods used to assess the responses of fish communities, especially native fish, to disturbances, with a focus on lakes. These methods include contemporary population surveys, manipulative experimental approaches, paleolimnological approaches and Indigenous Knowledge and social histories. We identify knowledge gaps, such as a lack of baseline data for native fish, an inability to assess the impact of historical disturbances, stressor response dynamics in contemporary multi-stressor environments, and natural disturbance regimes. Our assessment of the current methods highlights challenges to filling these knowledge gaps using the reviewed methods. We advocate strongly for the implementation of an integrative approach that combines emerging technologies (i.e. molecular-based techniques in contemporary surveys and paleolimnology) and underutilised knowledge streams (i.e. Indigenous Knowledge and social histories) which should be used in concert with conventional methods. This integrative approach will allow researchers to determine the key drivers of decline and the degree of change, which will enable more informed and successful management actions.

A taxonomy-free diatom eDNA-based technique for assessing lake trophic level using lake sediments.

Anthropogenic eutrophication is one of the most pressing issues facing lakes globally. Our ability to manage lake eutrophication is hampered by the limited spatial and temporal extents of monitoring records, stemming from the time-consuming and expensive nature of physiochemical and biological monitoring. Diatom-based biomonitoring presents an alternative to traditional eutrophication monitoring, yet it is restricted by the high degree of taxonomic expertise required. Environmental DNA metabarcoding, while providing a promising substitute for diatom community enumeration, is plagued by inadequate taxonomic coverage of reference databases and methodological bias, limiting its use for biomonitoring. Here we show that taxonomy-free diatom-biomonitoring, in which environmental DNA metabarcoding data is utilised but not assigned to specific taxonomic classes, presents an accurate, fast, and relatively automated alternative to taxonomically assigned eutrophication biomonitoring. Our taxonomy-free index accounted for 85% of trophic level variability across 89 lakes and had the lowest average prediction error of the three approaches tested. By not relying on taxonomic identification or metabarcoding reference databases, taxonomy-free biomonitoring maintains diatom diversity that is lost in taxonomic assignment using molecular approaches. Furthermore, by utilising lake sediments, the approach outlined here presents a time-integrated estimation of lake trophic level and thus does not require time-consuming seasonal sampling. Taxonomy-free biomonitoring addresses the limitations of traditional physicochemical eutrophication monitoring and taxonomic biomonitoring alternatives and can be used to extend the spatial and temporal extents of eutrophication monitoring.

Bioavailable iron concentrations regulate phytoplankton growth and bloom formation in low-nutrient lakes.

The growth of phytoplankton in lakes is thought to be primarily controlled by macronutrient concentrations, but the availability of trace metal micronutrients, such as iron (Fe), are increasingly recognised as important regulators of lake primary production. This study evaluates the role of Fe in regulating phytoplankton growth in lakes of different nutrient status in New Zealand. The results of this unique year-long study, combining highly sensitive trace metal concentration analysis of waters and particulates with advanced trace metal bioavailability and speciation modelling, constrains thresholds for bioavailable Fe and colloidal Fe of 0.8 nmol·L-1 and 30 nmol·L-1, respectively, below which phytoplankton growth-limitation occurs. These thresholds specifically control diatom bloom formation and termination in lakes, thereby exerting a strong influence on freshwater carbon sequestration, given the dominance of diatoms in lake bloom assemblages. Importantly, potentially toxic cyanobacteria thrived only after events of bottom water anoxia, when additional dissolved Fe in concentrations ≥4 nmol·L-1 was released into the water column. These new thresholds for bioavailable and colloidal Fe offer the potential to manage micronutrient levels in lakes for the purpose of regulating algal bloom formation and carbon sequestration, while at the same time, suppressing the formation of harmful cyanobacterial blooms.

Characterizing carotenoids in cyanobacterial cultures - Opportunities and implications for paleolimnological studies.

Cyanobacterial blooms are increasing in frequency and intensity globally, impacting lake ecosystem health and posing a risk to human and animal health due to the toxins they can produce. Cyanobacterial pigments preserved in lake sediments provide a useful means of understanding the changes that have led to cyanobacterial blooms in lakes. However, there is some uncertainty as to whether specific carotenoids are unique to certain genera or types of cyanobacteria. To fill this knowledge gap, we analyzed pigments in 34 cyanobacteria cultures and applied the findings to sediments from three New Zealand lakes. The cyanobacterial carotenoids canthaxanthin, echinenone and zeaxanthin were detected in all cultures, whereas myxoxanthophyll was only detected in ten cultures (Microcoleus, Planktothrix and the picocyanobacteria cultures; Synechococcaceae). The sum of the individual carotenoid concentrations provided the strongest relationship with cyanobacterial biomass (R2 = 0.58) and could be used in paleolimnology studies to evaluate general cyanobacterial abundance. Ratios of canthaxanthin, zeaxanthin and myxoxanthophyll relative to echinenone indicated that carotenoid ratios could be used to differentiate picocyanobacteria and bloom-forming cyanobacteria, to some degree. High zeaxanthin/echinenone ratios were measured in picocyanobacteria and low zeaxanthin/echinenone ratios were measured in bloom-forming cyanobacteria. The zeaxanthin/echinenone ratio was applied to sediment core samples where the cyanobacterial community was also evaluated by 16S rRNA gene metabarcoding, with the zeaxanthin/echinenone ratios showing similar patterns to those observed in the cultures. The preliminary assessment described here suggests that zeaxanthin/echinenone ratios could provide a valuable paleoecological proxy for evaluating historical shifts in cyanobacterial communities and warrants further exploration.

CRISPR-Cas-Based Biomonitoring for Marine Environments: Toward CRISPR RNA Design Optimization Via Deep Learning.

Almost all of Earth's oceans are now impacted by multiple anthropogenic stressors, including the spread of nonindigenous species, harmful algal blooms, and pathogens. Early detection is critical to manage these stressors effectively and to protect marine systems and the ecosystem services they provide. Molecular tools have emerged as a promising solution for marine biomonitoring. One of the latest advancements involves utilizing CRISPR-Cas technology to build programmable, rapid, ultrasensitive, and specific diagnostics. CRISPR-based diagnostics (CRISPR-Dx) has the potential to allow robust, reliable, and cost-effective biomonitoring in near real time. However, several challenges must be overcome before CRISPR-Dx can be established as a mainstream tool for marine biomonitoring. A critical unmet challenge is the need to design, optimize, and experimentally validate CRISPR-Dx assays. Artificial intelligence has recently been presented as a potential approach to tackle this challenge. This perspective synthesizes recent advances in CRISPR-Dx and machine learning modeling approaches, showcasing CRISPR-Dx potential to progress as a rising molecular tool candidate for marine biomonitoring applications.

Optimal sample type and number vary in small shallow lakes when targeting non-native fish environmental DNA.

Non-native fish have been shown to have deleterious impacts on freshwater ecosystems in New Zealand. Early detection is critical for their effective management. Traditional capture-based techniques may not detect newly introduced fish, especially if they are present in low abundance. Molecular techniques that target environmental DNA (eDNA) have been shown, in many instances, to be more sensitive, cost-effective and require lower sampling effort. However, appropriate sampling strategies are needed to ensure robust and interpretable data are obtained. In this study we used droplet digital PCR assays to investigate the presence of two non-native fish in New Zealand, the European perch (Perca fluviatilis) and rudd (Scardinius erythrophthalmus) in three small lakes. Samples were collected from water and surface sediment at near-shore and mid-lake sites. Probabilistic modelling was used to assess the occupancy of fish eDNA and develop guidance on sampling strategies. Based on the detection probability measures from the present study, at least six sites and five replicates per site are needed to reliably detect fish eDNA in sediment samples, and twelve sites with eight replicates per site for water samples. The results highlight the potential of developing monitoring and surveillance programs adapted to lakes, that include the use of assays targeting eDNA. This study focused on small shallow lakes, and it is likely that these recommendations may vary in larger, deeper, and more geomorphologically complex lakes, and this requires further research.

Growth conditions impact particulate absorption and pigment concentrations in two common bloom forming cyanobacterial species.

Remote sensing using satellite imagery has been promoted as a method to broaden the scale and frequency of cyanobacterial monitoring. This relies on the ability to establish relationships between the reflectance spectra of water bodies and the abundance of cyanobacteria. A challenge to achieving this comes from a limited understanding of the extent to which the optical properties of cyanobacteria vary according to their physiological state and growth environment. The aim of the present study was to determine how growth stage, nutrient status and irradiance affect pigment concentrations and absorption spectra in two common bloom forming cyanobacterial taxa: Dolichospermum lemmermannii and Microcystis aeruginosa. Each species was grown in laboratory batch culture under a full factorial design of low or high light intensity and low, medium, or high nitrate concentrations. Absorption spectra, pigment concentrations and cell density were measured throughout the growth phases. The absorption spectra were all highly distinguishable from each other, with greater interspecific than intraspecific differences, indicating that both D. lemmermannii and M. aeruginosa can be readily differentiated using hyperspectral absorption spectra. Despite this, each species exhibited different responses in the per-cell pigment concentrations with varying light intensity and nitrate exposure. Variability among treatments was considerably higher in D. lemmermannii than in M. aeruginosa, which exhibited smaller changes in pigment concentrations among the treatments. These results highlight the need to understand the physiology of the cyanobacteria and to take caution when estimating biovolumes from reflectance spectra when species composition and growth stage are unknown.

Assessment of the recovery and photosynthetic efficiency of Breviolum psygmophilum and Effrenium voratum (Symbiodiniaceae) following cryopreservation.

Many strains of Symbiodiniaceae have been isolated and their genetics, taxonomy, and metabolite production studied. Maintaining these cultures requires careful and regular sub-culturing that is costly with a high risk of species contamination or loss. Cryopreservation is a viable alternative for their long-term storage; however, there is uncertainty as to whether cryopreservation impacts the photosynthetic performance of Symbiodiniaceae. We investigated the growth rates and photosynthetic efficiency of two species, Breviolum psygmophilum and Effrenium voratum before and after cryopreservation. Rapid light curves (RLCs) produced using Pulse Amplitude Modulated (PAM) fluorometry were used to generate detailed information on the characteristics of photosystem II (PSII). The maximum electron transport rate (ETRmax) and the quantum yield (Fv/Fm) of the control (non-cryopreserved) and cryopreserved culture isolates were assessed across the growth cycle. The non-cryopreserved isolate of B. psygmophilum had a higher quantum yield than the cryopreserved isolate from day 12 to day 24, whereas there were no differences from day 28 to the late stationary phase. There were no significant differences in ETRmax. No significant differences were observed in quantum yield or ETRmax between the control and cryopreserved E. voratum isolates. The ability of cryopreserved strains to recover and regain their photosynthetic efficiency after freezing demonstrates the utility of this method for the long-term storage of these and other Symbiodiniaceae species.

Paleo-diatom records reveal ecological change not detected using traditional measures of lake eutrophication.

Lakes provide crucial ecosystem services and harbour unique and rich biodiversity, yet despite decades of research and management focus, cultural eutrophication remains a predominant threat to their health. Our ability to manage lake eutrophication is restricted by the lack of long-term monitoring records. To circumvent this, we developed a bio-indicator approach for inferring trophic level from lake diatom communities and applied this to sediment cores from two lakes experiencing eutrophication stress. Diatom indicators strongly predicted observed trophic levels, and when applied to sediment cores, diatom predicted trophic level reconstructions were consistent with monitoring data and land-use histories. However, there were significant recent shifts in diatom communities not captured by the diatom-based index or monitoring data, suggesting that conventional trophic level indices obscure important ecological change. New approaches, such as the one in this study, are critical to detect early changes in water quality and prevent the decline of lake ecosystems worldwide.

Does plastic type matter? Insights into non-indigenous marine larvae recruitment under controlled conditions.

Marine plastic debris (MPD) are a global threat to marine ecosystems. Among countless ecosystem impacts, MPD can serve as a vector for marine 'hitchhikers' by facilitating transport and subsequent spread of unwanted pests and pathogens. The transport and spread of these non-indigenous species (NIS) can have substantial impacts on native biodiversity, ecosystem services/functions and hence, important economic consequences. Over the past decade, increasing research interest has been directed towards the characterization of biological communities colonizing plastic debris, the so called Plastisphere. Despite remarkable advances in this field, little is known regarding the recruitment patterns of NIS larvae and propagules on MPD, and the factors influencing these patterns. To address this knowledge gap, we used custom-made bioassay chambers and ran four consecutive bioassays to compare the settlement patterns of four distinct model biofouling organisms' larvae, including the three notorious invaders Crassostrea gigas, Ciona savignyi and Mytilus galloprovincialis, along with one sessile macro-invertebrate Spirobranchus cariniferus, on three different types of polymers, namely Low-Linear Density Polyethylene (LLDPE), Polylactic Acid (PLA), Nylon-6, and a glass control. Control bioassay chambers were included to investigate the microbial community composition colonizing the different substrates using 16S rRNA metabarcoding. We observed species-specific settlement patterns, with larvae aggregating on different locations on the substrates. Furthermore, our results revealed that C. savignyi and S. cariniferus generally favoured Nylon and PLA, whereas no specific preferences were observed for C. gigas and M. galloprovincialis. We did not detect significant differences in bacterial community composition between the tested substrates. Taken together, our results highlight the complexity of interactions between NIS larvae and plastic polymers. We conclude that several factors and their potential interactions influenced the results of this investigation, including: (i) species-specific larval biological traits and ecology; (ii) physical and chemical composition of the substrates; and (iii) biological cues emitted by bacterial biofilm and the level of chemosensitivity of the different NIS larvae. To mitigate the biosecurity risks associated with drifting plastic debris, additional research effort is critical to effectively decipher the mechanisms involved in the recruitment of NIS on MPD.

Resolving 500 Years of Anthropogenic Impacts in a Mesotrophic Lake: Nutrients Outweigh Other Drivers of Lake Change.

Interactions among multiple stressors, legacies of past perturbations, and the lack of historical information make it difficult to determine the influence of individual anthropogenic impacts on lakes and separate them from natural ecosystem variability. In the present study, we coupled paleolimnological approaches, historical data, and ecological experiments to disentangle the impacts of multiple long-term stressors on lake ecosystem structure and function. We found that the lake structure and function remained resistant to the impacts of catchment deforestation and erosion, and the introduction of several exotic fish species. Changes in ecosystem structure and function were consistent, with nutrient enrichment being the primary driver of change. Significant and sustained changes in the lake diatom community structure (and their nutrient requirements), bacterial community function, and paleolimnological proxies of ecosystem function coincided with nitrogen and phosphorus fertilizers in the catchment. The results highlight that the effects of increased nutrient inputs are much stronger than the influence of other, potentially significant, drivers of ecosystem change, and that the degree of nutrient impact can be underestimated by environmental monitoring due to its diffuse and accumulative nature. Delineating the effects of multiple anthropogenic drivers requires long-term records of both impacts and lake ecosystem change across multiple trophic levels.

Human access and deterministic processes play a major role in structuring planktonic and sedimentary bacterial and eukaryotic communities in lakes.

Lakes provide habitat for a diverse array of species and offer a wide range of ecosystem services for humanity. However, they are highly vulnerable as they are not only impacted by adverse actions directly affecting them, but also those on the surrounding environment. Improving knowledge on the processes responsible for community assembly in different biotic components will aid in the protection and restoration of lakes. Studies to date suggested a combination of deterministic (where biotic/abiotic factors act on fitness differences amongst taxa) and stochastic (where dispersal plays a larger factor in community assembly) processes are responsible for structuring biotic communities, but there is no consensus on the relative roles these processes play, and data is lacking for lakes. In the present study, we sampled different biotic components in 34 lakes located on the South Island of New Zealand. To obtain a holistic view of assembly processes in lakes we used metabarcoding to investigate bacteria in the sediment and surface waters, and eukaryotes in the sediment and two different size fractions of the water column. Physicochemical parameters were collected in parallel. Results showed that deterministic processes dominated the assembly of lake communities although the relative importance of variable and homogeneous selection differed among the biotic components. Variable selection was more important in the sediment (SSbact and SSeuks) and for the bacterioplankton (Pbact) while the assembly of the eukaryotic plankton (SPeuks, LPeuks) was driven more by homogeneous selection. The ease of human access to the lakes had a significant effect on lake communities. In particular, clade III of SAR11 and Daphnia pulex were only present in lakes with public access. This study provides insights into the distribution patterns of different biotic components and highlights the value in understanding the drivers of different biological communities within lakes.

Cryopreservation of six Symbiodiniaceae genera and assessment of fatty acid profiles in response to increased salinity treatments.

Symbiodiniaceae are a diverse group of dinoflagellates, the majority of which are free-living and/or associated with a variety of protists and other invertebrate hosts. Maintenance of isolated cultures is labour-intensive and expensive, and cryopreservation provides an excellent avenue for their long-term storage. We aimed to cryopreserve 15 cultured isolates from six Symbiodiniaceae genera using dimethyl sulfoxide (DMSO) as the cryoprotectant agent (CPA). Under 15% DMSO, 10 isolates were successfully cryopreserved using either rapid freezing or controlled-rate freezing. Cultures that failed or had low survival, were subjected to (1) a reduction of CPA to 10%, or (2) increased salinity treatment before freezing. At 10% DMSO, three further isolates were successfully cryopreserved. At 15% DMSO there were high cell viabilities in Symbiodinium pilosum treated with 44 parts per thousand (ppt) and 54 ppt culture medium. An isolate of Fugacium sp. successfully cryopreserved after salinity treatments of 54 ppt and 64 ppt. Fatty acid (FA) analyses of S. pilosum after 54 ppt salinity treatment showed increased saturated FA levels, whereas Fugacium sp. had low poly-unsaturated FAs compared to normal salinity (34 ppt). Understanding the effects of salinity and roles of FAs in cryopreservation will help in developing protocols for these ecologically important taxa.

Using metabarcoding and droplet digital PCR to investigate drivers of historical shifts in cyanobacteria from six contrasting lakes.

The frequency and intensity of cyanobacterial blooms is increasing worldwide. Multiple factors are implicated, most of which are anthropogenic. New Zealand provides a useful location to study the impacts of human settlement on lake ecosystems. The first humans (Polynesians) arrived about 750 years ago. Following their settlement, there were marked landscape modifications which intensified after European settlement about 150 years ago. The aims of this study were to reconstruct cyanobacterial communities in six lakes over the last 1000 years and explore key drivers of change. Cyanobacterial environmental DNA was extracted from sediment cores and analysed using metabarcoding and droplet digital PCR. Cyanobacteria, including potentially toxic or bloom forming species, were already present in these lakes prior to human arrival, however their overall abundance was low. Total cyanobacteria abundance and richness increased in all lakes after European settlement but was very pronounced in four lakes, where bloom-forming taxa became dominant. These shifts occurred concomitant with land-use change. The catchment of one deteriorated lake is only moderately modified, thus the introduction of non-native fish is posited as the key factor driving this change. The paleolimnological approach used in this study has enabled new insights into timing and potential causes of changes in cyanobacterial communities.

Schindler's legacy: from eutrophic lakes to the phosphorus utilization strategies of cyanobacteria.

David Schindler and his colleagues pioneered studies in the 1970s on the role of phosphorus in stimulating cyanobacterial blooms in North American lakes. Our understanding of the nuances of phosphorus utilization by cyanobacteria has evolved since that time. We review the phosphorus utilization strategies used by cyanobacteria, such as use of organic forms, alternation between passive and active uptake, and luxury storage. While many aspects of physiological responses to phosphorus of cyanobacteria have been measured, our understanding of the critical processes that drive species diversity, adaptation and competition remains limited. We identify persistent critical knowledge gaps, particularly on the adaptation of cyanobacteria to low nutrient concentrations. We propose that traditional discipline-specific studies be adapted and expanded to encompass innovative new methodologies and take advantage of interdisciplinary opportunities among physiologists, molecular biologists, and modellers, to advance our understanding and prediction of toxic cyanobacteria, and ultimately to mitigate the occurrence of blooms.

Molecular and Pigment Analyses Provide Comparative Results When Reconstructing Historic Cyanobacterial Abundances from Lake Sediment Cores.

Understanding the historical onset of cyanobacterial blooms in freshwater bodies can help identify their potential drivers. Lake sediments are historical archives, containing information on what has occurred in and around lakes over time. Paleolimnology explores these records using a variety of techniques, but choosing the most appropriate method can be challenging. We compared results obtained from a droplet digital PCR assay targeting a cyanobacterial-specific region of the 16S rRNA gene in sedimentary DNA and cyanobacterial pigments (canthaxanthin, echinenone, myxoxanthophyll and zeaxanthin) analysed using high-performance liquid chromatography in four sediment cores. There were strong positive relationships between the 16S rRNA gene copy concentrations and individual pigment concentrations, but relationships differed among lakes and sediment core depths within lakes. The relationships were more consistent when all pigments were summed, which we attribute to different cyanobacteria species, in different lakes, at different times producing different suites of pigments. Each method had benefits and limitations, which should be taken into consideration during method selection and when interpreting paleolimnological data. We recommend this biphasic approach when making inferences about changes in the entire cyanobacterial community because they yielded complementary information. Our results support the view that molecular methods can yield results similar to traditional paleolimnological proxies when caveats are adequately addressed.

Cryoprotectant treatment tests on three morphologically diverse marine dinoflagellates and the cryopreservation of Breviolum sp. (Symbiodiniaceae).

Dinoflagellates are among the most diverse group of microalgae. Many dinoflagellate species have been isolated and cultured, and these are used for scientific, industrial, pharmaceutical, and agricultural applications. Maintaining cultures is time-consuming, expensive, and there is a risk of contamination or genetic drift. Cryopreservation offers an efficient means for their long-term preservation. Cryopreservation of larger dinoflagellate species is challenging and to date there has been only limited success. In this study, we explored the effect of cryoprotectant agents (CPAs) and freezing methods on three species: Vulcanodinium rugosum, Alexandrium pacificum and Breviolum sp. A total of 12 CPAs were assessed at concentrations between 5 and 15%, as well as in combination with dimethyl sulfoxide (DMSO) and other non-penetrating CPAs. Two freezing techniques were employed: rapid freezing and controlled-rate freezing. Breviolum sp. was successfully cryopreserved using 15% DMSO. Despite exploring different CPAs and optimizing the freezing techniques, we were unable to successfully cryopreserve V. rugosum and A. pacificum. For Breviolum sp. there was higher cell viability (45.4 ± 2.2%) when using the controlled-rate freezing compared to the rapid freezing technique (10.0 ± 2.8%). This optimized cryopreservation protocol will be of benefit for the cryopreservation of other species from the family Symbiodiniaceae.