A comparison of two gene regions for assessing community composition of eukaryotic marine microalgae from coastal ecosystems.

Two gene regions commonly used to characterise the diversity of eukaryotic communities using metabarcoding are the 18S ribosomal DNA V4 and V9 gene regions. We assessed the effectiveness of these two regions for characterising diverisity of coastal eukaryotic microalgae communities (EMCs) from tropical and temperate sites. We binned amplicon sequence variants (ASVs) into the high level taxonomic groups: dinoflagellates, pennate diatoms, radial centric diatoms, polar centric diatoms, chlorophytes, haptophytes and 'other microalgae'. When V4 and V9 generated ASV abundances were compared, the V9 region generated a higher number of raw reads, captured more diversity from all high level taxonomic groups and was more closely aligned with the community composition determined using light microscopy. The V4 region did resolve more ASVs to a deeper taxonomic resolution within the dinoflagellates, but did not effectively resolve other major taxonomic divisions. When characterising these communities via metabarcoding, the use of multiple gene regions is recommended, but the V9 gene region can be used in isolation to provide high-level community biodiversity to reflect relative abundances within groups. This approach reduces the cost of sequencing multiple gene regions whilst still providing important baseline ecosystem function information.

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.

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.

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.

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.

Production of Betacyanins in Transgenic Nicotiana tabacum Increases Tolerance to Salinity.

Although red betalain pigments (betacyanins) have been associated with salinity tolerance in some halophytes like Disphyma australe, efforts to determine whether they have a causal role and the underlying mechanisms have been hampered by a lack of a model system. To address this, we engineered betalain-producing Nicotiana tabacum, by the introduction of three betalain biosynthetic genes. The plants were violet-red due to the accumulation of three betacyanins: betanin, isobetanin, and betanidin. Under salt stress, betacyanic seedlings had increased survivability and leaves of mature plants had higher photochemical quantum yields of photosystem II (F v /F m ) and faster photosynthetic recovery after saturating light treatment. Under salt stress, compared to controls betacyanic leaf disks had no loss of carotenoids, a slower rate of chlorophyll degradation, and higher F v /F m values. Furthermore, simulation of betacyanin pigmentation by using a red filter cover improved F v /F m value of green tissue under salt stress. Our results confirm a direct causal role of betacyanins in plant salinity tolerance and indicate a key mechanism is photoprotection. A role in delaying leaf senescence was also indicated, and the enhanced antioxidant capability of the betacyanic leaves suggested a potential contribution to scavenging reactive oxygen species. The study can inform the development of novel biotechnological approaches to improving agricultural productivity in saline-affected areas.

Rapid Manipulation in Irradiance Induces Oxidative Free-Radical Release in a Fast-Ice Algal Community (McMurdo Sound, Antarctica).

Sea ice supports a unique assemblage of microorganisms that underpin Antarctic coastal food-webs, but reduced ice thickness coupled with increased snow cover will modify energy flow and could lead to photodamage in ice-associated microalgae. In this study, microsensors were used to examine the influence of rapid shifts in irradiance on extracellular oxidative free radicals produced by sea-ice algae. Bottom-ice algal communities were exposed to one of three levels of incident light for 10 days: low (0.5 µmol photons m-2 s-1, 30 cm snow cover), mid-range (5 µmol photons m-2 s-1, 10 cm snow), or high light (13 µmol photons m-2 s-1, no snow). After 10 days, the snow cover was reversed (either removed or added), resulting in a rapid change in irradiance at the ice-water interface. In treatments acclimated to low light, the subsequent exposure to high irradiance resulted in a ~400× increase in the production of hydrogen peroxide (H2O2) and a 10× increase in nitric oxide (NO) concentration after 24 h. The observed increase in oxidative free radicals also resulted in significant changes in photosynthetic electron flow, RNA-oxidative damage, and community structural dynamics. In contrast, there was no significant response in sea-ice algae acclimated to high light and then exposed to a significantly lower irradiance at either 24 or 72 h. Our results demonstrate that microsensors can be used to track real-time in-situ stress in sea-ice microbial communities. Extrapolating to ecologically relevant spatiotemporal scales remains a significant challenge, but this approach offers a fundamentally enhanced level of resolution for quantifying the microbial response to global change.

Mapping the in situ microspatial distribution of ice algal biomass through hyperspectral imaging of sea-ice cores.

Ice-associated microalgae make a significant seasonal contribution to primary production and biogeochemical cycling in polar regions. However, the distribution of algal cells is driven by strong physicochemical gradients which lead to a degree of microspatial variability in the microbial biomass that is significant, but difficult to quantify. We address this methodological gap by employing a field-deployable hyperspectral scanning and photogrammetric approach to study sea-ice cores. The optical set-up facilitated unsupervised mapping of the vertical and horizontal distribution of phototrophic biomass in sea-ice cores at mm-scale resolution (using chlorophyll a [Chl a] as proxy), and enabled the development of novel spectral indices to be tested against extracted Chl a (R2 ≤ 0.84). The modelled bio-optical relationships were applied to hyperspectral imagery captured both in situ (using an under-ice sliding platform) and ex situ (on the extracted cores) to quantitatively map Chl a in mg m-2 at high-resolution (≤ 2.4 mm). The optical quantification of Chl a on a per-pixel basis represents a step-change in characterising microspatial variation in the distribution of ice-associated algae. This study highlights the need to increase the resolution at which we monitor under-ice biophysical systems, and the emerging capability of hyperspectral imaging technologies to deliver on this research goal.

Community assembly in a modular organism: the impact of environmental filtering on bryozoan colony form and polymorphism.

Understanding community assembly is a key goal in community ecology. Environmental filtering influences community assembly by excluding ill-adapted species, resulting in communities with similar functional traits. An RLQ (a four-way ordination) analysis incorporating spatial data was run on a data set of 642 species of cheilostomes (Bryozoa) from 779 New Zealand sites, and results were compared to trends in other sessile, epibenthic taxa. This revealed environmental filtering of colony form: encrusting-cemented taxa were predominant in shallow environments with hard substrata (<200 m), while erect-rooted taxa characterized deeper environments with soft substrata (>200 m). Furthermore, erect taxa found in shallow environments with high current speeds were typically jointed. Polymorphism also followed environmental gradients. External ovicells (brood chambers) were more common in deeper, low-oxygen water than immersed and internal ovicells. This may reflect the oxygen needs of the embryo or increased predation intensity in shallow environments. Bryozoans with costae tended to be found in deeper water as well, while bryozoans with calcified frontal shields were found in shallow environments with a higher concentration of CaCO3 . Avicularia did not appear to be related to environmental conditions, and changes in pivot bar structure with depth likely represent a phylogenetic signal. The importance of substratum type as a strict environmental filter suggests that anchoring structures, like rootlets, may be "key innovations" for other sessile, epibenthic taxa like sponges and ascidians.

Multiple cyanotoxin congeners produced by sub-dominant cyanobacterial taxa in riverine cyanobacterial and algal mats.

Benthic cyanobacterial proliferations in rivers are have been reported with increasing frequency worldwide. In the Eel and Russian rivers of California, more than a dozen dog deaths have been attributed to cyanotoxin toxicosis since 2000. Periphyton proliferations in these rivers comprise multiple cyanobacterial taxa capable of cyanotoxin production, hence there is uncertainty regarding which taxa are producing toxins. In this study, periphyton samples dominated by the cyanobacterial genera Anabaena spp. and Microcoleus spp. and the green alga Cladophora glomerata were collected from four sites in the Eel River catchment and one site in the Russian River. Samples were analysed for potential cyanotoxin producers using polymerase chain reaction (PCR) in concert with Sanger sequencing. Cyanotoxin concentrations were measured using liquid chromatography tandem-mass spectrometry, and anatoxin quota (the amount of cyanobacterial anatoxins per toxigenic cell) determined using droplet digital PCR. Sequencing indicated Microcoleus sp. and Nodularia sp. were the putative producers of cyanobacterial anatoxins and nodularins, respectively, regardless of the dominant taxa in the mat. Anatoxin concentrations in the mat samples varied from 0.1 to 18.6 µg g-1 and were significantly different among sites (p < 0.01, Wilcoxon test); however, anatoxin quotas were less variable (< 5-fold). Dihydroanatoxin-a was generally the most abundant variant in samples comprising 38% to 71% of the total anatoxins measured. Mats dominated by the green alga C. glomerata contained both anatoxins and nodularin-R at concentrations similar to those of cyanobacteria-dominated mats. This highlights that even when cyanobacteria are not the dominant taxa in periphyton, these mats may still pose a serious health risk and indicates that more widespread monitoring of all mats in a river are necessary.

Differential strain response in alkaline phosphatase activity to available phosphorus in Microcoleus autumnalis.

Toxic, benthic cyanobacterial proliferations have increased in frequency and severity globally and can have negative impacts on aquatic ecosystems, recreation and human health. Microcoleus autumnalis has been associated with numerous animal fatalities and is causing increasing concern. It tends to grow in systems with moderate dissolved inorganic nitrogen and very low dissolved reactive phosphorus. Acquisition of nutrients, particularly phosphorus, from organic sources may explain how M. autumnalis can reach the high biomass in these relatively nutrient deplete environments. In the present study the effect of phosphorus concentration and source on alkaline phosphatase activity was investigated in toxic and non-toxic M. autumnalis strains. Toxic strains exhibited significantly higher alkaline phosphatase activity than non-toxic strains (p < 0.05), and alkaline phosphatase activity increased in all strains under phosphorus-depleted conditions (p < 0.05). Alkaline phosphatase activity was also present in environmental M. autumnalis mats, though at lower levels than in laboratory experiments. The presence of alkaline phosphatase activity indicates that the acquisition of phosphorus from organic phosphorus sources may contribute to the ability of M. autumnalis to grow in systems with low dissolved reactive phosphorus.

Modularity is the mother of invention: a review of polymorphism in bryozoans.

Modularity is a fundamental concept in biology. Most taxa within the colonial invertebrate phylum Bryozoa have achieved division of labour through the development of specialized modules (polymorphs), and this group is perhaps the most outstanding exemplar of the phenomenon. We provide a comprehensive description of the diversity, morphology and function of these polymorphs and the significance of modularity to the evolutionary success of the phylum, which has >21000 described fossil and living species. Modular diversity likely arose from heterogeneous microenvironmental conditions, and cormidia (repeated clusters of associated modules) are an emergent property of the cue thresholds governing zooid plasticity. Polymorphs in a colony have, during phylogeny, transitioned into associated non-zooidal structures (appendages), increasing colonial integration. While the level of module compartmentalization is important for the evolution of bryozoan polymorphism, it may be less influential for other colonial invertebrates.

Bait-attending amphipods of the Tonga Trench and depth-stratified population structure in the scavenging amphipod Hirondellea dubia Dahl, 1959.

BACKGROUND: The hadal zone encompasses the deepest parts of the world's ocean trenches from depths of ∼6,000-11,000 m. The communities observed at these depths are dominated by scavenging amphipods that rapidly intercept and consume carrion as it falls to the deepest parts of the trenches. New samples collected in the Tonga Trench provide an opportunity to compare the amphipod assemblages and the population structure of a dominant species, Hirondellea dubia Dahl, 1959, between trenches and with earlier data presented for the Tonga Trench, and other trenches in the South Pacific. METHODS: Over 3,600 individual scavenging amphipods across 10 species were collected in seven baited traps at two sites; in the Horizon Deep site, the deepest part of the Tonga Trench (10,800 m) and a site directly up-slope at the trench edge (6,250 m). The composition of the bait-attending amphipods is described and a morphometric analysis of H. dubia examines the bathymetric distribution of the different life stages encountered. RESULTS: The amphipod assemblage was more diverse than previously reported, seven species were recorded for the first time from the Tonga Trench. The species diversity was highest at the shallower depth, with H. dubia the only species captured at the deepest site. At the same time, the abundance of amphipods collected at 10,800 m was around sevenfold higher than at the shallower site. H. dubia showed clear ontogenetic vertical structuring, with juveniles dominant at the shallow site and adults dominant at the deep site. The amphipods of the deeper site were always larger at comparable life stage. DISCUSSION: The numbers of species encountered in the Tonga Trench is less than reported from the New Hebrides and Kermadec trenches, and six species encountered are shared across trenches. These findings support the previous suggestion that the fauna of the New Hebrides, Tonga and Kermadec Trenches may represent a single biogeographic province. The ontogenetic shift in H. dubia between the two Tonga Trench sites supports the hypothesis of interspecific competition at the shallower bathymetric range of the species, and the presence of competitive physiological advantages that allow the adults at the trench axis to exploit the more labile organic material that reaches the bottom of the trench.

Development and Application of a Quantitative PCR Assay to Assess Genotype Dynamics and Anatoxin Content in Microcoleus autumnalis-Dominated Mats.

Microcoleus is a filamentous cyanobacteria genus with a global distribution. Some species form thick, cohesive mats over large areas of the benthos in rivers and lakes. In New Zealand Microcoleus autumnalis is an anatoxin producer and benthic proliferations are occurring in an increasing number of rivers nationwide. Anatoxin content in M. autumnalis-dominated mats varies spatially and temporally, making understanding and managing proliferations difficult. In this study a M. autumnalis-specific TaqMan probe quantitative PCR (qPCR) assay targeting the anaC gene was developed. The assay was assessed against 26 non-M. autumnalis species. The assay had a detection range over seven orders of magnitude, with a limit of detection of 5.14 × 10-8 ng µL-1. The anaC assay and a cyanobacterial specific 16S rRNA qPCR were then used to determine toxic genotype proportions in 122 environmental samples collected from 19 sites on 10 rivers in New Zealand. Anatoxin contents of the samples were determined using LC-MS/MS and anatoxin quota per toxic cell calculated. The percentage of toxic cells ranged from 0 to 30.3%, with significant (p < 0.05) differences among rivers. The anatoxin content in mats had a significant relationship with the percentage of toxic cells (R² = 0.38, p < 0.001), indicating that changes in anatoxin content in M. autumnalis-dominated mats are primarily related to the dominance of toxic strains. When applied to more extensive samples sets the assay will enable new insights into how biotic and abiotic parameters influence genotype composition, and if applied to RNA assist in understanding anatoxin production.

Bacterial bioclusters relate to hydrochemistry in New Zealand groundwater.

Groundwater is a major source of New Zealand's water supply and supports base flows in rivers. Microbial communities in groundwater ecosystems mediate biogeochemical processes, and it is therefore crucial to understand microbial diversity in these ecosystems. We analysed bacterial assemblages from 35 New Zealand groundwater monitoring sites with varying hydrogeochemical conditions across the country. Proteobacteria was the most abundant phylum, and Variovorax represented the most common taxon. Pseudomonas, Burkholderia, Acidovorax, Janthinobacterium, Polaromonas and Caulobacter were the other common taxa. There was no Operational Taxonomic Unit (OTU) that was found in every one of the 35 samples. Here, we introduce a framework that has potential utility for groundwater ecosystem management, where the samples with similar microbial communities are grouped together into 'bioclusters'. Metabolic inferences derived from the taxonomic data were used to predict the oxygen requirements, metabolic potential and bacterial energy sources of each biocluster. Groundwater chemistry explains 59% of the variation in the relative abundance of all OTUs, with NO3-N, pH, DO, NH4-N, Fe, Br and SO4 displaying the strongest relationships to bioclusters. We propose that the biocluster framework, coupled with metabolic inferences derived from the taxonomic data, may have application outside New Zealand for on-going monitoring of the health of groundwater ecosystems.

Nutritional composition of the diet of the northern yellow-cheeked crested gibbon (Nomascus annamensis) in northeastern Cambodia.

This study measured the nutritional composition of foods consumed by the northern yellow-cheeked crested gibbon (Nomascus annamensis) in northeastern Cambodia. One group of N. annamensis was studied, and focal animal sampling was used to observe their feeding behavior. The study was conducted for 4 months (January-April 2015) in the dry season and 69 foods were collected for nutritional analyses. N. annamensis fed on 37 plant species, but only seven species made up more than 80% of feeding time. N. annamensis spent the majority of their time feeding on fruit (60.36%), followed by young leaves (22.60%), flowers (13.74%), and mature leaves (3.30%). Fruit had the highest concentrations of total non-structural carbohydrates, while young leaves had the highest concentration of crude protein compared to other food items. All food items had similar concentrations of lipids, except young leaves, which had lower levels than fruit and flowers. All plant parts consumed by N. annamensis had similar amounts of neutral detergent fiber, acid detergent fiber, and acid detergent lignin. Thirty-two percent of all food items contained condensed tannins, and they had a similar level of it. All foods also had similar energy contents. This study adds to our knowledge of the feeding ecology of N. annamensis by providing baseline data on the make-up of the foods they eat and may contribute to captive feeding programs, ultimately assisting the conservation of this species.

In situ light responses of the proteorhodopsin-bearing Antarctic sea-ice bacterium, Psychroflexus torques.

Proteorhodopsin (PR) is a wide-spread protein found in many marine prokaryotes. PR allows for the potential conversion of solar energy to ATP, possibly assisting in cellular growth and survival during periods of high environmental stress. PR utilises either blue or green light through a single amino acid substitution. We incubated the PR-bearing bacterium Psychroflexus torquis 50 cm deep within Antarctic sea ice for 13 days, exposing cultures to diurnal fluctuations in light and temperature. Enhanced growth occurred most prominently in cultures incubated under irradiance levels of ∼50 µmol photons m-2 s-1, suggesting PR provides a strong selective advantage. In addition, cultures grown under blue light yielded over 5.5 times more live cells per photon compared to green-light incubations. Because P. torquis expresses an apparently 'green-shifted' PR gene variant, this finding infers that the spectral tuning of PR is more complex than previously thought. This study supports the theory that PR provides additional energy to bacteria under sub-optimal conditions, and raises several points of interest to be addressed by future research.

High resolution microscopy reveals significant impacts of ocean acidification and warming on larval shell development in Laternula elliptica.

Environmental stressors impact marine larval growth rates, quality and sizes. Larvae of the Antarctic bivalve, Laternula elliptica, were raised to the D-larvae stage under temperature and pH conditions representing ambient and end of century projections (-1.6°C to +0.4°C and pH 7.98 to 7.65). Previous observations using light microscopy suggested pH had no influence on larval abnormalities in this species. Detailed analysis of the shell using SEM showed that reduced pH is in fact a major stressor during development for this species, producing D-larvae with abnormal shapes, deformed shell edges and irregular hinges, cracked shell surfaces and even uncalcified larvae. Additionally, reduced pH increased pitting and cracking on shell surfaces. Thus, apparently normal larvae may be compromised at the ultrastructural level and these larvae would be in poor condition at settlement, reducing juvenile recruitment and overall survival. Elevated temperatures increased prodissoconch II sizes. However, the overall impacts on larval shell quality and integrity with concurrent ocean acidification would likely overshadow any beneficial results from warmer temperatures, limiting populations of this prevalent Antarctic species.

The role of nitrogen and phosphorus in regulating Phormidium sp. (cyanobacteria) growth and anatoxin production.

Benthic proliferations of the cyanobacteria Phormidium can cover many kilometres of riverbed. Phormidium can produce neurotoxic anatoxins and ingestion of benthic mats has resulted in numerous animal poisonings in the last decade. Despite this, there is a poor understanding of the environmental factors regulating growth and anatoxin production. In this study, the effects of nitrogen and phosphorus on the growth of two Phormidium strains (anatoxin-producing and non-anatoxin-producing) were examined in batch monocultures. Cell concentrations were significantly reduced under reduced nitrogen (ca. <0.100 mM) and phosphorus conditions (ca. <0.003 mM). Cell concentrations and maximum growth rates were higher for the non-anatoxin-producing strain in all treatments, suggesting there may be an energetic cost to toxin production. Cellular anatoxin concentrations were lowest (169 fg cell(-1)) under the high-nitrogen and high-phosphorus treatment. This supports the growth-differentiation balance hypothesis that suggests actively dividing and expanding cells are less likely to produce secondary-metabolites. Anatoxin quota was highest (>407 fg cell(-1)) in the reduced phosphorus treatments, possibly suggesting that it is produced as a stress response to growth limiting conditions. In all treatments there was a 4-5-fold increase in anatoxin quota in the lag growth phase, possibly indicating it may provide a physiological benefit during initial substrate colonization.

Successional change in microbial communities of benthic Phormidium-dominated biofilms.

Benthic cyanobacterial blooms are increasing worldwide and can be harmful to human and animal health if they contain toxin-producing species. Microbial interactions are important in the formation of benthic biofilms and can lead to increased dominance and/or toxin production of one or few taxa. This study investigated how microbial interactions contribute to proliferation of benthic blooms dominated by the neurotoxin-producing Phormidium autumnale. Following a rainfall event that cleared the substrate, biofilm succession was characterised at a site on the Hutt River (New Zealand) by sampling every 2-3 days over 32 days. A combination of morphological and molecular community analyses (automated ribosomal intergenic spacer analysis and Illumina™ MiSeq sequencing) identified three distinct phases of succession in both the micro-algal and bacterial communities within P. autumnale-dominated biofilms. Bacterial composition shifted between the phases, and these changes occurred several days before those of the micro-algal community. Alphaproteobacteria and Betaproteobacteria dominate in the early phase; Alphaproteobacteria, Betaproteobacteria, Sphingobacteria and Flavobacteria in the mid-phase; and Sphingobacteria and Flavobacteria in the late phase. Collectively, the results suggest that succession is driven by bacteria in the early stages but becomes dependent on micro-algae in the mid- and late stages of biofilm formation.