cawrpt 2347 didymo phase 4 the influence of water ... · report no. 2347 didymo phase 4: the...

REPORT NO. 2347

DIDYMO PHASE 4: THE INFLUENCE OF WATER CHEMISTRY AND BIOFILM COMPOSITION ON DIDYMOSPHENIA GEMINATA ESTABLISHMENT

CAWTHRON INSTITUTE | REPORT NO. 2347 JUNE 2013

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EXECUTIVE SUMMARY

Didymosphenia geminata (didymo) was first detected in New Zealand in 2004. Despite extensive proliferation in some areas, the diatom remains absent from several areas where it is expected to be found. Both water chemistry and biofilm composition are thought to be important factors in the distribution and abundance of didymo. However, research has been limited by the lack of laboratory techniques for studying the diatom. During Phase 2 of this project, we developed a 96-well-plate-based method for assessing didymo survival in the laboratory under controlled conditions. In Phase 3, we found that water chemistry alone was insufficient to explain didymo distribution patterns and that substrate composition may play a critical role in didymo establishment. Building upon these previous findings, the current study, Phase 4, comprised experiments designed to explore the influence of water chemistry and substrate composition, as well as the potential influence of biofilms formed by other organisms on didymo establishment. Using a laboratory-based method for assessing didymo survival and attachment under controlled laboratory conditions, didymo was grown in river water systematically spiked with select nutrients at a range of concentrations. Based on results from these experiments, a synthetic medium that enables didymo attachment and cell division was developed. This is the first reported synthetic medium that supports the short term growth and attachment of didymo. Twelve substrates were evaluated for effects on didymo attachment; of these, Parafilm was found to enhance attachment over controls. Using standard microbiology techniques, 76 bacterial strains were isolated from didymo mats and 14 unique strains identified using molecular methods. Co-culturing experiments were undertaken and 3 strains that enhanced or decreased didymo survival were identified. Bacterial biofilm communities at geographically-similar sites with and without didymo were characterised using molecular fingerprinting techniques, revealing distinct differences in community structure between didymo and non-didymo sites. Key outcomes from the study were as follows:

1. The first ever chemically-defined synthetic growth medium that supports attachment of didymo cells was formulated.

2. Biofilm composition in didymo-infested versus didymo-free areas was found to differ markedly.

3. Preliminary progress towards the axenic culture of didymo was made.

4. Bacterial strains isolated from didymo mats were found to influence the establishment of didymo cells.

The results of these studies provide insight into factors that affect the establishment and survival of didymo, furthering our understanding of the invasive nature of this organism. The continued development of a laboratory-based method for the study of didymo provides potential to investigate thus-far unexplored aspects of this organism.


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TABLE OF CONTENTS

1. INTRODUCTION .............................................................................................................. 1

2. METHODS ........................................................................................................................ 3 2.1. Didymo and river water collection ................................................................................................................... 3

2.2. Multiple cell experiments ................................................................................................................................ 3 2.2.1. Method 1 ................................................................................................................................................... 3 2.2.2. Method 2 ................................................................................................................................................... 3 2.2.3. Method 3 ................................................................................................................................................... 3 2.2.4. Analysis of multiple cell experiments. ........................................................................................................ 4

2.3. Single cell experiments ................................................................................................................................... 4

2.4. River water spiking experiments ..................................................................................................................... 4

2.5. Synthetic media experiments.......................................................................................................................... 7 2.5.1. pH trials ..................................................................................................................................................... 7 2.5.2. Macronutrient base.................................................................................................................................... 7 2.5.3. Synthetic medium improvements .............................................................................................................. 7

2.6. Substrate experiments .................................................................................................................................... 8

2.7. Bacterial community comparisons .................................................................................................................. 8

2.8. Axenic culture of didymo .............................................................................................................................. 10

2.9. Co-cultivation experiments ........................................................................................................................... 11

3. RESULTS ....................................................................................................................... 13 3.1. River water spiking experiments ................................................................................................................... 13

3.2. Synthetic media experiments........................................................................................................................ 15

3.3. Substrate experiments .................................................................................................................................. 17

3.4. Bacterial community comparisons ................................................................................................................ 18

3.5. Axenic culture of experiments....................................................................................................................... 22

3.6. Co-cultivation experiments ........................................................................................................................... 22

4. DISCUSSION .................................................................................................................. 27

5. ACKNOWLEDGEMENTS ............................................................................................... 31

6. REFERENCES ............................................................................................................... 32

JUNE 2013 REPORT NO. 2347 | CAWTHRON INSTITUTE

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LIST OF FIGURES

Figure 1. Survival and attachment of didymo cells incubated for 15 days in filtered river water sourced from the Buller River spiked with increasing concentrations of various chemical compounds. ....................................................................................................... 14

Figure 2. Survival of didymo cells incubated in filtered river water sourced from the Buller River, Tasman, at various pH levels over 30 days. ..................................................................... 15

Figure 3. Mean percentage of didymo cells that were alive-free, alive-attached, or unhealthy after 14 days in basic synthetic medium supplemented with one of five sources of iron at 50 µM. ........................................................................................................................... 16

Figure 4. Mean percentage of didymo cells that were alive-free, alive-attached, or unhealthy after 14 days in basic synthetic medium supplemented with different concentrations of silicon. ............................................................................................................................... 17

Figure 5. Survival of didymo cells incubated in filtered river water sourced from the Buller River, Tasman, with the addition of various substrates. .............................................................. 18

Figure 6. Two-dimensional nonmetric multidimensional scaling ordination based on Bray-Curtis similarities of ARISA fingerprints from biofilm communities on didymo-covered rocks in the didymo-infested Buller River versus rocks in adjacent didymo-free tributaries. ......... 19

Figure 7. Comparison of physical parameters and water chemistry variables at didymo-infested rivers versus adjacent didymo-free creeks. ...................................................................... 21

Figure 8. Neighbour-joining phylogenetic tree showing the relationship between 24 bacterial strains showing unique RFPL banding patterns isolated from didymo mat sourced from the Buller River based on 16S rRNA gene sequences. ........................................... 23

Figure 9. Mean percentage of didymo cells that were alive-free, alive-attached, or unhealthy after eight days in 0.22 µm filter sterilised river water spiked with various dilutions of full-strength Luria Bertani medium. ................................................................................... 25

Figure 10. Mean percentage of didymo cells that were alive-free, alive-attached, or unhealthy after 14 days when co-cultured with various concentrations of seven bacteria strains [A) P6A, B) F5, C) F10, D) T18, E) T8, F) T14, G) T7] isolated from didymo mat.. ......... 26

LIST OF TABLES

Table 1. Compounds and corresponding nutrients assessed in river water spiking experiments. ... 6 Table 2. GenBank BLASTn search results for the 24 bacterial strains showing unique

restriction fragment length polymorphism banding patterns isolated from didymo mat sourced from the Buller River. .......................................................................................... 24


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1. INTRODUCTION

The Ministry of Primary Industries (MPI) contracted the Cawthron Institute (Cawthron) to investigate the influence of water chemistry and biofilm composition on the initial establishment and survival of Didymosphenia geminata (didymo). This project focuses on increasing knowledge on didymo using laboratory-based techniques. Didymo was first detected in New Zealand in 2004 and has since spread throughout the South Island. Despite extensive proliferation in many South Island rivers, the diatom is absent from groundwater-fed tributaries of these rivers and remains undetected in the North Island. Both water chemistry and biofilm composition are thought to be important in defining didymo distribution and abundance, however, research has been limited by the lack of laboratory techniques for studying the diatom. A laboratory-based method for assessing didymo survival and attachment has been developed (Phase 2: Kuhajek & Wood 2011a), allowing investigation of the organism under controlled conditions. Using this method, didymo cells were found to survive, attach, and divide in water sourced from rivers throughout New Zealand (Phase 3: Kuhajek & Wood 2011b). Although river water is suitable for preliminary laboratory studies of didymo, establishment of a defined synthetic growth medium is crucial for improving understanding of how physio-chemical variables influence attachment and growth of this organism. Successful establishment of didymo cells in water collected from areas where the diatom does not persist (e.g. ground-water fed tributaries), as well as unsuccessful establishment in water from areas where didymo is abundant suggests that water chemistry alone is insufficient to explain didymo distribution patterns (Phase 3: Kuhajek & Wood 2011b). Based on findings of enhanced attachment rates by didymo cells in the presence of the hydrophobic substance Parafilm (Phase 3: Kuhajek & Wood 2011b), substrate composition may be an important factor in determining whether didymo can adhere to a surface. Recent research on other freshwater diatoms has shown that soluble substances from bacterial biofilms impact diatom attachment, physiology, and extracellular polymeric substances. Investigation of how the chemical properties of a substrate and the corresponding bacterial biofilm influence didymo attachment are critical to furthering understanding of the unique distribution patterns of this organism. In this study, nutrient-spiking experiments were conducted to increase knowledge on the chemical requirements of didymo and to aid progress towards the formulation of a chemically-defined growth medium that supports attachment and division of didymo cells. The influences of substrate and biofilm composition were investigated, and attempts were made to create axenic (bacteria-free) cultures, as well as to co-culture didymo with bacteria isolated directly from didymo mats. The specific objectives were as follows:

Explore the chemical and nutrient requirements of didymo using nutrient spiking experiments


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Establish a chemically-defined growth medium in which didymo can be grown

Evaluate the attachment of didymo cells in the presence of various substrates

Compare bacterial biofilm communities at sites with and without didymo

Attempt to create axenic cultures of didymo

Conduct co-cultivation experiments between didymo and bacteria isolated from didymo mats.


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2. METHODS

2.1. Didymo and river water collection

Didymo was collected from the Buller River, Tasman (41° 47.119' S, 172° 48.600' E = Site 1), between October 2011 and January 2012, and October 2012 and January 2013. Due to poor health of didymo from this site, collections were made from various locations on the Monowai River, Southland, in February and March 2012, and the Buller River-Howard River junction, Tasman (41° 43.18' S, 172° 40.91' E = Site 2), in February 2013. Collected mats were placed into 1-L Schott bottles containing river water from the collection site, transported chilled, and received in the laboratory within 4 h (Buller River) or 24 h (Monowai River) of collection. Once in the laboratory, didymo was maintained at 5°C and bubbled with sterile air. Collections were made approximately every 3 wk to ensure healthy didymo cells were available for experiments. River water was collected from the collection site in conjunction with each didymo collection. River water samples were filtered (0.22 µm — axenic experiments; 0.45 µm — all other experiments; Millipore S-Pak Filters, 47 mm, Billerica, MA, USA) within 4 h of collection.

2.2. Multiple cell experiments

Over the course of this study, three methods were used for preparing multiple cell experiments.

2.2.1. Method 1

An aliquot (1 mL) of filtered river water (FRW) or test solution was added to each well of a 24-well plate (tissue culture treated polystyrene; Becton Dickenson), four wells per treatment; five test concentrations plus control per plate. Mats were triple-rinsed in FRW to remove other organisms, debris and dead cells and cut into sections (c. 0.5 cm2). For each treatment, three sections of mat were rinsed in well 1 and then placed in to wells 2-4, one section per well. After 48 h the mats were removed leaving multiple cells that had detached from the mats and settled to the bottom of the wells.

2.2.2. Method 2

Sections of mat (c. 0.5 cm2) were triple rinsed in FRW and then placed into wells of a 24-well plate each containing 1 mL of FRW. After 24 h at 18°C the mats were removed leaving multiple cells that had detached from the mats and settled to the bottom of the wells. Remaining cells in each well were rinsed with and then submerged in 1 mL of FRW (control) or test solution, four replicate wells per control or treatment.

2.2.3. Method 3

A section of didymo mat (c. 8 cm2) was rinsed with Milli-Q water (MQ) and then transferred to a sterile 200-mL Schott bottle containing 100 mL of MQ. The bottle was


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shaken vigorously by hand for 1 min. The contents of the Schott bottle were then poured through a 100-µm and then a 20-µm polyester mesh screen to remove debris. The 20-µm screen was rinsed with 1 L of MQ and then flushed with 5 mL of MQ into a polystyrene culture pottle (Biolab, NZ) containing 20 mL of FRW. Aliquots of approximately 100 µL, depending on cell concentration, were transferred to wells of a 24-well plate, each containing 900 µL of FRW. Plates were then placed at 18°C for 2 h after which the FRW was exchanged for the desired test solution.

2.2.4. Analysis of multiple cell experiments.

Cells from multiple cell experiments were monitored for percentage survival and attachment every four days for eight days using an inverted microscope at 100–200× magnification. Plates were maintained under standard conditions (40 ± 5 µmol photons m–2 s–1; 16:8 h light:dark; 18 ± 1°C).

2.3. Single cell experiments

Within 48 h of placing mats in wells, cells that had settled to the bottom of the wells of the 24-wells plate were transferred to 96-well plates (Becton Dickenson). Unless otherwise specified, tissue culture treated polystyrene plates were used. A small (c. 3 mm2) piece of Parafilm® M (Parafilm) was placed into the bottom of each well. The plates were heated for 3 min in a microwave (450 W) to soften the Parafilm and the Parafilm was pressed to the bottom of each well to prevent it floating to the surface upon addition of liquid. The plates were then sterilized by exposure to UV light (15 min). An aliquot (200 µL) of FRW or the appropriate treatment solution was then transferred into each well. For the substrate experiments, plates were prepared without Parafilm. A single didymo cell was transferred into each well of the 96-well plate using a micro-pipette while viewed through an inverted microscope. Unless specified, cells were transferred into wells containing the corresponding treatment water so that cells were exposed to the same water in both the 24- and 96-well plates. All wells were checked at 200× magnification to ensure that each contained a single didymo cell and that the cell was not damaged during transfer. Plates were maintained under standard conditions (see Multiple Cell Experiments) and cells were monitored for up to 30 days using an inverted microscope (100-200×). Cells were scored as dead or alive based on the presence of a visible chloroplast and survival (total number of live cells) was recorded. Attachment and stalk length were also noted.

2.4. River water spiking experiments

Filtered river water used in this experiment was collected from the Buller River (Site 1) on 15 February 2012. Stock solutions were prepared by dissolving each compound (Table 1) in MQ water at 10x the highest concentration to be tested. The pH of each stock solution was adjusted to match that of the FRW (7.45 ± 0.2). Solutions of


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disodium ethylenediaminetetraacetic acid dihydrate (EDTA) and EDTA ferric sodium salt (FeEDTA) were adjusted to pH 8.0 to achieve solubility. Where pH adjustment of the stock solution caused precipitation (FeCl3, FeSO4, ZnCl2), pH adjustments were made on the first test concentration rather than the stock solution. Initially, stock concentrations were selected based on 10,000x the highest concentration of the corresponding component found in New Zealand river waters evaluated in Phase 3 (Kuhajek & Wood 2011b). Concentrations were refined based on range-finding pilot studies using the 24-well plate format. Where possible, the lowest toxic concentration for each compound was established in range-finding studies and used as the highest test concentration in 96-well single cell tests. Test solutions were prepared by serial dilution of the stock solutions into FRW to give a range of concentrations between the level in the FRW and the lowest toxic concentration; five concentrations were prepared for each compound tested. Adjustments were made to account for the concentration of a given component naturally present in the FRW so that the test concentrations listed (Table 1) are the concentration of a given nutrient naturally present in the FRW plus additional nutrient spiked into the sample.


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Table 1. Compounds and corresponding nutrients assessed in river water spiking experiments. Concentrations tested in synthetic media experiments are also given. Concentrations in New Zealand river waters are data from Phase 3 (Kuhajek & Wood 2011b). Disodium EDTA dihydrate was included as a control for EDTA ferric sodium salt. EDTA = ethylenediaminetetraacetic acid; FRW = filtered river water, collected from the Buller River (Tasman) on 15 February 2012; MM = molecular mass; NT = not tested / no data available.

Compound Chemical formula Compound MM (g/mol) Nutrient

Nutrient MM (g/mol)

Concentrations in NZ rivers

(µM) Concentration in FRW (µM)

Concentration range tested (µM) River water spiking exp

Synthetic media exp

Calcium chloride dehydrate CaCl2·2H2O 147.01 Ca 40.08 50–400 100 200–1000 0.1–1000

Ferric chloride, anhydrous FeCl3 162.2

Fe 55.85 0.02–12 0.13

5–500 0.5–500

Ferrous sulphate heptahydrate FeSO4·7H2O 278.05 5–500 0.5–500

EDTA ferric sodium salt FeNaEDTA 367.05 0.5–5000 0.05–5000

Disodium EDTA dehydrate Na2EDTA · 2H2O 372.24 EDTA 292.24 NT NT 0.5–5000 NT

Magnesium chloride hexahydrate MgCl2·6H2O 203.31 Mg 24.31 15–350 13

75–1200 0.05–500

Magnesium sulphate heptahydrate MgSO4·7H2O 246.47 75–1200 0.01–1000

Potassium dihydrogen phosphate KH2PO4 136.09 PO4 94.97 0.1–0.25 0.08 5–475 0.0125–250

Sodium metasilicate pentahydrate Na2SiO3·5H2O 212.13 Si 28.09 40–820 68 105–1680 0.2–2000

Sodium bicarbonate NaHCO3 84.01 HCO3 61.01 NT 580 1200–6000 0–3000

Sodium nitrate NaNO3 85.00 NO3 62.01 0–30 0.04 124–12,400 0.025–500

Copper chloride dehydrate CuCl2·2H2O 170.48 Cu 63.55 0–0.04 0.01 0.02–1.0 0.001–1

Zinc chloride ZnCl2 136.32 Zn 65.38 0–0.12 0.03 0.6–3.0 0.001–1


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2.5. Synthetic media experiments

2.5.1. pH trials

An aliquot of FRW (pH 7.5), collected from the Buller River (Site 1) on 15 February 2012, was sequentially adjusted using 0.001–1 M hydrochloric acid to pH 7.4, 7.2, 7.0, 6.8 and 6.6. A second aliquot was sequentially adjusted using 0.001–1 M sodium hydroxide to pH 7.6, 7.8, 8.0, 8.2, 8.4, 8.6 and 8.8. When each target pH was reached, 5 mL of water was removed and set aside for testing and further pH adjustment was continued on the remaining solution. Cells were harvested using the 24-well plate format from wells containing 1 mL of FRW (no pH adjustment). Cells were transferred to wells of a 96-well plate, each containing 200 µL of pH-adjusted river water, eight wells per pH.

2.5.2. Macronutrient base

Stock solutions of nutrients (calcium chloride, FeEDTA, magnesium sulphate, potassium dihydrogen phosphate, sodium metasilicate, and sodium nitrate) were prepared by dissolving each compound in MQ at 10x the highest concentration to be tested and adjusting to pH 7.5 ± 0.2. Stock concentrations were selected based on results from river water spiking experiments. Test solutions were prepared by serial dilution of the stock solutions into MQ to give a range of concentrations (Table 1), five concentrations per compound tested per experiment. Nutrient solutions were tested in an array format using multiple cell experiments. Sequential experiments were conducted to evaluate serial addition of the six macronutrient solutions at multiple concentrations of each nutrient to establish a macronutrient base suitable for supporting successful attachment of didymo cells to the culture plate.

2.5.3. Synthetic medium improvements

To further optimise the macronutrient medium, four additional trials were conducted: iron source evaluation, silicon concentration reduction, and micronutrient evaluation. Using the 24-well format, additional experiments were conducted using the macronutrient base established above. Each well of the 24-well plate was filled with 900 µL of macronutrient base and then spiked with 100 µL of MQ (control) or test nutrient solution over a range of five concentrations. Unless specified, the macronutrient base was adjusted to pH 7.5 ± 0.2 prior to use. In the first experiment, the macronutrient base was prepared without iron and then spiked with a range of concentrations (0.5–500 µM) of one of five iron sources: ferrous sulphate, ferric chloride, ferrous sulphate + EDTA, ferric chloride + EDTA, or FeEDTA. In a second experiment, the macronutrient base was prepared with FeEDTA as the iron source and then spiked with varying concentrations (0.001–1.0 µM) of


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micronutrient solutions, including boric acid (H3BO3), cobalt chloride (CoCl2), potassium chromate (K2CrO4), copper sulphate (CuSO4), manganese chloride (MnCl2), sodium molybdate (Na2MoO4), nickel sulphate (NiSO4), selenous acid (H2SeO3), sodium orthovanadate (Na3VO4), and zinc sulphate (ZnSO4), one plate per micronutrient solution. In a third experiment, the macronutrient base was prepared without silicate and FeEDTA as the iron source and then spiked with a range of concentrations (0.2–2000 µM) of sodium metasilicate.

2.6. Substrate experiments

Tissue-culture treated polystyrene 96-well plates were used as the base for all substrate experiments except non-treated plate trials. Treated and non-treated polystyrene plates were used with no additional preparation. Parafilm treatments were prepared according to the procedure described for single cell experiments. Colloidal suspensions (1 mg/mL) of silica (SiO2), boehmite alumina (AlO(OH)), iron oxide (Fe3O4), and titanium dioxide (TiO2) were sonicated for 10 min to ensure homogeneity. Immediately after sonication the suspensions were transferred into wells of a 96-well plate, 32 µL per well to give an approximately 1 µm-thick film. The plate was then placed in a desiccator containing silica gel desiccant for 2 h. Powders (C18-bonded silica, washed sand, and quartz) and borosilicate glass (broken into 1 to 2-mm2 sections) were transferred into wells in sufficient quantity to provide a sparse covering

of the bottom of the well. A 0.1% solution of -poly-L-lysine (polylysine) was diluted

1:10 in MQ water and then added to wells, 50 µL per well (0.15 mL/cm2). Plates were rocked gently (1 h). The solution was then removed and the plates were dried at ambient temperature (2 h). Milli-Q water (200 µL) was added to each well for 24 h and then removed prior to sterilization. Glycerine leaf was cut into approximately 3-mm2 sections and placed into wells, one section per well. All plates were sterilized under UV light (15 min). Cells were harvested using the 24-well plate format from wells containing 1 mL of FRW. Cells were transferred to wells of prepared 96-well plates, each containing 200 µL of FRW, eight wells per substrate. Substrates were tested in duplicate or in some cases triplicate experiments.

2.7. Bacterial community comparisons

Four side streams along the Buller River were selected for evaluation of bacterial communities in didymo-infested versus didymo-free sites. Biofilm and water samples were collected from the confluence of the Buller River with the four inflowing tributaries:

Duckpond Stream (41° 47.50' S, 172° 49.09' E)

Speargrass Creek (41° 45.55' S, 172° 46.10' E)


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Howard River (41° 43.18' S, 172° 40.91' E)

Baigent Creek (41° 42.37' S, 172° 38.99' E).

Turbidity (2100P Portable Turbidimeter; Hach), water flow (Flo-mate 2000; Marsh McBirney), dissolved oxygen, temperature, and conductivity (600QS Multimeter; YSI) were measured in each river just prior to the confluence as well as at the confluence at each site. A water sample was collected from each river just prior to the confluence. Samples were transported chilled (4°C) to the laboratory. A subsample (250 mL) of each was sent to Hill Laboratories (Hamilton, NZ) for determination of pH, total alkalinity (titration), and bicarbonate (calculation) using methods in APHA (2005). The remaining volume of each sample was filtered (0.45 µm Millipore S-Pak Filters, 47 mm, Billerica, MA, USA) within 24 h of collection. A subsample (10 mL) of each filtered sample was preserved (2% nitric acid) and sent to University of Waikato (NZ) for measurement of element concentrations using an inductively coupled plasma mass spectrophotometer (ICP/MS; Perkin Elmer ELAN DRC II). A second subsample (250 mL) was sent to Water Care Laboratory Services (Auckland, NZ) for assessment of chloride, nitrate-N, nitrite-N, and sulphate (ion chromatography; APHA 2012); ammonium-N (colorimetry; MEWAM 1981), dissolved reactive phosphorus and reactive silica (colorimetry; APHA 2012); and dissolved organic carbon (non-dispersive infrared detection; APHA 2005). Two-tailed paired t-tests of physical parameters and water chemistry were conducted using Microsoft Excel (2010). Biofilm samples were taken by selecting three rocks from each didymo and didymo-free site. Rocks selected were of similar size and were taken from as close to confluence as possible. The entire surface of each rock was swabbed with a sterile Speci-spongeTM (Nasco). Sponges were placed in sterile bags and transport chilled (4°C) to the laboratory where they were frozen (-20°C) until use. Sponges were thawed and the sponges were placed in 40 mL of RNA/DNA free water (Invitrogen) followed by maceration using a Colworth 400 laboratory stomacher (AJ Seward, UK) for 90 s. Sponges were then squeezed to remove excess liquid and the resulting biofilm suspensions pelleted by centrifugation (3000 × g, 20 min). The supernatant was discarded and the DNA extracted from the pellet using the Power Biofilm DNA Isolation Kit (MoBio Laboratories) following the manufacturer’s instructions. A region of the internal transcribed spacer (ITS) region was amplified by polymerase chain reaction (PCR) (iCycler; Biorad) using bacterial-specific primers ITSF 5′-TCGTAACAAGGTAGCCGTA-3′ and ITSReub 5’-GCCAAGGCATCCACC-3’ (Cardinale et al., 2004). The PCR reactions were performed in 20-µL volumes with the reaction mixture containing 12.5 µl of i-Taq 2x PCR master mix (Intron Biotechnology, Korea), 0.4 µM of each primer, and 15-20 ng of the template DNA. The reaction mixture was held at 94°C for 3 min followed by 30 cycles of 94°C for 45 s, 52°C for 60 s, 72°C for 2 min, with final extension step at 72°C for 7 min. Amplified reactions were diluted (1:20) with MQ and sent to Waikato University (Hamilton, NZ) for automated


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ribosomal intergenic spacer analysis (ARISA). Amplicon lengths were resolved on an ABI 3130xI Genetic Analyzer (Applied Biosystems) and run under GeneScan mode at 15 kV for 45 min according to the manufacturer’s protocol. Each sample contained 0.25 µL of the internal GS1200LIZ ZyStandard (Applied Biosystems) to determine the size of the fluorescently-labelled fragments. Electropherograms were processed using the PeakScannerTM software v1.0 (Applied Biosystems) and an in-house pipeline modified from Abdo et al. (2006) written using Python 2.7.1 (Python Software Foundation) and R (http://www.r-project.org). Fragments with peaks heights greater than 30 relative fluorescence units and lengths between 100 and 1200 base pairs that comprised at least 0.1% of the entire signal were included in the analysis. Peaks were binned to the nearest one base pair. ARISA fragment lengths were converted to presence/absence and analysed using nonmetric multidimensional scaling (MDS) based on Bray-Curtis similarities using the PRIMER 6 software package (PRIMER-E Ltd.). Nonmetric MDS was undertaken with 100 random restarts and results were plotted in two-dimensions. Agglomerative, hierarchical clustering of the Bray-Curtis similarities was undertaken using the CLUSTER function of PRIMER 6 and plotted onto the two-dimensional MDS.

2.8. Axenic culture of didymo

The feasibility of producing an axenic culture of didymo was assessed by using chloramphenicol, gentamycin, penicillin, and streptomycin, as well as mixtures of chloramphenicol and penicillin. Antibiotics were evaluated at five concentrations each in the 24-well-plate multiple-cell format, one plate per antibiotic or antibiotic mix. Antibiotic stocks were prepared be dissolving each antibiotic into MQ and filter sterilizing (0.22 µm). Stocks were added to wells of separate 24-well plates at 0, 25, 50, 100, 200, and 400 µL per well, four replicate wells per volume. Concentration ranges tested were as follows: chloramphenicol = 0.625 to 5 mg/mL, gentamycin = 0.625 to 1 mg/mL, penicillin = 0.25 to 4 mg/mL, and streptomycin = 0.625 to 2 mg/mL. Plates were maintained under standard conditions (see Multiple Cell Experiments) and cells were monitored for percentage didymo survival. Initially cells were monitored after 24, 48, and 72 h using an inverted microscope. Axenicity was assessed using a nutrient-rich growth medium as well as by epifluorescence. An aliquot (100 µL) from each well of the treatment plates was transferred using sterile techniques to the corresponding well of a second 24-well plate containing 1 mL of Luria Bertani (LB) medium. These plates were placed at 25°C in the dark. Plates were visually assessed for cloudiness after 24, 48, and 72 h. A second aliquot (20 µL) of each well was diluted (1:50) into FSRW and then stained with SYBR Gold (Invitrogen). Bacterial cells were counted under blue-green light excitation at 1000× magnification using an epifluorescence microscope (Olympus


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BX51). Random fields (n = 10) were counted and the bacterial concentration calculated.

2.9. Co-cultivation experiments

A piece of didymo mat was collected in a sterile 50 mL tube from the Buller River (Site 1) on 7 October 2012 and transported chilled to the laboratory. Within 4 h of collection, the mat was wetted with MQ water (15 mL) and homogenised (Omni GLH, Omni International). The homogenate was diluted into LB medium in a four-fold 1:10 serial dilution. A 100-µL aliquot of each dilution was plated onto one of four types of agar plates: FW70, Reasoner’s 2A agar (R2A), plate count agar (PCA), and tryptic soy agar (TSA); 16 plates total. The plates were incubated in the dark at 18°C overnight. Individual colonies were then picked from each of the agar plates (approximately 20 per agar type) and streaked onto new plates of the same agar type. The plates were incubated in the dark at 18°C overnight. Single colonies were then transferred to sterile 15 mL conical centrifuge tubes containing 10 mL of LB medium and incubated in the dark with continuous shaking (300 rpm) at 37°C overnight. An aliquot (1 mL) of each culture was transferred to a cryotube, mixed with 225 µL of sterile 80% glycerol, and stored at -80°C. The remaining 9 mL of each culture was centrifuged (3000 × g, 15 min). The supernatant was discarded and DNA was extracted from the pellet (PureLink® Genomic DNA Kit, Invitrogen) following the manufacturer’s gram negative bacterial cell lysate protocol. The DNA was eluted in 50 µL of elution buffer; DNA concentrations was measured (NanoPhotometer P360, Implen). Dilutions were prepared as required using MQ to give a working concentration of 15–20 µg/µL. A region of the 16S rRNA gene was amplified by PCR (Master Cycler; Eppendorf) using bacterial-specific primers 27F 5'- AGA GTT TGA TCM TGG CTC AG -3' and 1518R 5'- AAG GAG GTG ATC CAN CCR CA -3' (Giovannoni 1991). PCR reactions were performed in 20 µL volumes with the reaction mixture containing 10 µL of i-Taq 2xPCR master mix (Intron Biotechnology, Korea), 0.4 µM of each primer, and 15–20 ng of template DNA. The reaction mixture was held at 94°C for 2 min followed by 30 cycles of 94°C for 20 s, 57°C for 20 s, 72°C for 1 min, with final extension step at 72°C for 7 min. PCR reactions were visualised using agarose gel electrophoresis (1 %) run at 70 V for 70 min (PowerPacBasic, Biorad Laboratories). Sequences were differentiated using restriction fragment length polymorphism (RFLP). An aliquot of each PCR product was then digested using the restriction enzyme HaeII; samples showing a non-unique banding pattern in the HaeII digestion were also digested using RsaI. The digestion was performed in 10 µL volumes with the reaction mixture containing 7.5 µL of MQ water, 1 µL of 10x NE Buffer 4 digestion buffer (New England Biolabs), 0.5 µL of restriction enzyme (10,000 U/mL; New England Biolabs), and 1 µL of PCR product. Digestions were held at 37°C for 1 h and


12

visualised using agarose gel electrophoresis (2 %) run at 70 V for 120 min. A representative of each banding pattern group was selected and the remaining 16S PCR product purified using an AxyPrep PCR Cleanup Kit (Axygen Biosciences) and submitted to the University of Otago (Dunedin, NZ) for sequencing. The 16S rRNA gene sequences were compared with sequences from the NCBI Genbank database using BLASTn (Benson et al. 2007). Sequences were aligned and phylogenetic trees created using the neighbour-joining algorithm with 1 000 bootstrap replicates (MEGA version 4.1; Tamura et al. 2007). Cryopreserved samples of unique strains (as confirmed by DNA sequencing of 16S) were inoculated into 10 mL of LB medium and grown overnight with continuous shaking at 37°C. The bacterial cultures were centrifuged (3000 × g, 3 min), the supernatant discarded, and the bacteria resuspended in 10 mL of FSRW. The process was repeated to completely remove all LB medium. Each culture diluted with FSRW in a 5 x 10-fold serial dilution series, and then co-cultured with didymo using the 24-well plate format. The remaining culture was stained with SYBR Gold (Invitrogen) and quantified using epifluorescence (Olympus BX51). Survival and attachment of didymo cells were monitored for 14 days relative to FSRW controls.


13

3. RESULTS

3.1. River water spiking experiments

In the 96-well format, most attachment and division of didymo cells placed in FRW occurred within the first 15–20 days of the assay (data not shown). The effects of nutrient supplementation of FRW were most notable during this period. Four compounds (CaCl2, KH2PO4, MgCl2, and FeEDTA) had a positive effect on didymo survival relative to survival in FRW with no additional nutrients added. Toxicity due to nutrient excess was taken as reduced survival of didymo compared with survival in FRW. No toxicity was seen for the first three compounds in the concentration range tested, however, FeEDTA had a toxic effect at the two highest concentrations tested. FeCl3 was toxic at the three lowest concentrations tested but had no effect at the two highest concentrations, likely due to precipitation of the iron at the high concentrations. FeSO4 had no effect at the concentrations tested; evaluation of higher concentrations of this compound was not possible due to solubility limits of the compound at the pH required. In contrast to results from initial 24-well multi-cell trials used to established concentration limited for the 96-well testing, ZnCl2 was toxic at all concentrations tested and requires re-evaluation at lower concentrations to determine the effects on didymo survival at lower concentrations (Figure 1A). In addition to improving didymo survival, two compounds (CaCl2 and KH2PO4) resulted in increased the rate of attachment relative to the controls. An additional four compounds (FeEDTA, MgCl2, FeSO4 and MgSO4) gave moderate increases in attachment. All compounds except CaCl2 and KH2PO4 inhibited attachment at one or more concentrations (Figure 1B). None of the different concentrations of the 13 compounds had a consistent effect on stalk length (data not shown).


14

Figure 1. Survival (A) and attachment (B) of didymo cells incubated for 15 days in filtered river

water (FRW) sourced from the Buller River (Tasman) spiked with increasing concentrations of various chemical compounds (Table 1). Cells were assessed in a 96-well single cell format, 8 wells per concentration per compound. The shaded section of the graph indicates the average (± standard deviation) survival (7.1 ± 2.5) or attachment (5.0 ± 2.9) of didymo cells in the control, FRW with no additional chemicals added (n = 15, control only).

FeEDTA

CaCl2

KH2PO4

MgCl2

FeSO4

Na2SiO3

NaNO3

CuCl2

MgSO4

NaHCO3

EDTA

ZnCl2

FeCl3

FeEDTA

CaCl2

KH2PO4

MgCl2

FeSO4

Na2SiO3

NaNO3

CuCl2

MgSO4

NaHCO3

EDTA

ZnCl2

FeCl3


15

3.2. Synthetic media experiments

Cell division was greatest in water between pH 7.6 and 8.2. After Day 15, new divisions approximately equalled cell death in water between pH 7.8 and 8.2 but continued almost linearly in pH 7.6. Although cell division did occur, no net increase in the number of cells occurred over the course of the experiment in water below pH 7.4 or above 8.4 (Figure 2).

Figure 2. Survival of didymo cells incubated in filtered river water (FRW) sourced from the Buller

River, Tasman, at various pH levels over 30 days. Cells were assessed in a 96-well single cell format, eight wells per pH. The dashed line indicates the number of alive cells (8) placed in each pH treatment when the experiment commenced.

A formulation of sodium metasilicate, potassium dihydrogen phosphate, calcium chloride, magnesium sulphate, and FeEDTA was identified in which successful attachment and colony formation by didymo cells was achieved. Further experiments were then undertaken to improve growth in the medium. The addition of a nitrate source (sodium nitrate) resulted in a decrease in cell health compare to the base medium (data not shown). When different iron sources were tested over a range of

0

5

10

15

20

25

30

1 5 10 15 20 25 30

Total N

umber of Alive Cells

Days

6.6

6.8

7.0

7.2

7.4

7.6

7.8

8.0

8.2

8.4

8.6

8.8


16

concentrations, survival and attachment of didymo were highest when iron was provided at 50 µM for all five sources of iron tested (data not shown) and were similar regardless of source (Figure 3). The largest colonies were observed in medium supplemented with FeEDTA at 50 µM (data not shown).

Figure 3. Mean percentage of didymo cells (sourced from the Buller River) that were alive-free (), alive-attached ( ), or unhealthy () after 14 days in basic synthetic medium supplemented with one of five sources of iron at 50 µM. Cells were assessed in a 24-well format. Results are an average of four replicates; error bars are ± 1 standard error.

Using FeEDTA as the iron source, the macronutrient medium with a silicon concentration of 200 µM gave the greatest survival and attachment of didymo cells (Figure 4). None of the micronutrients tested had any obvious impacts on didymo survival or attachment relative to the control when added to the basic macronutrient medium at up to 1 µM (data not shown).

FRW FeEDTA FeSO4 FeSO4 FeCl3 FeCl3

+EDTA +EDTA


17

Figure 4. Mean percentage of didymo cells (sourced from the Buller River) that were alive-free (), alive-attached ( ), or unhealthy () after 14 days in basic synthetic medium supplemented with different concentrations of silicon. Cells were assessed in a 24-well format. Results are an average of four replicates; error bars are ± 1 standard error.

3.3. Substrate experiments

Tissue-culture treated plates plus Parafilm gave the highest survival of didymo over the 30 d; the effect was evident within 5 d. Parafilm was the only substrate to improve survival of didymo over that in control wells. Adding quartz, sand, glass or C18 had no effect on didymo survival relative to control wells. Colloidal SiO2, TiO2, AlO(OH) and Fe3O4 reduced survival relative to the control. Glycerine and polylysine were toxic to didymo with 100% cell death within three days (Figure 5). Survival in non-treated plates was similar to that in treated plates, but attachment was delayed in non-treated plates (data not shown).


18

Figure 5. Survival of didymo cells incubated in filtered river water (FRW) sourced from the Buller

River, Tasman, with the addition of various substrates. Cells were assessed in a 96-well single cell format, eight wells per substrate. Error bars indicate standard deviation in the survival of cells in the controls, wells in which no additional substrate was added (n = 3). Nontx = non-treated polystyrene plates.

3.4. Bacterial community comparisons

Multivariate analysis of ARISA data from biofilm communities on didymo-covered rocks in a didymo-infested river versus rocks in an adjacent didymo-free tributary showed similarity groupings influenced primarily by location at Duckpond Creek and Baigent Creek, and the presence or absence of didymo at Speargrass Creek and the Howard River (Figure 6).

Control

Parafilm

Glass

Quartz

Sand

C18

Silica

TiO2

AlO(OH)

Fe3O4

Nontx

Glycerine

Polylysine


19

Figure 6. Two-dimensional nonmetric multidimensional scaling (MDS) ordination (stress = 0.19)

based on Bray-Curtis similarities of ARISA fingerprints from biofilm communities on didymo-covered rocks in the didymo-infested Buller River () versus rocks in adjacent didymo-free tributaries (). Points enclosed by dashed lines cluster at 25% similarity.

No significant differences were found in the physical parameters (conductivity, temperature, dissolved oxygen, turbidity, and flow) measured at the paired didymo-infested versus adjacent didymo-free sites (Figure 7A). Nor did bicarbonate concentrations, total alkalinity, or total hardness differ (Figure 7B). Significant differences between the paired sites were found for concentrations of six chemical parameters: silicon, sodium, and magnesium were higher and concentrations of strontium, arsenic, and sulphate lower in the didymo-free waters (Figure 7C–G). Silicate concentrations were also higher in the didymo-free waters, but due to a disproportionately high concentration at the Baigent Creek site (a relatively sandy site), the difference was not significant (data not shown). The proportion of magnesium relative to calcium, sodium, and potassium was also lower in didymo-free waters (Figure 7H).


20


21

Figure 7. Comparison of physical parameters (A) and water chemistry variables (B–H) at didymo-infested rivers (●) versus adjacent didymo-free creeks ().Symbols indicate the average value for four sites; error bars indicate the maximum and minimum values measured. Values for water chemistry variables (B–G) are in µg/L. DO = dissolved oxygen; DOC = dissolved organic carbon; DIN = dissolved inorganic nitrogen; DRP = dissolved reactive phosphorus. The maximum value for mercury (Hg),11.77 µg/L measured at Baigent Creek, is off-scale. Parameters with an asterisk (*) were significantly different between the didymo-infested versus didymo-free sites by paired two-tailed t-test (p < 0.05).


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3.5. Axenic culture of experiments

In initial antibiotic trials, streptomycin and gentamycin were toxic to didymo cells at all concentrations tested (0.00625–1.0 mg/mL). Chloramphenicol had no impact on didymo health relative to FRW controls at less than 0.7 mg/mL, and penicillin had no impacts at less than 2 mg/mL. When didymo was exposed to chloramphenicol and penicillin in combination, no additive toxic effects on survival or attachment were observed. When aliquots of didymo cultures spiked with antibiotic were transferred to LB medium, the broth became cloudy within 3 d; control medium without antibiotics became cloudy overnight. SYBR Gold staining further confirmed the presence of bacteria in the antibiotic-containing cultures (data not shown).

3.6. Co-cultivation experiments

Using four different agar types, 76 bacterial colonies were isolated from didymo mat collected from the Buller River. Following RFLP analysis of DNA extracted from the colonies, 24 unique banding patterns were identified. Sequencing of the 16S rRNA gene and subsequent phylogenetic analysis identified 13 different bacterial strains (Figure 8). Evaluation using BLASTn revealed these strains belonged to eight different genera (Table 2). Seven strains were co-cultured with didymo using the 24-well format.


23

Figure 8. Neighbour-joining phylogenetic tree showing the relationship between 24 bacterial strains showing unique RFPL banding patterns isolated from didymo mat sourced from the Buller River (Tasman) based on 16S rRNA gene sequences. The bootstrap values are inferred from 1000 replicates and branches corresponding to partitions reproduced in less than 20% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. E. coli was used as an outgroup.


24

Table 2. GenBank BLASTn search results for the 24 bacterial strains showing unique restriction fragment length polymorphism (RFLP) banding patterns isolated from didymo mat sourced from the Buller River (Tasman).

Code Highest GenBank BLASTn match Strain Access. no.

Similarity (%)

F5 Aeromonas popoffii S_T_TSA_64 JX860592 100

R10 Aeromonas popoffii S_T_TSA_51 JX860612 100

T11 Aeromonas salmonicida QL102 KC130963 99

R7 Aeromonas sp. REm-amp_251 JX899627 99

P2 Carnobacterium maltaromaticum LMA28 HE999757 99

T18 Deefgea rivuli S_T_TSA_63 JX860598 99

T3 Exiguobacterium antarcticum IARI-L-70 JX429003 99

F6 Exiguobacterium undae Su-1 AB669472 100

R11A Exiguobacterium sp. 7-3 DQ019168 100

T7 Exiguobacterium sp. 7-3 DQ019168 99

F11 Micrococcus luteus KUDC1784 KC355291 99

P1 Staphylococcus aureus ST228 HE579073 98

P12 Staphylococcus aureus ST228 HE579073 99

R8 Staphylococcus aureus ST228 HE579073 99

R9 Staphylococcus aureus ST228 HE579073 100

R12A Staphylococcus aureus ST228 HE579073 99

T14 Staphylococcus aureus ST228 HE579073 100

T16 Staphylococcus aureus ST228 HE579073 99

P6A Staphylococcus warneri SG1 CP003668 99

F10 Staphylococcus sp. N2 HF545642 100

T13 Pseudomonas fluorescens RBE2CD-72 EF111238 95

P6 Pseudomonas saponiphila ME BHU9 JN033556 100

T8 Pseudomonas sp. RHLB15-1 JX949420 99

P5 Yersinia ruckeri NBRC 102019 AB681666 99


25

Figure 9. Mean percentage of didymo cells (sourced from the Buller River) that were alive-free (), alive-attached ( ), or unhealthy () after eight days in 0.22 µm filter sterilised river water (FSRW) spiked with various dilutions of full-strength Luria Bertani (LB) medium. Cells were assessed in a 24-well format. Results are an average of four replicates; error bars are ± 1 standard error.

Initial experiments revealed that LB medium was toxic to didymo cells when diluted up to 100-fold and inhibited attachment when diluted 1000-fold. Survival and attachment were not affected in the presence of LB medium diluted 50,000-fold or 100,000-fold, while attachment was slightly higher at 10,000-fold diluted (Figure 9). At high concentrations (greater than 107–108 bacterial cells/mL) all bacterial strains inhibited survival and attachment of didymo cells relative to the controls (Figure 10A–G), while at low concentrations (104–105 cells/mL), one strain (P6) increased didymo survival (Figure 10A), one strain (Figure 10) increased didymo attachment (Figure 10C), and one strain increased both survival and attachment (Figure 10B).


26

Figure 10. Mean percentage of didymo cells (sourced from the Buller River) that were alive-free (), alive-attached ( ), or unhealthy () after 14 days when co-cultured with various concentrations of seven bacteria strains [A) P6A, B) F5, C) F10, D) T18, E) T8, F) T14, G) T7] isolated from didymo mat. Control wells were 0.22 µm filter sterilised river water. Cells were assessed in a 24-well format. Results are an average of four replicates; error bars are ± 1 standard error.

Per

cent

age

of A

live

Ce

lls

Alive-free

Alive-attached

Unhealthy


27

4. DISCUSSION

As our understanding of didymo grows, laboratory techniques to study this diatom are constantly improving. Over the course of this study, three methods for harvesting free cells and conducting multiple cell experiments were used. The initial method developed (Method 1) involved placing didymo mats directly into solutions to be tested. In some cases, this led to inhibition of the release of cells from the mats. The method was improved by incorporating a step to allow cell release in FRW (Method 2), providing more reliable cell release and more reproducible test conditions. Both Methods 1 and 2 required incubation of the mats for up to 48 h to achieve release of sufficient numbers of didymo cells to conduct multiple cell experiments. The method was further improved by adding a shake and filter step (Method 3) to allow the harvest of free cells within 2 to 3 h. In parallel with the development of laboratory techniques, the establishment of a defined synthetic growth medium is crucial for robust study of didymo in the laboratory. Spiking individual nutrients into a river water base allowed the upper tolerance threshold of those nutrients present in limiting amounts to be determined. Established thresholds were then used as starting points for the design of a synthetic growth medium. When individually supplemented into river water, four compounds (CaCl2, KH2PO4, MgCl2, and FeEDTA) improved survival of didymo relative to FRW controls. A combination of these four compounds, pH-adjusted to between 7.6 and 8.2 and with the addition of a silicon source, crucial to diatoms for synthesis of the frustule, were found to be sufficient to support attachment and growth of didymo in culture short term. This is the first ever recorded attachment of didymo cells in a synthetic medium. Further investigation of the source of iron for use in the medium was undertaken since inorganic iron is available in two forms: ferric (Fe+3) or ferrous (Fe+2). Bioavailability of the two forms can differ depending on the organism, with some organisms preferring one type over the other. Iron is insoluble in water at near neutral pH and consequently is typically present in culture medium in an unusable form. Bioavailability of iron can be improved by the addition of a chelator such as EDTA, however, EDTA alone is toxic to didymo because it degrades stalk material (pers. comm. M. Gretz, Michigan Technological University), as confirmed by river water spiking experiments conducted in this study. The addition of EDTA in conjunction with an iron source did not improve the survival or attachment of didymo relative to iron without EDTA. However, EDTA did improve the solubility of iron, facilitating preparation of the medium. Citrate is a potential alternative to EDTA, although it was not investigated because iron-citrate complexes are less stable than those of EDTA. When iron was added in the form of FeEDTA (optimal concentration 50 µM), attachment frequency was highest. With the basic nutrients and optimal iron source for survival and attachment of didymo cells in culture identified, further nutrients were evaluated with the goal of improving


28

survival duration and attachment. Typical micronutrients, nutrients required in minute quantities, recognised as beneficial in the culture of many other algae were investigated. Initial experiments showed little effect of any of these nutrients on general survival or attachment of didymo relative to controls. However, further experiments investigating higher concentrations using the 24-well format are necessary, as well as examination of fine-scale effects using the 96-well format. During trials to further optimise the macronutrient growth medium, precipitation of silicon became problematic. At the initially established silicon concentration, a large pH adjustment was necessary to achieve a pH suitable for didymo survival. Efforts were focused on reducing the concentration of silica needed for didymo growth in culture. Through these efforts, a 10-fold reduction in the silicon concentration from 2000 to 200 µM was achieved. In addition to the establishment of a synthetic media for culturing didymo, identification of the surface properties required for cell attachment will assist in understanding this organism. Substrate composition is of interest not only to advance understanding of didymo distribution in nature, but it may also assist in improving the success of culturing didymo. Results from Phase 3 (Kuhajek & Wood 2011b) provide evidence that cell attachment is a critical step in the establishment of didymo colonies and subsequent bloom formation. Both substrate and biofilm composition are predicted to play a role in cell attachment. Cells must attach to a substrate within approximately 20 d of release from a stalk or they will die (Kuhajek & Wood, pers. obs.). Previous trials have shown that the presence of Parafilm (a hydrophobic substance) enhances attachment and thus survival of didymo cells (Kuhajek & Wood 2011b). Other diatoms have also been found to attach more strongly to hydrophobic surfaces than to hydrophilic ones (Krishnan et al. 2006). Eleven additional substrates were evaluated, but only Parafilm had a positive effect on cell survival. The delayed attachment on non-treated plates, which are hydrophobic, compared to hydrophilic tissue culture-treated surfaces suggests that the factors influencing attachment are complex and require further study.

In river ecosystems, most substrates available to didymo as attachment sites are covered in a biofilm, usually containing a mixed assemblage of organic compounds, detritus, heterotrophic bacteria, cyanobacteria, and photoautotrophic algae. Recent research on other diatoms has shown that soluble substances from bacterial biofilms impact diatom attachment, physiology, and extracellular polymeric substances. Higher didymo abundance (Flöder & Kilroy 2009) and greater attachment (Bergey et al. 2010) on colonized versus un-colonized surfaces suggests that didymo cells may require a biofilm to attach successfully and that biofilm composition may be a contributing factor to successful attachment.


29

The ARISA and subsequent MDS analysis showed distinct differences in bacterial community structure in the biofilm communities from didymo versus adjacent non-didymo sites. Although further investigation is required, including larger sample sets and a more in-depth analysis of the species present, these initial findings suggest that the presence of specific bacterial species may be associated with didymo establishment and/or survival. The difference in bacterial community may also be a function of variations in other parameters, for example, water chemistry. No differences in the physical parameters were observed, however, minor differences in water chemistry between the paired sites were found. In addition to influencing bacterial community composition, differences in water chemistry may also affect the didymo distribution. Rost et al. (2011) showed that in Sierra Nevada streams levels of sulphate were higher and levels of magnesium and sodium were lower in didymo relative to non-didymo sites. Lindstrøm & Skulberg (2008) reported that the presence of didymo in Norway is associated with a minimum sulphate concentration of 2.5 mg/L (26 µM). The data collected in the current study suggest that the lower limit for the Buller River may be closer to 2.0 mg/L. Rost et al. (2011) proposed that the positive correlation between sulphate with didymo presence may reflect the biochemical composition of the stalk, which is primarily sulphated polysaccharide (Gretz 2008). Significant amounts of sulphur may be required to support stalk production during bloom conditions and sulphur-limitation may inhibit stalk formation as observed in the stalk forming marine diatom Achnanthes longipes when grown in the absence of sulphur (Johnson et al. 1995). In combination with higher sulphur, lower concentrations of magnesium, as well as proportion of magnesium relative to other cations, in didymo areas may be related to interactions between magnesium and calcium. As demonstrated in river water spiking and synthetic medium experiments performed in this study, calcium and magnesium are both essential nutrients for didymo. Although specific functions of magnesium are less well known, established functions of calcium in diatoms include stalk requirements (Gretz, 2008), motility (Cooksey & Cooksey, 1980; Cohn & Disparti, 1994), and adhesion (Cooksey, 1981). Higher calcium concentrations was one factor that distinguished didymo from non-didymo streams in the Sierra Nevada mountains (Rost et al. 2011), and surveys in Norway have shown that didymo does not occur when calcium concentrations are below 2 mg/L (Lindstrøm & Skulberg 2008). In this study, calcium concentrations did not differ between didymo and non-didymo streams, however, calcium was greater than 2 mg/L at all sites with the lowest concentration (3 mg/L) at Baigent Creek. Investigation of biofilm differences and potential water chemistry drivers between didymo and non-didymo streams is aided by the study of didymo in the absence of biofilm-forming organisms. Axenic cultures free of bacteria and other organisms may be useful for elucidating the relationship between didymo survival and associated microorganisms, as well as for further molecular investigations of didymo. Antibiotic


30

treatment was selected as the most promising method for creation of axenic cultures of didymo. Initial experiments were conducted to evaluate the toxicity of four antibiotics to didymo cells. Although streptomycin and gentamycin were found to be toxic to didymo cells at bactericidal concentrations, the maximum useful concentrations of chloramphenicol (0.7 mg/mL) and penicillin (2 mg/mL) were established. Treatment of didymo cells with a combined dose of chloramphenicol plus penicillin at the maximum established dosages was insufficient to remove all bacteria from the culture, as confirmed by spiking an aliquot of each culture into broth, as well as by SYBR Gold staining. Investigation of alternative options including successive treatment with each antibiotic, analysis of additional antibiotics, and attempts to begin with didymo containing fewer other microorganisms are warranted. Further attempts towards achieving axenic culture are best conducted in August through October when didymo mats tend to be relatively free of other organisms. The production of axenic cultures is complemented by the study of the effect of individual microbial inhabitants of didymo mats and survival and attachment of the diatom. Didymo mats are inhabited by a diverse array of micro-organisms, as evidenced by the isolation of 14 phylogenetically distinct bacterial strains representing eight different genera from a single section of mat. Initial co-culturing experiments revealed that LB medium was toxic to didymo, necessitating careful removal of this growth medium from bacterial cultures prior to co-culturing assays. The toxicity of LB medium is consistent with previous findings that the nutrient concentration of typical culture media is above the tolerances of didymo in vitro (Kuhajek et al. 2011). Didymo survived poorly when co-cultured with each of the isolated bacterial strains above a given concentration; the concentration was similar for all seven strains. Improved survival and attachment in the presence of at least some of the isolated strains provides evidence in support of the role of biofilm bacteria in the establishment of didymo in a given area. The successful formulation of a basic synthetic medium that supports the growth and attachment of didymo cells in the lab is a fundamental step in the study of this enigmatic organism. Further optimisation of the medium prioritising long-term culture of didymo in conjunction with in-depth nutrient-limitation studies to gain complete understanding of the nutritional requirements this organism are recommended. Regular monitoring of nutrient fluctuations in the field and changing nutritional requirements of didymo mats throughout the growing season will serve to complement nutritional studies in the laboratory. Co-culturing studies to determine effects of the remaining eight bacterial strains (two strains already tested were from the same group) on didymo survival and attachment, as well as evaluation of fine-scale effects of these bacteria using the 96-well format will help drive understanding of didymo-biofilm relationships. Finally, establishing axenic cultures of didymo will aid in elucidating the molecular sequence of the didymo genome, which may ultimately assist in understanding the distribution of this diatom.


31

5. ACKNOWLEDGEMENTS

The authors gratefully acknowledge that contributions of Marion Lemoine, Sam Drew and Josephin Brandes, who undertook the experiments described in this report and thank Philippe Gerbeaux (Department of Conservation), Cathy Kilroy (NIWA), and Max Bothwell (National Water Research Institute, Canada) for helpful discussions and advice.


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cawrpt 2347 didymo phase 4 the influence of water ... · report no. 2347 didymo phase 4: the...

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