didymosphenia geminata in river waters

REPORT NO. 2060
SURVIVAL AND ATTACHMENT OF
DIDYMOSPHENIA GEMINATA IN RIVER WATERS
FROM AROUND NEW ZEALAND
CAWTHRON INSTITUTE | REPORT NO. 2060
DECEMBER 2011
EXECUTIVE SUMMARY
The aim of Phase 3 of this study was to investigate whether the absence of Didymosphenia
geminata (didymo) from North Island waterways and groundwater-fed creeks is related to
water chemistry. River water was collected from 16 North Island and 17 South Island rivers
and 4 groundwater-fed creeks. Water chemistry and nutrient concentrations were determined
and survival and attachment of didymo cells was monitored in each water. Experiments were
undertaken with and without the inclusion of Parafilm to aid didymo attachment, and were
conducted using didymo from two disparate sources within New Zealand, the Buller River
(Tasman) and the Hurunui River (Canterbury). Principle component analysis was used to
explore relationships between didymo survival and water chemistry.
Didymo cells survived, attached and divided in river waters with a wide range of elemental
and nutrient concentrations. Results from multivariate analysis of water chemistry and cell
survival data indicated that optimal concentration ranges of specific elements or nutrients
may be required for didymo to establish in a given area. In general, didymo did best in water
from sites with moderate water chemistries, although testing of more sites throughout New
Zealand is required to confirm this trend.
The key findings from the study were as follows:
1.
Attachment of didymo cells to a substrate is a prerequisite for cell division.
2.
The addition of Parafilm (a hydrophobic substance) greatly enhances attachment and
cell viability, suggesting that substrate composition may play an important role in didymo
adhesion.
3.
Didymo cells can survive, attach and divide in water with a wide range of elemental and
nutrient concentrations, including water from North Island rivers and groundwater-fed
creeks.
4.
Results from multivariate analysis of water chemistry and cell survival data indicate that
didymo may survive but be unable to attach or divide (i.e., bloom) when certain
elements/nutrients are present above or below a given optimal concentration range.
We recommend further investigation of the nutrient requirements of didymo as well as indepth evaluation of substrate properties that favour attachment. In light of our findings, we
recommend that measures presently in place within New Zealand to limit to spread of didymo
be continued.
iii
CAWTHRON INSTITUTE | REPORT NO. 2060
DECEMBER 2011
TABLE OF CONTENTS
1. INTRODUCTION .............................................................................................................. 1 2. 2.5. METHODS ........................................................................................................................ 2 River and groundwater-fed creeks sampling and processing ......................................................................... 2 Didymo collection ........................................................................................................................................... 4 Single cell survival and attachment experiments ............................................................................................ 4 Multiple cell survival and attachment experiments.......................................................................................... 5 Statistical analysis .......................................................................................................................................... 5 3. RESULTS ......................................................................................................................... 6 2.1. 2.2. 2.3. 2.4. 3.1. Water chemistry and nutrient concentrations ................................................................................................. 6 3.2. Single-cell 96-well plate experiments, without Parafilm .................................................................................. 7 3.3. Single-cell 96-well plate experiments, with Parafilm ....................................................................................... 7 3.4. Hurunui experiment ........................................................................................................................................ 8 3.5. Multi-cell experiment ....................................................................................................................................... 8 3.6. Statistical analysis .......................................................................................................................................... 8 4. CONCLUSIONS AND RECOMMENDATIONS ............................................................... 15 5. ACKNOWLEDGEMENTS ............................................................................................... 16 6. REFERENCES ............................................................................................................... 17 7. APPENDICES ................................................................................................................. 19 LIST OF FIGURES
Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Locations of sampling sites. ................................................................................................ 3 Survival, attachment and division of didymo cells (sourced from the Buller River) in
river and groundwater-fed creek water sourced from across New Zealand (see Figure
1 for location of sites). ....................................................................................................... 10 Survival, attachment and division of didymo cells (sourced from the Buller River) in
river and groundwater-fed creek water sourced from across New Zealand (see Figure
1 for location of sites). ....................................................................................................... 11 Survival, attachment and division of eight didymo cells (sourced from the Hurunui
River) in selected river and groundwater-fed creek water sourced from across New
Zealand (see Figure 1 for location of sites). ..................................................................... 12 Mean percentage of didymo cells (sourced from the Buller River) that were alive after
20 days in selected river and groundwater-fed creek water sourced from across New
Zealand (see Figure 1 for location of sites). ..................................................................... 13 Principal component analysis ordination showing the number of alive didymo cells in
96-well plates containing Parafilm at day 30 in relation to elemental and nutrient
concentrations of water samples. ..................................................................................... 14 v
DECEMBER 2011
REPORT NO. 2060 | CAWTHRON INSTITUTE
LIST OF TABLES
Table 1. Mean elemental and nutrient concentrations in water samples from North Island rivers
(N), South Island rivers (S) and groundwater-fed creeks (G). ............................................ 6 LIST OF APPENDICES
Appendix 1. Elemental and nutrient concentrations in water samples collected from 16 North Island
rivers (see Figure 1 for location of sites). .......................................................................... 19 Appendix 2. Elemental and nutrient concentrations in water samples collected from 17 South Island
rivers (see Figure 1 for location of sites). .......................................................................... 20 Appendix 3. Elemental and nutrient concentrations in water samples collected from 4 groundwaterfed creeks (see Figure 1 for location of sites). .................................................................. 21 vi
CAWTHRON INSTITUTE | REPORT NO. 2060
1.
DECEMBER 2011
INTRODUCTION
The Department of Conservation contracted Cawthron Institute to test the
survival of Didymosphenia geminata (didymo) in water collected from various
rivers and groundwater-fed creeks from throughout New Zealand.
Didymo was first detected in New Zealand in October 2004, in the lower
Waiau River, Southland (Kilroy 2004). By the end of 2007 the diatom had
become established over most of the South Island and its distribution
continues to expand, albeit more slowly, with more than 280 sites within 150
South Island rivers now infested (www.didymosamplesdb.org.nz). During the
expansion phase of didymo in New Zealand interesting distribution patterns
have been observed. Notably, didymo is currently absent from some key
habitats where it is expected to be found. For example, didymo remains
undetected in the North Island, despite the presence of numerous habitats
predicted to be suitable and the high potential for didymo introductions to the
North Island (Kilroy et al. 2005, 2007). Furthermore, didymo grows prolifically
in many rivers throughout the South Island, yet is absent in the groundwaterfed tributaries of these rivers, despite known introductions (Kilroy & Bothwell
2010). Additionally, field observations have demonstrated that the presence of
didymo cells in a river does not always result in blooms. Using a net sampling
technique, didymo cells have been repeatedly identified in some rivers but no
macroscopic or microscopic evidence of didymo growth has been found
(Kilroy & Urwin 2011). This patchy distribution suggests that some waterways
may be resistant to didymo colonisation.
Relationships between didymo and physical requirements for proliferation
have advanced with research demonstrating that blooms are more likely in
cooler rivers (Kumar et al. 2009) with moderate or low water velocities and
stable substrates (Sherbot & Bothwell 1993, Kilroy & Dale 2006, Kirkwood et
al. 2007, Spaulding & Elwell 2007) and high light intensity (Lindstrøm &
Skulberg 2008). The absence of extensive water-chemistry datasets at
regional and continental scales has prevented the inclusion of chemical
parameters in habitat-suitability modelling (Kumar et al. 2009). However,
water chemistry is known to influence the distribution of other diatom species
(Stoermer & Smol 2001, Potapova & Charles 2003) and is widely
acknowledged as a likely important parameter in defining didymo distribution
and abundance.
Field-based studies have been invaluable in increasing knowledge on didymo,
however, controlling for variables such as light, temperature, water velocity,
substrate type and the presence and abundance of other organisms creates
challenges when studying the effect of water chemistry and nutrients on cell
viability and attachment. Laboratory-based studies have been constrained by
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DECEMBER 2011
REPORT NO. 2060 | CAWTHRON INSTITUTE
an inability to culture didymo. In Phase 1 and 2 of this study, we successfully
cultured didymo and observed that cells remain viable, attach and produce
stalks when kept in filtered river water (Kuhajek et al. 2011, Kuhajek & Wood
2011). In these studies a 96-well-plate-based method for assessing didymo
survival in the laboratory under controlled conditions was developed (Kuhajek
& Wood 2011), providing a unique platform to assess the influence of water
chemistry on didymo survival and attachment. The aim of Phase 3 of this
project was to explore didymo distribution patterns in New Zealand by testing
the following hypothesis: Variations in elemental and/or nutrient
concentrations are critical in determining the survival and initial adhesion of
didymo cells.
To test our hypothesis river water was collected from 16 North Island and 17
South Island rivers and four groundwater-fed creeks. Water chemistry and
nutrient concentrations were determined and survival and attachment of
didymo cells was monitored. Experiments were undertaken, with and without
the inclusion of a small piece of Parafilm in each well to aid didymo
attachment, using didymo from two disparate sources within New Zealand.
Principle component analysis was used to explore relationships between
didymo survival and water chemistry.
2.
METHODS
2.1. River and groundwater-fed creeks sampling and processing
Water samples (500 mL or 1 L) were collected from 16 North Island and 17
South Island rivers and four South Island groundwater-fed creeks (Figure 1).
Samples were transported chilled (4 ºC) to the laboratory where they were
filtered (0.45 µm) within 48 h of collection. A sub-sample (250 mL) of each
filtered sample was preserved (2% nitric acid) and stored at 4 ºC for later
elemental analysis. A second sub-sample (250 mL) was stored in a sterile
container at 4 ºC in the dark for use in survival experiments. From the Buller
River and groundwater-fed creek samples, an additional sub-sample (500 mL)
was frozen for later nutrient analysis. Nutrient levels (ammonium, dissolved
reactive phosphorus and nitrate) in the Buller River and groundwater-fed creek
samples were measured using a Lachat QuickChem® Flow Injection Analyser
(FIA+ 8000 Series; Zellweger Analytics, Inc.) according to methods given in
APHA (2005). The river samples, with the exception of the Buller River sample,
were collected as part of the National Water Quality Monitoring Programme
undertaken by the National Institute of Water and Atmospheric (NIWA). For
these samples the nutrient data supplied by this programme was used. Further
information about these sites and historical water quality data is available at
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CAWTHRON INSTITUTE | REPORT NO. 2060
DECEMBER 2011
https://secure.niwa.co.nz/wqis/index.do. Elemental concentrations in each
water sample were measured using inductively coupled plasma mass
spectrophotometer (ICP/MS) by the ICP Laboratory at the University of Waikato
(Hamilton, New Zealand).
Figure 1.
Locations of sampling sites.
Codes used for river sites correspond to those used in the National Water
Quality Monitoring Programme. Further information about these sites can be
obtained from https://secure.niwa.co.nz/wqis/index.do
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2.2. Didymo collection
Didymo was collected on 7 September 2011 from the Buller River, Nelson
(41º 47.119' S, 172º 48.600' E) and on 14 September 2011 from the Hurunui
River, Canterbury (47º 46.0601' S, 172º 33.0679' E). Mats were scraped off
rocks and placed in 1-L Schott bottles containing river water from the
collection site. Once in the laboratory didymo mats were maintained at 5 ºC
and bubbled with sterile air.
2.3. Single cell survival and attachment experiments
Within 48 hours of collection a small (c. 0.5-cm2) section of didymo mat was
placed into individual wells of a 24-well plate (BD Falcon™ 24-well Multiwell
Plate), each containing 1 mL of river or groundwater-fed creek water, one well
per water sample (= treatment). Twenty-four hours later the mats were
removed and cells which had detached from the mats and settled to the
bottom of the wells were used for further experiments.
Two separate single-cell survival experiments were set up in 96-well plates
(BD Falcon™ Clear 96-well Microtest™ Plate). In the first experiment, no
additional substrate was added to the wells. In the second experiment, a small
(c. 3-mm2) piece of Parafilm was placed into the bottom of each well. Previous
experiments had shown that the presence of Parafilm in culture vessels
improved the attachment rate of didymo cells when grown in culture (Kuhajek
et al. 2011, Kuhajek & Wood 2011). The plates were heated for c. 3 minutes 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. These plates were sterilised by exposure to UV (15 minutes). Three 96well plates were prepared for each experiment.
An aliquot (200 µL) of river or creek water was added to each well of the six
96-well plates; eight replicate wells per treatment. For each experiment one
treatment of GR- medium (UTEX algal culture collection, University of Texas,
USA), diluted 1:10 with MQ-water, was included for comparison. In initial
culturing trials in Phase 1 of this project, didymo grew well in various dilutions
of GR- medium but not in a range of other freshwater algal media (Kuhajek et
al. 2011)
A single didymo cell was transferred from the 24-well plate into each well of
the 96-well plate using a micro-pipette and an inverted microscope (CK2,
Olympus). Cells were transferred into wells containing the corresponding
treatment water so that cells were exposed to the same water in both the 24and 96-well plates. All wells were checked at 200× magnification (CK2,
Olympus) to ensure that each contained a single didymo cell and that the cell
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CAWTHRON INSTITUTE | REPORT NO. 2060
DECEMBER 2011
was not damaged during transfer. Plates were maintained under standard
conditions (35 ± 10 µmol photons m–2 s–1; 16:8 hours light:dark; 12 ± 1 °C).
Survival, attachment, stalk production and cell division were monitored every
2-3 days for 30 days using an inverted microscope (CK2, Olympus). Based on
the number of alive cells at day 30 in the Parafilm experiment, each treatment
was placed in to one of four groups (Group 1 ≥ 14 cells, Group 2 = 9-13 cells,
Group 3 = 5-8 cells, Group 4 ≤ 4 cells) for statistical analysis.
To determine whether the responses observed in the above experiments were
specific to didymo sourced from the Buller River the experiments were
repeated with mats collected from the Hurunui River, Canterbury. Twelve
treatment waters were selected for this experiment, six from the North Island
(HM1, RO1, RO6, WA5, GS2 and WN5) and six from the South Island,
including two groundwater-fed creeks (CH3, TK1, AX1, DN2, Wash Creek and
Upper Oreti). These sites represented a range of responses in the initial
experiments.
2.4. Multiple cell survival and attachment experiments
An aliquot of each treatment water (1 mL) was added to each of 4 wells of a
24-well plate. Thirteen days after collection (20 September 2011) sections (c.
0.5-cm2) of didymo mat collected from the Buller River, three sections per
treatment, were washed in well 1 and then placed in to wells 2-4, one section
per well. After two days the mats were removed leaving multiple cells
(estimated between 50-200) which had detached from the mats and settled to
the bottom of the wells. The percentage of surviving cells in each well was
recorded every three to four days for 20 days.
2.5. Statistical analysis
Mean elemental and nutrient concentrations were calculated for North Island
rivers, South Island rivers and groundwater-fed creeks. One-way ANOVA
followed by a Tukey’s HSD post hoc test (Zar 1974) was used to test for
significant differences in water chemistries and nutrient concentrations among
sample types at a 95% confidence interval. Pearson correlations were
undertaken between each individual elemental and nutrient concentration and
the number of alive cells at day 30 in the 96-well plate Parafilm experiment.
Elemental and nutrient data were normalised and principal component
analysis (PCA) conducted using PRIMER 6 software (PRIMER-E Ltd., UK).
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DECEMBER 2011
3.
REPORT NO. 2060 | CAWTHRON INSTITUTE
RESULTS
3.1. Water chemistry and nutrient concentrations
Mean elemental and nutrient concentrations differed among sites (Table 1,
Appendices 1-3). Silicon and vanadium were higher in North Island rivers
versus South Island rivers, while barium was higher in North Island rivers
versus both South Island rivers and groundwater-fed creeks.
Table 1.
6
Mean elemental and nutrient concentrations in water samples from North Island
rivers (N), South Island rivers (S) and groundwater-fed creeks (G).
All values are given in µg L-1 except silicon (mg L-1). Results given are from a
one-way ANOVA followed by a Tukey test (α = 0.05) where sites sharing an
underline are significantly different from each other. DRP = dissolved reactive
phosphorus.
Element
or
Nutrient
North
Island
rivers
South
Island
rivers
Groundfed
creeks
F 2,34
P
B
Na
Mg
Al
Si
P
K
Ca
V
Cr
Fe
Mn
Co
Ni
Cu
Zn
As
Se
Sr
Cd
Ba
Pb
U
DRP
NH3
NO3
69.7
5880
1800
37.6
10200
4.55
1560
5960
0.61
0.04
0.07
4.83
0.04
0.37
0.79
1.32
2.37
0.30
47.2
0.02
9.46
0.05
0.01
9.05
3.14
270
13.1
5250
1830
34.2
3120
6.59
789
6630
0.23
0.07
0.08
3.45
0.05
0.58
0.88
1.62
0.43
0.31
54.4
0.01
4.92
0.05
0.02
5.25
19.40
268
9.7
3210
3790
3.3
5550
<0.01
450
4950
0.44
0.36
<0.01
0.52
<0.01
1.02
0.07
0.65
<0.01
<0.01
30.0
0.01
2.27
0.01
<0.01
12.25
6.00
956
1.48
0.33
2.00
0.79
14.33
0.43
2.78
0.55
3.82
3.92
0.62
1.16
0.67
1.45
2.96
0.78
0.80
2.15
2.02
0.64
6.11
0.27
1.71
2.69
0.72
4.06
0.24
0.72
0.15
0.46
<0.01
0.65
0.08
0.58
0.03
0.03
0.54
0.33
0.52
0.25
0.07
0.47
0.46
0.13
0.15
0.54
0.01
0.77
0.20
0.08
0.49
0.03
Tukey
test
NSG
NSG
NGS
GNS
NGS
CAWTHRON INSTITUTE | REPORT NO. 2060
DECEMBER 2011
Chromium and nitrate were higher in groundwater-fed creeks compared with
North and South Island rivers, with no difference in the concentrations of these
components between North and South Island rivers (Table 1). Two sites (RO1
and DN1) had markedly different water chemistries from all other sites
(Appendix 1). Water sampled from RO1 had high conductivity (504 μS cm-1 at
25 °C) and elevated concentrations of the cations magnesium (>12 000 µg L1
), sodium (7 400 µg L-1), silicon (21 000 mg L-1) and potassium (7 100 µg L-1).
Water from DN1 had high concentrations of the metals lead (0.4 µg L-1),
manganese (22 µg L-1) and iron (0.7 µg L-1).
3.2. Single-cell 96-well plate experiments, without Parafilm
On day 10 of the experiment all treatments contained at least one alive cell
and in nine of the treatments all eight cells remained healthy. Attachment was
apparent in three treatments (GS2, DN1 and DN10); cell division occurred in
one of these (DN1; Figure 2A). The cell that divided had attached to a fibre
which had been accidentally introduced during the plate set-up. By day 20 all
cells had died in three treatments (RO6, WN5 and TK1) and were either dead
or unhealthy in two treatments (CH1 and TK3). Cell attachment was evident in
an additional five treatments (RO4, WA2, TK4, AX3 and Flaxy Creek) and
seven new cell divisions were recorded across four treatments (RO4, GS2,
TK4, DN1; Figure 2B). By day 30 attachment was observed in an additional
two treatments (TU1, NN2) and one further cell division was observed in one
treatment (GS2). In total, attachment was observed in 10 treatments. The
greatest number of alive cells (10) was seen in GS2 (Figure 2C).
3.3. Single-cell 96-well plate experiments, with Parafilm
On day 10 of the experiment all treatments contained at least two healthy cells
and twenty treatments contained at least eight alive cells. Attachment had
occurred in 24 treatments; 19 of these showed cell division (Figure 3A). By
day 20 all cells had died in AX3 and one healthy cell remained in RO1.
Attachment had occurred in waters from five additional sites (NN2, NN3, TK1,
TK2, and Wash Creek). Cell division was observed in two additional
treatments (HV4 and WN2) and further divisions occurred in 14 treatments
(Figure 3B). Over the 30 day experiment, no attachment was observed in five
of the treatments (WN5, TK4, AX1, AX3 and DN10) and no cell division had
occurred in eleven treatments (RO4, RO6, WA5, WN2, WN5, GS3, TK1, TK2,
TK4, AX1, AX3, DN10; Figure 3A-C). The highest number of alive cells (22)
occurred in DN9; no cells died in this treatment during the 30 day experiment.
The next highest number of alive cells on day 30 occurred in CH3 (21)
followed by GR- Medium and GS2 (18; Figure 3C).
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3.4. Hurunui experiment
By day 30, attachment was observed in four treatments without Parafilm
(RO1, GS2, CH3 and DN2) and all twelve treatments with Parafilm (Figure 4).
Division occurred in four of the treatments without Parafilm (RO1, CH3 and
DN2) and nine treatments with Parafilm (HM1, RO1, RO6, WA5, GS2, WN5,
CH3, TK1 and Upper Oreti). All the cells were unhealthy by day 10 in TK1
without Parafilm, and by day 20 one alive cell remained in RO6 without
Parafilm (data not shown). By day 30, all cells in TK1 and AX1 without
Parafilm were dead. Eight or more cells remained throughout the course of the
experiment in four treatments without Parafilm (HM1, CH3, DN2 and Upper
Oreti) and seven treatments with Parafilm (RO1, WA5, GS2, WN5, CH3, TK1
and Upper Oreti). The highest number of alive cells (29) occurred in CH3 with
Parafilm (Figure 4).
3.5. Multi-cell experiment
On day 20, the average percentage survival was greater than 40% for all
treatments. In six treatments (RO1, RO6, GS2, TK3, DN6 and Flaxy Creek),
the average percentage survival was equal to or greater than 90% (Figure 5).
The lowest survival (41%) occurred in TU1 and DN5 (Figure 5). Attachment
and stalk production was observed in at least one replicate of most
treatments. The treatments with the most prolific attachment and stalk
production were HM1, RO1, GS2, TK4, DN9, Upper Oreti and Wash Creek
(data not shown).
3.6. Statistical analysis
When individual elemental and nutrient concentrations were compared to the
number of alive cells in each treatment on day 30 of the single-cell experiment
with Parafilm, no statistically significant Pearson correlation coefficients were
found (data not shown; all correlations coefficients were 0.0000). Principal
components 1 (PC1) and 2 (PC2) explained only 34% of the observed
variation (Figure 6). Phosphate, copper, aluminium, iron, manganese, cobalt,
lead, potassium, zinc and cadmium all made strong (< -0.2) negative
contributions to PC1. PC2 had a more even distribution with DRP, silicon,
boron and vanadium making strong (< -0.2) negative contributions and
selenium, calcium, uranium and sulphur making strong (> 0.2) positive
contribution (Figure 6). The PCA analysis showed that samples from similar
geographic locations tended to cluster close to one another (e.g. WA5 and
WA8), however, no distinct separation between North and South Island river
sites was evident (Figure 6). In contrast, the groundwater-fed creek sites
formed a relatively tight cluster. No obvious relationship was found between
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CAWTHRON INSTITUTE | REPORT NO. 2060
DECEMBER 2011
survival of didymo (based on designated cell survival groups) and water
chemistry (Figure 6). Survival of didymo varied among several sites with
similar elemental and nutrient compositions (e.g. DN9 and TK1; Wash Creek
and Flaxy Creek). In general, water in which didymo did best was from sites
with moderate water chemistries (e.g. CH3, GS2, TU1); these sites tended to
cluster towards the centre of the PCA analysis. In water from sites with
relatively high concentrations of one or more components, didymo either failed
to divide (e.g. AX1, RO6, TK1) or died (e.g. AX3, RO1, WA8).
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DECEMBER 2011
Figure 2.
10
REPORT NO. 2060 | CAWTHRON INSTITUTE
Survival, attachment and division of didymo cells (sourced from the Buller River)
in river and groundwater-fed creek water sourced from across New Zealand (see
Figure 1 for location of sites).
With the exception of GR- medium, an externally-sourced soil extract included
for comparison, waters to the left of the vertical dashed line were from the North
Island and to the right from the South Island. A sub-sample (200 µL) of filtered
water was added to each well of a 96-well plate, eight replicate wells per
treatment (as indicated by the horizontal dashed red line). The experiments were
conducted without Parafilm.
CAWTHRON INSTITUTE | REPORT NO. 2060
Figure 3.
DECEMBER 2011
Survival, attachment and division of didymo cells (sourced from the Buller River)
in river and groundwater-fed creek water sourced from across New Zealand (see
Figure 1 for location of sites).
With the exception of GR- medium, an externally-sourced soil extract included
for comparison, waters to the left of the vertical dashed line were from the North
Island and to the right from the South Island. A sub-sample (200 µL) of filtered
water was added to each well of a 96-well plate, eight replicate wells per
treatment (as indicated by the horizontal dashed red line). In this experiment a c.
3-mm2 piece of Parafilm was placed into the bottom of each well to provide an
attachment substrate.
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DECEMBER 2011
Figure 4.
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REPORT NO. 2060 | CAWTHRON INSTITUTE
Survival, attachment and division of eight didymo cells (sourced from the Hurunui
River) in selected river and groundwater-fed creek water sourced from across
New Zealand (see Figure 1 for location of sites).
A sub-sample (200 µL) of filtered water was added to each well of a 96-well
plate, eight replicate wells per treatment (as indicated by the horizontal dashed
red line). Results are shown for day 30 only. Treatments to the left of the vertical
dashed line were without Parafilm and to the right with Parafilm.
CAWTHRON INSTITUTE | REPORT NO. 2060
Figure 5.
DECEMBER 2011
Mean percentage of didymo cells (sourced from the Buller River) that were alive
after 20 days in selected river and groundwater-fed creek water sourced from
across New Zealand (see Figure 1 for location of sites).
With the exception of GR- medium, an externally-sourced soil extract included
for comparison, waters to the left of the vertical dashed line were from the North
Island and to the right from the South Island. A small section of didymo mat was
washed and placed in to a well of 24-well plates containing 1 mL of treatment
water. After two days the mats were removed. The health of the remaining cells,
which had detached from the mats, was monitored for 20 days. Results are
shown for day 20 only and are an average of three replicates; error bars are ± 1
standard deviation.
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DECEMBER 2011
Figure 6.
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Principal component analysis ordination showing the number of alive didymo
cells in 96-well plates containing Parafilm at day 30 in relation to elemental and
nutrient concentrations of water samples.
Group 1 S ≥ 14 cells alive, „ 9-13 cells alive, Group 3 ¡ 5-8 cells alive, Group
4 ▼ ≤ 4 Closed symbols are South Island sites, open symbols are North Island
sites and black symbols are groundwater-fed creek sites. Labels correspond to
sampling sites given in Figure 1. RO1 and DN1 were removed from the analysis
as their extreme water chemistries prevented analysis of other sites.
CAWTHRON INSTITUTE | REPORT NO. 2060
4.
DECEMBER 2011
CONCLUSIONS AND RECOMMENDATIONS
The aim of Phase 3 of this study was to investigate whether the absence of
didymo from North Island waterways and groundwater-fed creeks is related to
water chemistry. The key finding from Phase 3 was that didymo cells can
survive, attach and divide in water sourced from throughout New Zealand,
including water from North Island rivers and groundwater-fed creeks, with a
wide range of elemental and nutrient concentrations. Multivariate analysis of
water chemistry and cell survival data indicates that didymo may survive but
be unable to attach or divide (i.e. bloom) when certain elements/nutrients are
present above or below a given optimal concentration range. Based on these
findings, we recommend that measures presently in place within New Zealand
to limit to spread of didymo be continued.
In general, in single-cell assays didymo did best in water from sites with
moderate water chemistries. In water from sites with relatively high
concentrations of one or more components, didymo either failed to divide or
died. Testing of more sites throughout New Zealand is required to confirm this
trend. In conjunction, we recommend further investigation of the nutrient
requirements of didymo. We propose nutrient spiking experiments in which
select nutrients are systematically spiked into filtered river water at a range of
concentrations and the survival, attachment and stalk production of didymo
monitored. Data gathered may help guide predictive models through improved
understanding didymo distribution patterns.
As observed in Phase 2 (Kuhajek & Wood 2011), this study confirms that
attachment of didymo cells to a substrate is a prerequisite for cell division. The
addition of Parafilm (a hydrophobic substance) greatly enhanced attachment
and cell viability, suggesting that substrate composition may play an important
role in didymo attachment and subsequent survival and establishment at a
given site. Although attachment was enhanced by the presence of Parafilm,
the didymo cells did not necessarily attached to the Parafilm itself, suggesting
a potential chemical aspect to the attachment process. We propose that
attachment be further investigated through in-depth evaluation of the initial
stages of attachment using a range of hydrophobic/philc substrates.
In a river ecosystem, most substrates available to didymo as attachment sites
are covered in a bacterial biofilm, usually composed of a mixed assemblage of
heterotrophic bacteria, cyanobacteria and algae. Biofilm composition may
contribute a key component in the success of didymo establishment.
Evaluation and comparison of biofilm compositions from various sites
throughout New Zealand, particularly sites within close geographic proximity
(e.g. didymo-infested rivers and associated didymo-free groundwater-fed
creeks) is recommended.
15
DECEMBER 2011
REPORT NO. 2060 | CAWTHRON INSTITUTE
In general, cells survived better in the multi-cell experiments compared with
the single-cell experiments. Attachment in the multi-cell experiments was
prolific, even without Parafilm. This could reflect the importance of biofilm in
the attachment process given the potential for a more rapid establishment of a
biofilm layer from a grouping of didymo cells verses a single cell. Alternatively,
the improved success of didymo in the multi-cell format could be because of
additional nutrients contributed by the sections of mat initially placed in the
wells of the multi-cell experiment. A third possibility is that didymo does better
in presence of other didymo cells. Didymo may also survive better in the
presence of other organisms. In preliminary studies, didymo co-cultured with
Phormidium sp. showed enhanced survival and attachment compared with
didymo cultured alone.
Work towards a synthetic growth medium is still required to definitively further
our understanding of didymo in a laboratory setting. Initial synthetic media
trials based on the elemental and nutrient composition of GR- medium failed
(Kuhajek & Wood 2011). Nutrient spiking experiments proposed above will
form a basis for further work toward establishing a synthetic medium in which
didymo can grow.
5.
ACKNOWLEDGEMENTS
The authors thank Marion Lemoine who undertook the cell experiments
described in this report. The authors are grateful for assistance provided by
Philippe Gerbeaux (Department of Conservation), Cathy Kilroy (NIWA), Max
Bothwell (National Water Research Institute, Canada), and Kati Doehring and
Karen Shearer (Cawthron Institute). Thank you to National Institute of Water
and Atmospheric Research staff and Stu Sutherland (Fish and Game,
Southland) for collection of water samples and Steve Cameron (Waikato
University) for ICP-MS analysis of water samples.
16
CAWTHRON INSTITUTE | REPORT NO. 2060
6.
DECEMBER 2011
REFERENCES
APHA, AWWA & WEF 2005. Standard Methods for the Examination of Water
and Wastewater, 21st Edition. American Public Health Association
(APHA), American Water Works Association (AWWA) & Water
Environment Federation (WEF). 1368 p.
Kilroy C 2004. A new alien diatom, Didymosphenia geminata (Lyngbye)
Schmidt: its biology, distribution, effects and potential risks for New
Zealand fresh waters. NIWA Client Report, CHC2004-128. 34 p.
Kilroy C, Bothwell ML 2010. Factors affecting the growth and survival of
Didymosphenia geminata in rivers and their spring fed tributaries:
update of experimental investigations to June 2010. NIWA Client
Report 2010-094. 44 p.
Kilroy C, Dale M 2006. A comparison of sampling methods for the detection of
the invasive alga Didymosphenia geminata in New Zealand. NIWA
Client Report CHC2006-078. 48 p.
Kilroy C, Leathwick J, Dey K, Blair N, Roulston H, Sykes J, Sutherland D
2007. Predicting the suitability of New Zealand river and lake habitats
for colonisation and growth of the invasive, non-indigenous diatom,
Didymosphenia geminata. NIWA Client Report CHC2007-062. 93 p.
Kilroy C, Snelder T, Sykes J 2005. Likely environments in which the nonindigenous freshwater diatom, Didymosphenia geminata, can survive,
in New Zealand. NIWA Client Report 2005-043. 43 p.
Kilroy C, Unwin M 2011. The arrival and spread of the bloom-forming
freshwater diatom Didymosphenia geminata in New Zealand. Aquatic
Invasions 6: 349-362.
Kirkwood AE, Shea T, Jackson L, McCauley E 2007. Didymosphenia
geminata in two Alberta headwater rivers: an emerging invasive
species that challenges conventional views on algal bloom
development. Canadian Journal of Fisheries and Aquatic Science
64:1703–1709.
Kuhajek J, Adamson J, Wood S 2011. Experimental trials resulting in the
successful culturing of Didymosphenia geminata. Prepared for
Department of Conservation. Cawthron Report No. 1890. 15 p. plus
appendices.
Kuhajek J, Wood S 2011. Purifying and up-scaling of Didymosphenia
geminata cultures and characterisation of soil extract growth media.
Prepared for Department of Conservation. Cawthron Report No. 1949.
13 p. plus appendices.
17
DECEMBER 2011
REPORT NO. 2060 | CAWTHRON INSTITUTE
Kumar S, Spaulding SA, Stohlgren TJ, Hermann K, Schmidt T, Bahls TJ 2009.
Potential habitat distribution for freshwater diatom Didymosphenia
geminata in the continental United States. Frontiers in Ecology and
Environment 7(8): 415–420.
Lindstrøm EA, Skulberg O 2008. Didymosphenia geminata—a native diatom
species of Norwegian rivers coexisting with the Atlantic salmon. In
Bothwell ML. & SA. Spaulding (eds), Proceedings of the 2007
International Workshop on Didymosphenia geminata. Canadian
Technical Report on Fisheries and Aquatic Sciences 2795: 35–40.
Potapova M, Charles DF 2003. Distribution of benthic diatoms in U.S. rivers in
relation to conductivity and ionic composition. Freshwater Biology
48,1311–1328.
Sherbot DMJ, Bothwell ML 1993. Didymosphenia geminata
(Gomphonemaceae). A review of the ecology of D. geminata and the
physicochemical characteristics of endemic catchments on Vancouver
Island. NHRI Contribution No. 93005. National Hydrology Research
Institute, Environment Canada, Saskatoon, Saskatchewan.
Spaulding S, Elwell L 2007. Increase in nuisance blooms and geographic
expansion of the freshwater diatom Didymosphenia geminata:
Recommendations for response. White Paper. USEPA Region 8 and
Federation of Fly Fishers. (11 Sept 2009; http://www.epa.gov/region8/
water/didymosphenia/White%20Paper%20Jan%202007.pdf)
Stoermer EF, Smol JP 2001. The diatoms: applications for the environmental
and earth sciences. Cambridge University Press, England. 484 p.
Zar JH 1974. Biostatistical Analysis. Prentice-Hall Inc, Englewood Cliffs, New
Jersey, USA.
18
CAWTHRON INSTITUTE | REPORT NO. 2060
7.
DECEMBER 2011
APPENDICES
Appendix 1. Elemental and nutrient concentrations in water samples collected from 16 North Island rivers (see Figure 1 for location of sites).
All values are given in µg L-1 except silicon (mg L-1). Exceptionally high values are shaded. DRP = dissolved reactive phosphorus.
Element or
Nutrient
Boron - B
HM1
RO1
RO3
RO4
RO6
TU1
TU2
WA2
WA5
WA8
GS2
GS3
HV4
HV6
WN2
WN5
7
618
32
57
153
12
30
7
20
19
14
18
57
44
15
12
Sodium - Na
4596
12000
7388
5612
10672
4925
4432
4300
4767
8513
5287
4516
3621
4753
4774
3912
Magnesium - Mg
913
7397
1628
1214
2476
1375
2220
1103
1395
2309
1308
1205
984
1195
1089
916
Aluminium - Al
41
6
16
52
3
35
16
22
122
52
24
122
15
21
29
24
Silicon - Si
9185
20984
23081
14365
10886
9255
12859
9137
4803
4508
8581
9862
6484
11688
4528
3635
Phosphorus - P
4.61
0.00
7.05
19.94
0.00
1.66
6.82
0.00
7.64
17.75
0.00
7.36
0.00
0.00
0.00
0.00
Potassium - K
1385
7128
2338
1549
1705
1304
1268
1584
853
1487
1107
1035
456
943
458
379
Calcium - Ca
2127
8440
3822
3246
5194
5601
5817
3524
8137
11440
12568
5110
5482
6499
3970
4429
Vanadium - V
0.34
0.39
0.50
0.57
1.05
0.57
2.45
0.88
0.41
0.45
0.31
0.65
0.36
0.54
0.06
0.22
Chromium - Cr
0.00
0.44
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.11
0.09
0.00
0.00
0.00
0.00
0.00
Iron - Fe
0.140
0.023
0.057
0.094
0.001
0.130
0.010
0.096
0.100
0.130
0.055
0.240
0.004
0.022
0.011
0.006
Manganese - Mn
4.18
1.97
2.64
4.94
0.44
6.54
1.58
19.65
3.80
6.36
7.60
14.56
0.86
1.25
0.44
0.41
Cobalt - Co
0.01
0.00
0.00
0.02
0.00
0.03
0.00
0.17
0.07
0.11
0.06
0.09
0.00
0.00
0.00
0.00
Nickel - Ni
0.09
0.21
0.07
0.29
0.10
0.43
0.33
0.21
0.86
0.95
1.01
0.64
0.16
0.15
0.14
0.28
Copper - Cu
0.92
1.37
0.30
0.68
1.29
0.45
0.15
1.93
1.81
1.25
0.32
0.67
0.38
0.26
0.44
0.36
Zinc - Zn
1.17
2.42
0.75
1.15
0.91
0.70
0.91
1.10
3.34
3.40
0.30
0.90
0.67
0.96
1.16
1.34
Arsenic - As
0.00
28.45
0.00
0.00
8.46
0.09
0.26
0.00
0.15
0.37
0.00
0.00
0.12
0.05
0.00
0.04
Selenium - Se
0.43
1.37
0.30
0.32
0.20
0.00
0.00
0.35
0.19
0.07
0.52
0.27
0.00
0.00
0.35
0.49
49.67
Strontium - Sr
23.72
46.75
29.40
35.54
29.92
46.51
33.85
32.85
61.67
94.25
99.96
50.11
36.21
42.27
42.21
Cadmium - Cd
0.01
0.01
0.00
0.09
0.01
0.01
0.00
0.01
0.04
0.03
0.01
0.01
0.01
0.00
0.02
0.01
Barium - Ba
9.73
14.62
13.61
5.37
5.06
8.88
3.36
7.79
4.64
12.89
25.03
17.44
3.47
3.48
7.27
8.68
Lead - Pb
0.00
0.00
0.00
0.05
0.01
0.06
0.03
0.00
0.24
0.20
0.00
0.11
0.02
0.01
0.00
0.01
Uranium - U
0.00
0.03
0.01
0.01
0.00
0.01
0.01
0.00
0.05
0.04
0.03
0.01
0.00
0.01
0.00
0.00
DRP
7.6
2.7
22.7
23.6
1.4
9.5
15.4
7.1
3.8
8.8
11.2
10.9
3.6
9.9
4.1
2.7
1
0
3
1
1
7
1
8
0
7
7
8
0
2
2
1
464
0
1124
191
4
467
89
230
54
580
362
388
11
304
25
31
Ammonia - NH3
Nitrate - NO3
19
DECEMBER 2011
REPORT NO. 2060 | CAWTHRON INSTITUTE
Appendix 2. Elemental and nutrient concentrations in water samples collected from 17 South Island rivers (see Figure 1 for location of sites).
All values are given in µg L-1 except silicon (mg L-1). Exceptionally high values are shaded. DRP = dissolved reactive phosphorus.
Element or
Nutrient
Boron - B
20
Buller
River
NN2
NN3
CH1
CH3
TK1
TK2
TK3
TK4
AX1
AX3
DN1
DN2
DN5
DN6
DN9
DN10
20
6
12
30
39
19
16
12
6
3
4
12
7
13
8
11
4
Sodium - Na
1854
1773
1588
2798
36347
5087
4378
2079
1501
1415
1113
6714
5862
6306
2931
5124
2318
Magnesium - Mg
391
6436
517
906
4771
2081
1904
812
607
759
1100
2288
1591
2102
1323
2604
888
Aluminium - Al
13
20
17
9
26
15
6
7
24
11
27
274
62
34
7
21
8
Silicon - Si
2070
4624
2958
2505
2939
3909
4176
2439
1994
1159
1759
3310
3640
5296
4694
3614
1886
Phosphorus - P
0.00
15.53
0.00
0.00
0.00
8.16
0.67
0.00
0.00
0.00
0.00
0.00
73.70
1.95
12.03
0.00
0.00
Potassium - K
967
165
372
438
1567
906
935
459
560
688
588
2258
714
1124
546
626
507
Calcium - Ca
4834
3644
5050
7609
8440
10124
8931
4318
6727
9845
15110
5237
2560
5769
4063
8036
2355
Vanadium - V
0.10
0.12
0.16
0.12
0.24
0.23
0.19
0.11
0.18
0.07
0.13
0.69
0.16
0.40
0.18
0.73
0.09
Chromium - Cr
0.10
0.68
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.02
0.25
0.09
0.00
0.00
0.11
0.00
0.007
Iron - Fe
0.001
0.006
0.011
0.003
0.009
0.004
0.005
0.032
0.015
0.001
0.016
0.670
0.290
0.140
0.035
0.071
Manganese - Mn
2.97
0.53
0.66
0.38
5.14
1.16
1.06
1.05
0.64
0.49
2.13
22.10
9.70
4.93
3.74
1.55
0.36
Cobalt - Co
0.03
0.01
0.01
0.00
0.03
0.04
0.03
0.01
0.00
0.00
0.04
0.31
0.22
0.06
0.02
0.01
0.00
Nickel - Ni
0.38
3.24
0.34
0.17
0.28
0.35
0.35
0.20
0.17
0.35
0.51
1.21
0.95
0.72
0.11
0.44
0.09
Copper - Cu
2.57
0.26
0.04
0.55
0.90
1.02
0.94
0.75
0.30
0.24
0.70
2.15
1.43
1.18
0.48
1.00
0.44
Zinc - Zn
7.82
2.63
0.95
0.98
1.41
0.67
0.45
0.33
0.53
0.00
0.11
3.05
2.78
2.58
0.93
1.79
0.52
Arsenic - As
4.31
0.07
0.07
0.00
0.31
0.05
0.00
0.00
0.48
0.22
0.90
0.68
0.18
0.00
0.00
0.00
0.00
Selenium - Se
0.00
0.00
0.00
0.26
1.01
0.31
0.29
0.06
0.48
0.35
0.47
0.42
0.41
0.47
0.24
0.44
0.12
Strontium - Sr
46.84
32.63
54.70
71.27
69.50
76.65
65.83
33.01
73.60
63.15
112.81
54.32
23.55
46.41
29.82
52.41
17.85
Cadmium - Cd
0.03
0.01
0.01
0.00
0.02
0.00
0.00
0.00
0.00
0.01
0.01
0.03
0.01
0.01
0.01
0.02
0.01
Barium - Ba
2.07
1.45
5.10
10.52
1.63
6.18
6.19
3.01
2.27
3.95
3.11
12.71
6.26
7.77
3.24
5.39
2.84
Lead - Pb
0.10
0.02
0.04
0.00
0.06
0.01
0.00
0.02
0.05
0.00
0.01
0.41
0.06
0.02
0.00
0.01
0.00
Uranium - U
0.00
0.00
0.00
0.00
0.02
0.01
0.01
0.01
0.03
0.02
0.10
0.06
0.01
0.00
0.00
0.02
0.07
DRP
25
4.3
5.5
2.2
3.2
5.1
5.6
1.8
1.3
1.0
1.1
9.4
3.8
12.5
5.5
1.2
0.9
Ammonia - NH3
240
0
0
2
1
3
7
2
0
1
5
9
4
44
7
2
1
Nitrate - NO3
160
28
30
23
83
717
1375
190
26
25
16
94
34
1202
322
228
3
CAWTHRON INSTITUTE | REPORT NO. 2060
DECEMBER 2011
Appendix 3. Elemental and nutrient concentrations in water samples collected from 4 groundwater-fed creeks (see Figure 1 for location of sites).
All values are given in µg L-1 except silicon (mg L-1). Exceptionally high values are shaded. DRP = dissolved reactive phosphorus.
Element or
Nutrient
Boron - B
Bright
Water
Flaxy
Creek
Upper
Oreti
Wash
Creek
4
7
19
8
Sodium - Na
2301
2027
4143
4381
Magnesium - Mg
913
569
8129
5543
3
5
2
3
Silicon - Si
4228
3435
6723
7798
Phosphorus - P
0.00
0.00
0.00
0.00
Potassium - K
385
275
538
602
7652
Aluminium - Al
Calcium - Ca
2926
2549
6686
Vanadium - V
0.18
0.22
0.69
0.69
Chromium - Cr
0.00
0.00
1.08
0.35
Iron - Fe
0.001
0.001
0.001
0.001
Manganese - Mn
0.35
0.76
0.56
0.41
Cobalt - Co
0.00
0.00
0.02
0.00
Nickel - Ni
0.05
0.42
3.10
0.50
Copper - Cu
0.00
0.00
0.22
0.08
Zinc - Zn
0.96
0.73
0.47
0.42
Arsenic - As
0.00
0.01
0.00
0.00
Selenium - Se
0.00
0.00
0.00
0.00
Strontium - Sr
19.63
15.84
39.99
44.60
Cadmium - Cd
0.01
0.00
0.01
0.00
Barium - Ba
0.91
0.99
4.51
2.66
Lead - Pb
0.02
0.01
0.00
0.02
Uranium - U
0.00
0.00
0.00
0.00
DRP
9
15
6
19
Ammonia - NH3
7
5
5
7
130
1800
94
1800
Nitrate - NO3
21