M3-HABs
Updated version of the common M3HABs
protocol concerning
Ostreopsis monitoring (for summer 2015)
May 2015
CONTENTS
1. INTRODUCTION ...................................................................................................................... 3
2. “Classical methods” - Benthic and planktonic Sampling ........................................................ 3
2.1
2.2
When? Timing for sampling ...................................................................................... 3
How? ......................................................................................................................... 3
3. Deployment of artificial substrates ……….…………………………………………………………………….…….4
3.1
3.2
Set-up of artificial substrates……………………………………………………………….……………….4
Incubation and sampling………………………………………………………………….…………………..4
4. Treatment and storage .......................................................................................................... 5
4.1
4.2
4.3
4.4
How to preserve samples? ........................................................................................ 5
How to separate benthic Ostreopsis from substrates? ............................................. 5
How long between sample collection and processing? ............................................ 6
Specific focus on intercalibration between counting methods………………………………6
5. Counting procedures……………………………………………………………………………….…………………………7
5.1
5.2
5.3
How to count using classical microscopy methods? ................................................. 7
Alternative methods for counting .............................. Error! Bookmark not defined.
What to count ........................................................................................................... 8
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1. INTRODUCTION
Inter-calibration sessions between partners of the M3-HABs ENPI-CBCMED project were conducted in
2014, during meetings in Rome (Italy) and Nice (France). They gave the opportunity for partners of the
project to share their experience. One of the main deliverable was the definition of a common protocol
for monitoring of Ostreopsis blooms using classical methods (macroalgae sampling and planktonic
sampling). An alternative sampling method using artificial substrates was also tested and some
optimizations of this approach were shared with partners. Finally, main improvements were performed
for the elaboration of counting methods that are alternative to classical counts done with light
microscopy. They are related to automatic optical counts and molecular counts using qPCR. Acquisition of
data from Ostreopsis blooms in 2014 allowed for the first tests of intercalibration between counting
methods. All these procedures of sampling, preserving and counting techniques are detailed below and
associated recommendations are listed. This includes the classical common protocol that should be used
by all partners for the survey of Ostreopsis blooms during summer 2015.
2. “CLASSICAL METHODS” - BENTHIC AND PLANKTONIC SAMPLING
2.1
When? Timing for sampling
The sampling frequency has to be adapted to known seasonal variations of Ostreopsis blooms in each
survey area. For partners located in the North Part of the Mediterranean Sea, blooms are often occurring
between July and August, but extend in September-October along the North-Adriatic Sea. Monitoring of
such blooms should be performed from June to September (or October for later occurring blooms areas),
with a sampling frequency of at least one time per week as soon as “Ostreopsis blooming season” starts.
In the bloom period the frequency should be increased (every 2-3 days). For partners in Lebanon, samples
will be collected weekly in the blooming period, but a monthly sampling will be maintained all year long.
Partners agreed to fix sampling time between 8 am and 10 am for sampling during summer 2015. Morning
sampling ensures a better accessibility to sampling stations and enough time to process samples during
the afternoon. Furthermore, keeping a similar sampling time during bloom survey allows avoiding
fluctuations due to potential diel variations between benthic and planktonic pools.
2.2 How?
2.2.1
Benthic versus planktonic sampling
Collection of both benthic and planktonic samples is of great interest and should be performed as often
as possible. For each station, these samples have to be collected in a specific order: planktonic samples
before benthic ones in order to avoid a mechanical re-suspension of benthic cells that might artificially
increase planktonic concentrations.
2.2.2
Depth
Partners recommend a sampling of benthic substrates at – 0.5 m depth and a collection of planktonic
samples at 20 cm above the substrate (0.3 m depth).
2.2.3
Choice of substrate for benthic sampling
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As a recommendation, only the most abundant species of macroalgae should be used for benthic sampling
because it has to be considered as the most representative species of the site. Try to sample only one
macroalgal species (avoid mix of several species), take note of it and try to sample the same species at
each sampling time. However, if a strong modification of the macroalgal community structure is noted
during the survey, the sampling effort should be shifted towards the new strongly dominant species of
the community.
At least three stations have to be considered for each site in order to ensure replication: at each sampling
station a single replicate sample will be collected. Environmental agencies, having a large number of sites
in their monitoring programs, will sample only one station for each site. The optimal distance between
replicated stations within a site is dependent on the geography and complexity of each considered site.
However, considering stations 10m apart should work for most of the cases.
2.2.4
Material
Benthic and planktonic sampling of Ostreopsis can be performed using plastic (or glass) bottles (250 mL).
Partners agreed to define the optimal quantity of substrate for benthic sampling at 5 to 10 g fresh weight
of macroalgae.
3. DEPLOYMENT OF ARTIFICIAL SUBSTRATES
During the last intercalibration session, several partners showed their interest for the testing of artificial
substrates during monitoring of Ostreopsis blooms. A common protocol will have to be followed during
the summer 2015 in order to help for comparison of data sets. For this purpose, similar material were
provided to partners (located in Italy, Lebanon, Tunisia and France) who agreed to test artificial substrates
during their survey. Detailed information about how to deploy and sample artificial substrates were
presented during the Summer School that was hold in Tunisia in May 2015.
3.1 Set-up of artificial substrates
Artificial substrates that will be tested for bloom monitoring correspond to pieces of fiberglass screens
fixed on a rigid frame. Each frame has to be attached to a weight and a small subsurface float, so that the
device is moving freely in the water flow (see Figure 1). Pieces of artificial substrate with a width of 2.5
cm allow for an easy sampling using 250 mL plastic bottles. A length of 16.5 cm of artificial substrate is
exposed to water currents when using frames that will be provided to partners.
3.2 Incubation and sampling
Artificial substrates can be deployed in any type of environment. As a common protocol, they should be
settled for 24h at about 50 cm depth. If returning to the field two days in a row creates too much
constrains, an incubation duration of 8h (from morning to late afternoon) can be used; the impact of
deployment duration (4h, 8h, 12h and 24h) on cell collection will be characterized by the partner LOV
during summer 2015, allowing for a comparison of such data sets with the rest of the data collected after
24h of incubation. As defined for macroalgal sampling, a deployment of three devices per site (stations
10 m apart should work) will help for taking into account spatial variability of Ostreopsis abundances.
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For retrieval, the substrate is cut with scissors on one side and carefully put into a plastic bottle filled with
ambient seawater. Then, the other side of the substrate is cut and the sampling bottle is capped under
water. All samples can be fixed with 1% acidic lugol.
Figure 1: Description of the set-up for artificial substrate deployment
4. TREATMENT AND STORAGE
4.1 How to preserve samples?
For classical methods, each partner has been using a specific preservative in order to keep benthic and
planktonic samples before processing: it could be either formaldehyde or Lugol, at various concentrations.
If defining a common preserving protocol is not essential for ecological and monitoring studies, it does
have an importance for the completion of the Work Package 4, dealing with the optimization of an optoelectronic system for cell identification and counting, and Work Package 6 for the intercalibration
between molecular and microscopy methods for counting Ostreopsis abundances. For this purpose,
samples have to be fixed with 1% (vol/vol) of acidic Lugol solution (final concentration). For benthic
samples from biotic substrates, this fixative concentration is adapted to fast treatment of samples, when
the separation step (between macroalgal substrate and epiphytic microalgal cells) is done few minutes
after Lugol addition, however. If the separation step has to be delayed, macroalgae quickly absorb part of
the Lugol and an extra-addition of Lugol should be performed. More or less, the benthic samples of
isolated Ostreopsis cells should have at least a “tea color”.
4.2 How to separate benthic Ostreopsis from substrates?
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First tests analysing the influence of fixative addition on efficiency of separation between macroalgal
substrate and epiphytic microalgal cells were conducted during summer 2014. Additional data have to be
collected during summer 2015, however, in order to define recommendations regarding the most suitable
time for fixative addition (before or after the separation step). For the common protocol that will guide
sampling during summer 2015, it is recommended to add acidic Lugol fixative before the separation step
because it might help for making the mucilage looser. On the contrary, if the used fixative is formaldehyde,
it should be added after the separation step.
For isolation of epiphytic Ostreopsis cells, benthic samples should be vigorously shaken for 10 sec, then
rinsed two times with 100 mL of filtered seawater (0.2 µm) and shaken again. Similar protocols can be
used for both biotic and artificial substrates. The use of a sieve of 500 µm is useful for removing macroalgal
fragments or artificial substrates. However, in presence of macroflocs of Ostreopsis, the use of sieving
may lead to loss of cells, making the sieving step discretionary. The total volume (sample + rinsing water)
should be measured to allow for estimation of Ostreopsis densities. Abundances have to be normalized
per unit of macroalgal weight or surface of artificial substrates. In order to characterize biotic substrate
quantity, only fresh weight of macroalgal substrate has been usually measured. However, both fresh and
dry weights of macroalgal substrate quantity should be performed in order to get an appropriate
estimation for standardization of benthic Ostreopsis concentrations. A suitable drying of high weighted
samples of macroalgae might require more than 48h at 70°C.
4.3 How long between sample collection and processing?
The duration of the storage period before sample processing is dependent on the type of studies
conducted: it is restricted to 1 or 2 days for monitoring, but can be extended to several months for
ecological studies, if stored in dark conditions at +4°C. For molecular studies the maximum storage period
is one month to avoid material deterioration.
4.4 Specific focus on intercalibration between counting methods
All partners agreed to take sub-samples, at different phases of the bloom (development, peak and decline
of the bloom), in order to help for optimization of counting methods and intercalibration between them.
These sub-samples have to be stored according to the standard protocol: they have to be fixed with 1%
(vol/vol) of acidic Lugol solution (final concentration), stored at +4°C and sent for analyses in less than one
month in order to avoid any material deterioration. The same field samples (benthic or planktonic) have
to be analysed by the three different methods: classical microscopy method, automatic optical counting
and molecular qPCR method. The field sampling has to be split in three subsamples:
1- one subsample is kept for classical microscopy counting,
2- one subsample has to be sent to Massimo Vassalli and Francesca Sbrana (IBF-CNR, Italy) for automatic
optical counting,
3- one subsample has to be sent to Antonella Penna and Silvia Casabianca (CoNISMa, Italy) for molecular
counting.
A total number of 10 samples (five samples of benthic macroalga and five samples of seawater) per each
geographical area (i.e. Genoa, Villefranche, Nice, Ancona, Tunis, Batroun) during the monitoring activity
of summer 2015, will be analysed at the same time by the three methods: these samples must be collected
at 5 different periods by the blooming season. Collection of different samples is an advantage for
optimization of counting techniques and intercalibration. All measurements (optical, automatic and
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molecular) have to be performed in triplicates and counted by the same operator. This will help for
standardization of protocols and comparison between methods. For example, a planktonic sample for
intercalibration should be counted by the three methods:
a- 50-100 ml per replicate for classical optical method (three sedimentation events have to be
performed for each sample in order to get three replicated values)
b- a total of 200 ml for automatic counting (enough volume for three replicated measurements)
c- a total of 50 ml for molecular counting (enough volume for three replicated measurements).
-For automatic optical counting (Francesca Sbrana IBF-CNR) it is required a volume of 50 ml per each
macroalgal wash seawater subsample and 200 ml per each seawater subsample; these volumes will allow
three automatic counts per each category of sample.
-For molecular counting (Antonella Penna and Silvia Casabianca, CoNISMa Italy) it is required a volume of
50 mL per macroalgal wash seawater sample and 50 mL per seawater sample; these volumes will allow
three molecular counts per each category of sample.
It is important providing the fresh weight of macroalgae and washing volume of macroalgae per each
sample. Please, for shipping utilize a fast mail delivery and include ice-packs in the box for transport.
5. COUNTING PROCEDURES
5.1 How to count using classical microscopy methods?
In order to optimize time for sampling and processing, partners agreed to limit the number of counting
per sample to one (except for the samples dedicated to intercalibration): the maximum effort should be
focused on analyzing variability at small scale, monitoring as much stations as possible and analyzing
benthic and planktonic populations separately as often as possible.
The easiest way to count benthic populations requires the use of a Sedgewick Rafter Counting cell (1 mL)
and a classical microscope. Some partners use the Utermöhl method, where a subsample (1-25 mL) is
poured in a cylinder/chamber complex and left to settle before observation at the inverted microscope.
For planktonic samples, all partners use the Utermöhl method settling volumes from 10 to 100 mL. For
monitoring, a sedimentation volume of 50 mL is recommended, with a 24h settling time. For all samples,
at least 200 cells have to be counted per sample during the bloom duration.
5.2 Alternative methods for counting
5.2.1
Automatic optical counts
The development of an automatic opto-electronic system for counting Ostreospsis cell abundances is part
of Work Package 4. Good progress has been made in 2014 for this approach, including the definition of
an algorithm capable of recognising Ostreopsis cells. This methodology involved three main steps: the
segmentation (identifying objects out of the background and main debris), the identification (including
species identification) and the counting. M. Vassalli and E. Landini (Istituto di Biofisica, Italy) pointed out
potential errors in cell quantification associated with one or two of these steps. In particular, in aggregates
of cells, some algorithms can fail to identify objects and lead to an underestimation of cell numbers.
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However, the work done using 3D approaches showed promising results, with efficient separation
features and better species identification in mixed samples where different species of similar shape are
present (e.g. Ostreopsis cf. ovata and Prorocentrum lima).
5.2.2
Counts using molecular techniques
Field samples collected during summer blooms in 2014 were used to run qPCR identification and
quantification of Ostreopsis spp. These molecular analyses were applied to Ostreopsis cf. ovata and
Ostreopsis cf. siamensis, as part of the Work Package 6. For qPCR analysis, LSU rDNA was used as target
sequences and SYBR Green as the fluorescent dye for DNA quantification. Testing the efficiency of this
approach, qPCR showed a high sensitivity, high specificity and high rapidity. As a main advantage, qPCR
method allows species-specific identification and a counting efficiency often more sensitive than light
microscopy.
The qPCR assays run from benthic, planktonic and aerosol samples collected along the Mediterranean
coast showed that mainly O. cf. ovata was found in the study sites. Quantification of O. cf. ovata cells was
obtained using environmental site-specific standard curves generated in order to obtain O. cf. ovata LSU
copy number/cell at each site. This 1st set of values was compared to counting results provided by M3HABs
partners who used light microscopy for Ostreopsis cell enumeration showing comparable results.
5.2.3
Intercalibration among counting methods
During monitoring of summer 2014, sub-samples were collected by all partners in order to help for
optimization of counting methods and intercalibration among them. For the opto-electronic system, the
material already sent allowed for the definition of a digital representation of an Ostreospsis cell, which
was essential for species identification. However, more field material (preserved according to the
distributed protocol, intercalibration section) is required to continue the research effort in improving this
automatic counting system. Additional sub-samples should also be collected during summer 2015 in order
to increase the data set available for comparison between molecular counts and light microscopy results.
5.3 What to count?
For both benthic and planktonic samples, all partners are currently analyzing the abundance of Ostreopsis
species and of other benthic microalgal taxa, with particular attention to potentially toxic dinoflagellates
(e.g., Coolia, Prorocentrum, Amphidinium). Moreover, microphytobenthos dominant taxa (e.g., diatoms,
cyanobacteria etc.) are also counted in some laboratories, to collect additional and crucial information
about the environmental conditions promoting or limiting Ostreopsis bloom development. If such
sampling efforts are currently made, they should be maintained (if possible) for ecological studies and
monitoring performed in 2015.
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