RESEARCH ARTICLE
Cell-speci¢c b-N -acetylglucosaminidase activity in cultures and ¢eld
populations of eukaryotic marine phytoplankton
Alena Štrojsová1,2 & Sonya T. Dyhrman1
1
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts; and 2Institute of Hydrobiology, Biology Centre AS CR, and
Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic
Correspondence: Sonya T. Dyhrman,
Biology Department, Woods Hole
Oceanographic Institution, Woods Hole,
Massachusetts 02543, USA. Tel.: 11 508 289
3608; fax: 11 508 457 2134; e-mail:
[email protected]
Received 23 September 2007; revised 23
January 2008; accepted 6 February 2008.
First published online 21 April 2008.
DOI:10.1111/j.1574-6941.2008.00479.x
Editor: Riks Laanbroek
Keywords
chitin cycle; diatoms; dinoflagellates; DOM; ELF
method; N -acetylglucosamine.
Abstract
It is widely appreciated that eukaryotic marine phytoplankton can hydrolyze a
variety of compounds within the dissolved organic matter (DOM) pool in marine
environments. Herein, cultures and field populations of marine phytoplankton
were assayed for b-N-acetylglucosaminidase activity, a terminal enzyme of chitin
degradation. A traditional bulk assay, which can assess hydrolytic rate, but is not
cell-specific, was complemented with a cell-specific assay that images the activity
associated with single cells using an enzyme labeled fluorescence (ELF) substrate.
b-N-acetylglucosaminidase activity was widespread across various taxa of marine
phytoplankton, and activity was observed both under controlled culture conditions and in field populations. The number of cells with enzyme activity varied
with the nutritional physiology of the test species in three of the 17 cultures tested.
In these three cases the number of cells with activity in the low nutrient medium
was higher than in nutrient replete medium. Taken together, these data suggest
that a broad group of marine phytoplankton may be a relevant part of chitin-like
DOM degradation and should be incorporated into conceptual models of chitin
cycling in marine systems.
Introduction
Microbial hydrolytic ectoenzymes are an important, if not
crucial, part of nutrient fluxes in aquatic environments due
to their capability to efficiently cleave dissolved organic
matter (DOM). This is significant because DOM is one of
the largest dynamic pools of organic carbon and other
nutrients in the ocean (Hedges, 1992) and it is well understood that the turnover rate of DOM influences primary
production (Benitez-Nelson & Buesseler, 1999). Marine
heterotrophic bacteria and cyanobacteria have long been
known to hydrolyze DOM (Cho & Azam, 1988; Hoppe et al.,
1993). Eukaryotic marine phytoplankton are also known to
hydrolyze DOM, however, organic matter utilization and
hydrolysis is less well-understood in this group than in the
prokaryotic microorganisms. The main groups of microbial
ectoenzymes involved in degradation of DOM include
phosphatases, proteases, lipases, nucleases and glycosidases
such as a- and b-glucosidase, chitinase and b-N-acetylhexosaminidase (Jansson et al., 1988; Karner & Herndl, 1992;
FEMS Microbiol Ecol 64 (2008) 351–361
Christian & Karl, 1995; Martinez et al., 1996; Hoppe, 2003;
Vrba et al., 2004). However, in cultures and field populations
of eukaryotic phytoplankton, only phosphatases and proteases have been extensively studied, (Jansson et al., 1988;
Berges & Falkowski, 1996; Hoppe, 2003) and relatively little
is known about other enzymes that could be involved in
eukaryotic degradation of DOM.
One important group of ectoenzymes are those involved
in chitin biodegradation (Gooday, 1990a; Nedoma et al.,
1994). Chitin, composed of 1,4-b-linked units of N-acetylglucosamine (about 7% nitrogen – atomic concentration),
is widespread in the marine environment (Jeuniaux & VossFoucart, 1991) and has been identified in some marine
algae, such as chromophytes, diatoms and prymnesiophytes
(Blackwell et al., 1967; Smucker & Dawson, 1986; Round
et al., 1990; Mulisch, 1993; Chrétiennot-Dinet et al., 1997).
Chitin is rapidly recycled in most environments, potentially
serving as an important source of both carbon and nitrogen
nutrition to organisms capable of breaking it down (Gooday, 1990a; Nedoma et al., 1994). Chitinolytic activity in
2008 Federation of European Microbiological Societies
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c
352
aquatic environments is best studied in bacteria, but has
been characterized in other groups including arthropods
(Vrba et al., 1993; Vrba & Macháček, 1994; Cottrell et al.,
1999; Baty et al., 2000). The chitin polymeric chain is
hydrolyzed by chitinase (E.C. 3.2.1.14) to small oligosaccharides, including dimers composed of b-N-acetylglucosamine units that can be further hydrolyzed by b-Nacetylhexosaminidase (E.C. 3.2.1.52) to a final product, Nacetylglucosamine. In some cases b-N-acetylhexosaminidase
can also act weakly as an exochitinase, cleaving N-acetylglucosamine residues from the nonreducing ends of longer
chitin chains (Gooday, 1990b). Here, we refer to b-Nacetylhexosaminidase as b-N-acetylglucosaminidase, which
is more commonly used in both field and culture studies and
is synonymous with the IUBMB Enzyme Nomenclature.
It has been suggested that diatoms might significantly
contribute to bulk b-N-acetylglucosaminidase activity during diatom blooms in freshwater (Vrba et al., 1996, 1997,
2004), and some diatoms may also take up free dissolved Nacetylglucosamine (Smucker, 1991; Nedoma et al., 1994).
Consistent with these studies, the genome of the centric
marine diatom Thalassiosira pseudonana includes genes for
both chitin synthesis and degradation (Armbrust et al.,
2004), and the pennate diatom Phaeodactylum tricornutum
contains two chitin synthase gene sequences (Durkin et al.,
2007).
Despite the potential significance of chitin and its recycling in aquatic systems, little is known about the presence
of chitinolytic enzymes in marine phytoplankton. Much of
our understanding of these enzymes in marine phytoplankton stems from a single study. In this study the authors
assayed b-N-acetylglucosaminidase activity of eight unialgal, but not axenic, marine eukaryotic phytoplankton
species using the fluorogenic substrate methylumbelliferone-N-acetyl-b-D-glucosaminide (Sherr & Sherr, 1999).
Relatively high activity was found in all cultures, although
this has not been broadly examined in cultures or field
populations, and the contribution of heterotrophic bacteria
to the observed activities is not well constrained (Sherr &
Sherr, 1999). In fact, part of the challenge with using
traditional fluorometric assays of ectoenzyme activity, such
as that employed by Sherr & Sherr (1999), is the difficulty in
distinguishing between activity in heterotrophic bacteria
and eukaryotic phytoplankton. However, the development
of enzyme labeled fluorescence (ELF) now allows researchers
to assay for the presence of certain enzyme activities in a
cell-specific manner. Gonzáles-Gil et al. (1998) first developed and applied an ELF assay for direct detection of
alkaline phosphatase activity in cultures of marine phytoplankton. This assay has now been used in many studies to
identify alkaline phosphatase activity in cultures and field
populations of marine and freshwater phytoplankton
(Dyhrman & Palenik, 1999; Rengefors et al., 2001, 2003;
2008 Federation of European Microbiological Societies
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c
A. Štrojsová & S. T. Dyhrman
Dyhrman et al., 2002; Nedoma et al., 2003, 2007; Štrojsová
et al., 2003, 2005; Dignum et al., 2004; Lomas et al., 2004;
Cao et al., 2005; Ruttenberg & Dyhrman, 2005; Dyhrman &
Ruttenberg, 2006; Nicholson et al., 2006; Štrojsová & Vrba,
2006). Application of the ELF assay is limited by ELF type
substrate availability and to date application of the ELF
technology in marine systems has been limited to alkaline
phosphatase assays.
In this study, we investigated cultures and field populations of marine phytoplankton for the presence of b-Nacetylglucosaminidase activity. Herein, we complemented
traditional bulk assays of activity with the ELF approach for
imaging the activity associated with single cells. To our
knowledge, this is the first study on direct detection of bN-acetylglucosaminidase activity in marine phytoplankton
cultures and field populations using the ELF method.
Materials and methods
Field site and sampling
Salt Pond (41149.9 0 N, 69158.0 0 W), a coastal embayment,
was sampled weekly from March 29 to May 19, 2006. Net
tow samples (20 mm) were collected from near-surface water
and the concentrated phytoplankton sample was split for
analyses of chlorophyll a (Chla), bulk enzyme activity, and
cell-specific enzyme activity. Samples for enzyme activity
were transported on ice in a cooler to the laboratory and
processed immediately. Two 50 mL aliquots from the net
tow were collected onto GF/F filters and stored at 20 1C
for Chla analysis. The embayment was also sampled for
nutrient analyses. Nutrient samples were taken with a
modified integrated water sampler as described by Franks
(1995) at four sampling sites. The integrated sample was
filtered through a precombusted GF/F filter and stored at
20 1C before analysis, as highlighted below.
Culture strains and conditions
The screening for b-N-acetylglucosaminidase activity was
performed with seventeen species of marine phytoplankton
(Table 1) obtained from Provasoli-Guillard National Center
for Culture of Marine Phytoplankton or kindly provided by
D. Anderson of the Woods Hole Oceanographic Institution.
Batch cultures were grown at 19 1C (Chaetoceros gracilis,
Chaetoceros neogracilis, axenic T. pseudonana, axenic Thalassiosira weissflogii, Alexandrium minutum, A. cf. fundyense,
Gymnodinium catenatum, axenic Lingulodinium polyedra,
Prorocentrum minimum, Isochrysis galbana, axenic Aureococcus anophagefferens, Chattonella antiqua, Chattonella
marina, Heterosigma akashiwo) or 15 1C (Pseudo-nitzschia
multiseries and axenic Scrippsiella trochoidea) or 24 1C
(Karenia brevis) according to the strain preferences using a
14:10 h light–dark cycle provided by cool white fluorescent
FEMS Microbiol Ecol 64 (2008) 351–361
353
Chitinolytic activity in marine phytoplankton
Table 1. Eukaryotic marine phytoplankton cultures assayed for b-Nacetylglucosaminidase activity, and expressed as the percentage of ELFNAG labeling under replete, nitrogen deficient ( N), or phosphorus
deficient conditions ( P)
ELF-NAG labeling (%)
Strain
Bacillariophyceae
Chaetoceros gracilis
Chaetoceros neogracilis
Pseudo-nitzschia multiseries
Thalassiosira pseudonana
Thalassiosira weissflogii
Dinophyceae
Alexandrium minutum
Alexandrium cf. fundyense
Gymnodinium catenatum
Karenia brevis
Lingulodinium polyedra,w
Prorocentrum minimum
Scrippsiella trochoidea,w
Pelagophyceae
Aureococcus
anophagefferens ,w
Prymnesiophyceae
Isochrysis galbana
Raphidophyceae
Chattonella antiqua
Chattonella marina
Heterosigma akashiwo
ELF positive species; total = 17
Replete
N
P
Unknown
0
Unknown
0
CLN-34
21
CCMP1335 0
CCMP1336 5.7
0.5
0.5
74
0
11
0
0
86
NA
NA
AMBOP006 0
SPE10-03
20
CCMP1937 88
WILSON
0
CCMP2021 86
CCMP1329 0
CCMP1599 0
81
12
95
39
73
30
0
70
31
63
NA
91
76
38
CCMP1984
0
0
0
T-ISO
0
0
0
22
19
0
7
0.6
25
0
12
HK
HK
HK
3.8
6.5
0
9
Axenic culture.
w
L-1 trace metals.
bulbs with c. 200 mmol quanta m2 s1. The species were
grown in f/2 medium with silica (Bacillariophyceae and
Dinophyceae) or f/2 medium with out silica (Pelagophyceae,
Prymnesiophyceae and Raphidophyceae) in 0.2-mm filtered
local seawater containing natural concentrations of chitin
and other DOM (Guillard, 1975). Selected replicate cultures
(C. gracilis, C. neogracilis, I. galbana) were also grown in
medium with artificial seawater (Lyman & Fleming, 1940)
without natural chitin or DOM. Quadruplicate treatments
were grown for each species in nutrient replete (replete),
nitrogen deficient ( N), and phosphorus deficient ( P)
medium. Replete cultures were grown in medium with
36.3 mM NaH2PO4 and 880 mM NaNO3 and f/2 trace metals
(Guillard & Ryther, 1962), although L-1 trace metals were
used for A. anophagefferens, L. polyedra, and S. trochoidea
(Guillard & Hargraves, 1993). Low-nitrogen ( N) medium
was prepared as described above, but with 40 mM NaNO3
and low-phosphate ( P) medium was prepared with 1 mM
NaH2PO4. For the species that required silica, NaSiO3 9H2O
was added to the medium at a final concentration of
107 mM. In all cases, filter-sterilized f/2 vitamins (vitamin
B12, biotin, thiamin; Guillard, 1975) were added after
FEMS Microbiol Ecol 64 (2008) 351–361
autoclaving. Growth of all cultures was monitored by
relative fluorescence with a 10-AU fluorometer (Turner
Designs), or with direct cell counts. The activity of the bN-acetylglucosaminidase was measured in exponential or
late-exponential phase of the culture growth, when growth
in the N, P cultures was diminished relative to the
replete culture.
Cell-specific detection of b- N acetylglucosaminidase activity
An ELF substrate for b-N-acetylglucosaminidase was used to
examine cell-specific activity in phytoplankton cultures and
field populations. The soluble fluorogenic ELF97 N-acetylb-glucosaminide substrate (ELF-NAG, Invitrogen E22011)
is hydrolyzed by b-N-acetylglucosaminidase into an insoluble fluorescent ELF-alcohol which precipitates at the site of
enzyme activity. The precipitation of the ELF-alcohol can be
sensitive to pH. Here, we note that in previous work we have
demonstrated that the presence and fluorescence of the ELFalcohol is stable at pH 8, but can be diminished at pH 4 9
(Štrojsová et al., 2003). Many studies have applied ELFbased assays in seawater without pH adjustment (Dyhrman
& Palenik, 1999; Baty et al., 2000; Dyhrman et al., 2002;
Lomas et al., 2004; Dyhrman & Ruttenberg, 2006; Nicholson
et al., 2006; Nedoma et al., 2007), but care should be taken in
the application of this approach in systems where the pH
may be higher than that of seawater. The protocol for ELF
labeling of b-N-acetylglucosaminidase activity was modified
from Baty et al. (2000). The ELF-NAG was dissolved in
dimethyl sulfoxide at a stock concentration of 10 mM, and
stored at 20 1C before use. For each sample, 10 mL of
ELF-NAG stock was added to duplicates of 2 mL of culture
(replete, N and P) or into duplicate field samples with
a typical pH range of 7.9–8.1; the final concentration of ELFNAG was 50 mM. The samples were incubated in the dark at
20 1C for 3–5 h and the presence of b-N-acetylglucosaminidase was examined immediately in subsamples of this
incubation. Incubation times were determined empirically
for each sample. Cells were imaged immediately after
incubation using a Zeiss Axioplan 2 (Zeiss, Germany)
epifluorescence microscope, equipped with a X-Cite 120
lamp (EXFO Electro-Optical Engineering, Canada) and
a DAPI long-pass filter set. Color-scale images were
collected with an AxioCam color camera and AxioVision
4.2 image analysis system (Zeiss, Germany). Both autofluorescence of Chla (red) and the fluorescence (green) of
ELF-alcohol precipitates were visible in the DAPI filter
set. At least 100 cells from cultures and as many cells
as possible up to 100 of each type from the phytoplankton field samples were scored as either ELF positive or
negative and the percentages of ELF positive cells were
calculated.
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354
Bulk b- N -acetylglucosaminidase activity
Bulk enzyme activity was determined using a colorimetric
substrate 4-nitrophenyl N-acetyl-b-D-glucosaminide (NPNAG, Sigma-Aldrich N-9376). Briefly, cells from cultures
or from the field were centrifuged for 5–10 min at o 1400 g
and cell pellets were resuspended in 100 mL of 0.1 M
potassium phosphate buffer. These cell resuspensions were
examined microscopically, with no evidence of broken cells,
and were determined to be gentle enough to avoid breaking
cells and losing activity in this step of the protocol. NP-NAG
(100 mL at 2.92 mM) was added and the samples were
incubated for 5 h in the dark at 20 1C. After this incubation,
1 mL of 0.2 M borate buffer was added and the cell suspension was immediately spun at 10 000 g for 5 min to remove
cellular debris. The supernatant was assayed in a UV-1700
Shimadzu spectrophotometer at 410 nm. Blanks contained
autoclaved distilled water and the NP-NAG. Calibration
curves were obtained using 4-nitrophenol (Sigma) with
concentrations ranging from 0.05 to 20 mM in 0.2 M borate
buffer. Enzyme activity was normalized to Chla concentration (in the net tow) for field samples or cell number for
cultures.
Nutrient and Chla analyses
Concentrations of nitrate and nitrite (NO
3 , NO2 ), dissolved
inorganic phosphorous (DIP), ammonium (NH1
4 ), and
)
were
measured
colorimetrically,
using a
silicate (SiO
2
Lachat nutrient auto-analyzer with four-channel continuous
flow injection system (Zellweger Analytics, Quickchem 8000
Series). Dissolved inorganic nitrogen (DIN) is reported here
1
as the sum of NO
3 , NO2 and NH4 . Chla concentration was
determined in duplicate after extraction in 90% acetone
using a Turner Designs Aquafluor fluorometer; the concentration was calculated with the acid correction (Lorenzen,
1967).
Results
Bulk b-N-acetylglucosaminidase activity was variable, but
detectable in all phytoplankton net tow samples taken from
Salt Pond (Table 2). The activity ranged from 0.02 to
0.17 mmol mg Chla1 h1. DIN and DIP also varied through
the sampling period and in all cases the DIN:DIP ratio was
below Redfield (Table 2). There was no significant correlation between DIN, DIP, silicate, or DIN : DIP ratio and bulk
b-N-acetylglucosaminidase activity (data not shown).
Cell-specific b-N-acetylglucosaminidase activity was present in some phytoplankton species from every Salt Pond
sample tested (Table 3). As with the bulk activity, there was
no significant correlation between the percentage of ELF
labeling within a taxa and the concentration of any of the
nutrients (data not shown). The percentage of ELF labeling
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c
A. Štrojsová & S. T. Dyhrman
Table 2. Selected chemical parameters and bulk b-N-acetylglucosaminidase activity in Salt Pond during the spring sampling period
Parameters
Mean (min–max)
DIP (mM)
DIN (mM)
DIN : DIP
SiO
2 (mM)
Salinity
b-N-acetylglucosaminidase (mmol mg Chla1 h1)
0.68 (0.11–1.87)
2.47 (0.30–8.47)
3.8 (0.6–8.4)
9.86 (4.40–19.80)
28.96 (20.0–30.5)
0.079 (0.02–0.17)
The enzyme activity was determined from 4 20 mm samples and
normalized to Chla.
Table 3. ELF-NAG labeling of phytoplankton species from Salt Pond
ELFpositive
Bacillariophyceae
Asterionellopsis sp.
Chaetoceros spp.
Cylindrotheca sp.
Gyrosigma sp.
Melosira sp.
Pseudo-nitzschia sp.
Skeletonema sp.
Stephanopyxis sp.
Thalassionema sp.
Thalassiosira spp.
Pennate diatoms
Dictyochophyceae
Dictyocha sp.
Dinophyceae
Alexandrium cf. fundyense
Ceratium sp.
Protoperidinium sp.
Unidentif. dinoflagellates
Total
ELFnegative
Total
0
23
7
26
0
2
14
0
3
19
5
41
409
117
76
94
58
0
72
90
547
132
41
432
124
102
94
60
14
72
93
566
137
0
24
24
27
2
5
9
142
188
0
0
68
1916
215
2
5
77
2058
Labeled
(%)
NA
5.3
5.6
25.5
0
3
NA
0
3.2
3.4
3.6
NA
13
NA
NA
12
6.0
NA the percentage of labeled cells were calculated if total cell number
was 4 60.
within diatoms and dinoflagellates (the two dominant
eukaryotic phytoplankton groups) was not remarkably high,
typically o 10% of total observed cells, except for April 12,
where more than 30% of dinoflagellate cells were positive for
b-N-acetylglucosaminidase activity (Fig. 1). The cell-specific activity did not track directly with the bulk enzyme
activity (Fig. 1).
Of the 14 algal taxa observed over the course of the field
sampling, most (10 out of 14) exhibited ELF labeling on one
or more occasions (Table 3). Moreover, several positive
unidentified pennate diatoms and dinoflagellates were observed with labeling. Typically, ELF labeling resulted in
intense green fluorescence over most of the cell (Fig. 2f–j),
although there was some variability between species. Not
every cell of a particular species had activity within individual samples. Of the diatoms, Gyrosigma sp. had the highest
FEMS Microbiol Ecol 64 (2008) 351–361
355
% of labeled cells
40
0.3
Dinoflagellates
Diatoms
30
0.2
20
0.1
10
ND
0
0
10
20
ND
30
40
0.0
β-N-acetylglucosaminidase
activity (µmol µg Chla−1 h−1)
Chitinolytic activity in marine phytoplankton
50
Day
Fig. 1. The percentage of ELF-NAG labeled diatom or dinoflagellate cells
and 4 20 mm bulk b-N-acetylglucosaminidase activity in a coastal
embayment (March 29–May 19, 2006). Error bars – SEM; ND – no data.
average percentage of cells with activity (Table 3; Fig. 2h). Of
the dinoflagellates, Alexandrium fundyense had the highest
average percentage of cells with activity (Table 3, Fig. 2g).
Within different diatom taxa, usually 3–5% of the counted
cells were ELF labeled, whereas 12–13% were positive within
dinoflagellates (Table 2).
To examine the presence of bulk and cell-specific activity
in different cultured taxa and to assess the sensitivity of the
activity to nutrient physiology, 17 cultures were grown in
three different nutrient conditions: replete, N and P, all
in the presence of natural concentrations of chitin and
DOM. Several of the cultures were also tested in medium
made with artificial seawater, and lacking in natural chitin
or DOM. The patterns in both bulk enzyme activity and ELF
labeling were the same for all the culture treatments tested in
media with or without naturally occurring DOM.
Bulk b-N-acetylglucosaminidase activity was detected in
15 of 17 cultured species (Table 4). These bulk data were
compared with the cell-specific data to ascertain in which
cases the phytoplankton may be contributing to that activity. In the five axenic cultures tested, bulk activity was
detected in T. weissflogii, L. polyedra, S. trochoidea in
conjunction with the detection of cell-specific activity (Table
4). The highest activities were detected in the cultures of the
diatom T. weissflogii (1524.2 nmol L1 h1) and the dinoflagellate L. polyedra (655.5 nmol L1 h1). No bulk or cellspecific activity was detected in T. pseudonana or A. anophagefferens (Table 4). As most of the cultures were not axenic,
enzyme activity in the other species may be attributed to
heterotrophic bacteria, phytoplankton, or some combination of the two without the benefit of cell-specific ELFdetected data. In all cases, the ELF results were consistent
with the bulk results. For example, the P. minimum culture,
bulk activity was detected in both N and P, but not in
the replete condition and the same result was obtained with
the ELF assay (Table 1).
FEMS Microbiol Ecol 64 (2008) 351–361
The cell-specific b-N-acetylglucosaminidase activity was
detected in 13 cultures of 17 inspected (Table 1; Fig. 2),
although we note that activity was rarely detected in an
additional two species of Chaetoceros. Cell-specific activity
was typically present in one or more treatments within the
diatoms and dinoflagellates (Table 1). Only four species had
no detectable labeling across all treatments: T. pseudonana,
I. galbana (Fig. 2d), A. anophagefferens and H. akashiwo
(Table 1). Overall, higher percentages of ELF labeling were
detected within the Dinophyceae, while in Bacillariophyceae,
Pelagophyceae and Raphidophyceae, the labeling was lower
except for the diatom P. multiseries (Table 1; Fig. 2j). The
highest percentage of positive cells were observed in cultures
of the dinoflagellate G. catenatum (Table 1, in replete and
N), and L. polyedra (Table 1, in replete and P, Fig. 2a).
High percentages of labeled cells in N and P conditions
and no labeled cells in replete was found in dinoflagellate
cultures of A. minutum and P. minimum (Table 1). A similar
trend of increased ELF-labeling in the N and P
cultures, was also observed for the diatom, P. multiseries.
Other than these three cases (two dinoflagellates, one
diatom) there was not a clear trend in the percentage of
ELF labeling between the different treatments. We note that
under the culture conditions used in this study, 100%
labeling was never detected, although it reached over 75%
of the culture in the case of P. multiseries, A. minutum,
G. catenatum, L. polyedra, and P. minimum (Table 1).
Discussion
b-N-acetylglucosaminidase activity is not well characterized
in marine eukaryotic phytoplankton despite its potential
importance to chitin remineralization and DOM cycling.
The ELF b-N-acetylglucosaminidase substrate has recently
been used to discriminate the activity of bacteria cultured on
chitin and silicon surfaces and within rotifers in a freshwater
reservoir (Baty et al., 2000; Štrojsová & Vrba, 2005), yet there
has been no application of this substrate in marine systems.
Applying the ELF assay with eukaryotic marine phytoplankton, we directly linked the presence of b-N-acetylglucosaminidase activity with a particular phytoplankton species,
and thus examined patterns of DOM hydrolysis at the single
cell level in near real-time.
b-N -acetylglucosaminidase activity in marine
phytoplankton
Table 4 summarizes the eukaryotic marine phytoplankton
species that have been examined for b-N-acetylglucosaminidase activity to date. It is apparent that the activity
is widespread across various taxa in culture and in field
populations. In our study, we combined cell-specific assays
with traditional approaches to localize the presence of the
activity within the different eukaryotic species. In axenic
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356
A. Štrojsová & S. T. Dyhrman
Fig. 2. Micrographs of cells from cultures (a) Lingulodinium polyedra, (b) Scrippsiella trochoidea, (c) Chattonella marina, (d) Isochrysis galbana, (e)
Pseudo-nitzschia multiseries) and from field samples (f) Protoperidinium sp., (g) Alexandrium cf. fundyense, (h) Gyrosigma sp., (i) Skeletonema sp., (j)
Pseudo-nitzschia sp. Left panels are brightfield images, right panels are taken with a DAPI long-pass filter set, where red indicates chlorophyll
autofluorescence and green indicates b-N-acetylglucosaminidase activity. Note the two examples of unlabeled species, without activity (c and d). The
white scale bar is 10 mm.
cultures the cell-specific observations supported the bulk
assay data. In some cases, our nonaxenic cultures had
detectable bulk b-N-acetylglucosaminidase activity, but
were not ELF labeled (Table 4). Here, the bulk activity is
likely due to heterotrophic bacteria. In diatoms, there was
considerable heterogeneity in labeling, and bulk enzyme
activity, between species. ELF positive cells were detected in
both replete and N treatments of T. weissflogii, with no
detectable bulk or cell-specific activity in T. pseudonana.
These observations are consistent with other studies that
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c
have employed the bulk assay alone on T. pseudonana and
T. weissflogii (González et al., 1993; Sherr & Sherr, 1999).
Genes encoding at least 22 putative chitinolytic enzymes
were found in the T. pseudonana genome (Armbrust et al.,
2004), suggesting that chitinolytic activity might be dynamic and possibly regulated at different life-cycle stages or
under other conditions not examined here. In the nonaxenic
cultures C. gracilis and C. neogracilis, we detected bulk
b-N-acetylglucosaminidase activity, but given the negligible
percentage of labeling and the presence of this activity in the
FEMS Microbiol Ecol 64 (2008) 351–361
357
Chitinolytic activity in marine phytoplankton
Table 4. Summary of all marine phytoplankton species assayed for b-N-acetylglucosaminidase activity
b-N-acetylglucosaminidase activity
Substrate
Bacillariophyceae
Asterionellopsis sp.
Chaetoceros gracilis Schuettw
Chaetoceros neogracilis Van Landinghamw
Chaetoceros spp.
Cylindrotheca sp.
Gyrosigma sp.
Melosira sp.
Pseudo-nitzschia multiseries (Hasle) Haslew
Pseudo-nitzschia sp.
Stephanopyxis sp.
Thalassionema sp.
Thalassiosira pseudonana (Hustedt) Hasle et Heimdalz
Thalassiosira weissflogii (Grun.) Fryxell et Haslez
T. weissflogii (Grun.) Fryxell et Haslew
Thalassiosira spp.
Cryptophyceae
Chroomonas salina (Wislouch) Butcher w
Cryptomonas profunda Butcherw
Dictyochophyceae
Dictyocha sp.
Dinophyceae
Alexandrium minutum Halimw
Alexandrium cf. fundyensew
A.cf. fundyense
Ceratium sp.
Gymnodinium catenatum Grahamw
Gymnodinium simplex (Lohmann) Kofoid et Swezyw
Heterocapsa triquetra Steinw
Karenia brevis (Davis) Hansen et Moestrupw
Lingulodinium polyedra (Stein) Dodgez
Prorocentrum minimum (Pavillard) Schillerw,z
Protoperidinium sp.
Scrippsiella trochoidea (Stein) Loeblich IIIz
Scrippsiella sp.w,‰
Pelagophyceae
Aureococcus anophagefferens Hargraves et Sieburthz
A. anophagefferens Hargraves et Sieburthz
Prasinophyceae
Dunaliella tertiolecta Bucherw,‰
Prymnesiophyceae
Isochrysis galbana Parkew
I. galbana Parkew
Emiliania huxleyi (Lohmann) Hay et Mohlerw
Raphidophyceae
Chattonella antiqua (Hada) Onow
Chattonella marina (Subrahmanyan) Hara et Chiharaw
Heterosigma akashiwo (Hada) Hadaw
ELF-NAG
NP-NAG/ELF-NAG
NP-NAG/ELF-NAG
ELF-NAG
ELF-NAG
ELF-NAG
ELF-NAG
NP-NAG/ELF-NAG
ELF-NAG
ELF-NAG
ELF-NAG
NP-NAG/ELF-NAG
NP-NAG/ELF-NAG
MUF-NAG
ELF-NAG
MUF-NAG
MUF-NAG
fmol cell1 h1
nmol L1 h1
0.01
0.02
4.38
24.31
0.10
8.47
0
7.6
1140
0
1524.2
ELF-NAG
NP-NAG/ELF-NAG
MUF-NAG
MUF-NAG
NP-NAG/ELF-NAG
NP-NAG/ELF-NAG
NP-NAG/ELF-NAG
ELF-NAG
NP-NAG/ELF-NAG
MUF-NAG
Reference
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Sherr & Sherr (1999)
This study
Yes
40
108
Sherr & Sherr (1999)
Sherr & Sherr (1999)
ELF-NAG
NP-NAG/ELF-NAG
NP-NAG/ELF-NAG
ELF-NAG
ELF-labeled
0.17
10.4
5.78
12.78
146.1
0
290
1.29
91.4
0
21.92
1.59
655.5
0
42.2
8550
4.43
0
0
No
This study
Yes
Yes
Yes
This study
This study
This study
Yes
Yes
This study
This study
Sherr & Sherr (1999)
Sherr & Sherr (1999)
This study
This study
This study
This study
This study
Sherr & Sherr (1999)
Yes
Yes
Yes
Yes
Yes
NP-NAG/ELF-NAG
MUF-NAG
0
0
No
This study
Berg et al. (2003)
MUF-NAG
150
NP-NAG/ELF-NAG
MUF-NAG
MUF-NAG
0.003
16
83
5.66
No
This study
Sherr & Sherr (1999)
Sherr & Sherr (1999)
NP-NAG/ELF-NAG
NP-NAG/ELF-NAG
NP-NAG/ELF-NAG
3.89
1.7
0.98
17.6
3.82
86.66
Yes
Yes
No
This study
This study
This study
Sherr & Sherr (1999)
Field.
w
Nonaxenic culture.
Axenic culture.
‰
Measured at pH 4.5.
z
ELF-labeled in N medium, bulk activity was detected in the N and P medium.
The substrate used for the assay is noted. The enzyme activity in cultures was measured in nutrient-replete medium and is expressed in fmol cell1 h1
and nmol L1 h1.
z
FEMS Microbiol Ecol 64 (2008) 351–361
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
358
replete treatment where no ELF labeling was detected, the
b-N-acetylglucosaminidase activity was likely from heterotrophic bacteria. The pennate diatom, P. multiseries, was
labeled at high percentages in the N and P cultures,
indicating the presence of the enzyme activity in these
treatments. In this case, activity may be increased as part of
a general nutrient stress response, although this relationship
requires further examination.
All of the dinoflagellate species tested in culture had
detectable activity in at least one or more condition. Two
species, G. catenatum and L. polyedra, showed a consistently
high percentage of ELF labeling in all the treatments and
thus the activity did not appear to be sensitive to nutrient
status. Because dinoflagellates are well known for their
mixotrophic capacities (Stoecker, 1999), it is possible that
dinoflagellate species could employ b-N-acetylglucosaminidases to digest chitin compounds. In A. minutum and
P. minimum, where there was no activity in replete and a
relatively high percentage of positive cells in N and P
treatments, b-N-acetylglucosaminidase activity may be increased in response to nutrient stress, or stress in general.
The inconsistencies in the apparent nutrient-related trends
in b-N-acetylglucosaminidase activity between species may
be the result of many different factors, including life cycle
stage, mixotrophy, and nutritional physiology, depending
on the species or strain in question.
Species from Prymnesiophyceae and Raphidophyceae
groups rarely showed cell-specific enzyme activity. Cultures
of C. antiqua and C. marina, both harmful algal bloom
(HAB) forming species, were positive, but at low percentages and no consistent changes with nutrients were
apparent. The axenic culture of the HAB species
A. anophagefferens did not display any activity, which is
consistent with the findings of Berg et al. (2003) who did not
detect any extracellular b-N-acetylglucosaminidase activity
in an axenic culture.
b- N -acetylglucosaminidase activity in a coastal
embayment
Using a combination of ELF and bulk assays, phytoplankton
b-N-acetylglucosaminidase activity was detected in all samples during a 50-day-study of a coastal embayment. Bulk
b-N-acetylglucosaminidase activity ranged from 0.02 to
0.17 mmol mg Chla1 h1. Although variable, the hydrolytic
rates did not correlate with DIN, DIP, silicate, or DIN : DIP
ratio. Given that the activity may only be present in certain
species which shifted through time, and that the activity
may also be influenced by cellular physiology, it is not
surprising that the bulk activity did not correlate with these
parameters. It may be that the enzyme activity in phytoplankton is sensitive to other parameters such as chitin or
DOM concentration, which could be addressed in future
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
A. Štrojsová & S. T. Dyhrman
field work. Cell-specific b-N-acetylglucosaminidase activity
was present in at least some phytoplankton species from
every Salt Pond sample tested. The percentage of ELF
labeling within diatoms and dinoflagellates, the two dominant eukaryotic phytoplankton groups, was fairly low and
did not trend with the bulk enzyme activity. Although we
can discern that some of this bulk activity may be attributed
to diatoms and dinoflagellates, we cannot link the percentage of cells with activity to a bulk rate. This is in part
because different species may have very different hydrolytic
rates, even with the same qualitative presence of ELF
labeling. We also cannot control for the contribution of
particle-associated heterotrophic bacteria which may be
present in the net tow samples. Despite these caveats, it is
clear that some of the bulk activity can be attributed to
dinoflagellates and diatoms in this system. This underscores
the potential significance of eukaryotic phytoplankton in the
cycling of chitin.
In studies of freshwater systems, Vrba et al. (1996, 1997,
2004) repeatedly found a significant relationship between
the activity of b-N-acetylglucosaminidase and biomass of
certain diatom species (Asterionella formosa, Fragilaria crotonensis, Stephanodiscus sp., Cyclotella sp.), when the species
formed dominant or subdominant populations. There were
numerous diatom species with b-N-acetylglucosaminidase
positive cells in Salt Pond, but the percentage of labeled cells
at a given time was low. This lack of diatom cells with
enzyme activity relative to the total population appears to be
different than the freshwater studies highlighted above;
although, individual diatom species never formed the
dominant population in Salt Pond during our study.
The percentage of ELF-labeled dinoflagellates in the
spring phytoplankton was typically around 10% or less,
except for April 12, where more than 30% of dinoflagellate
cells were positive for b-N-acetylglucosaminidase activity.
This trend was largely driven by Alexandrium species, which
dominated the dinoflagellate community. Why more cells
were labeled on April 12 is unclear, as there was no
correlation between this cell-specific activity and nutrient
concentration (N, P, or Si) or other parameters that would
explain this change in the labeling percentage. In the culture
work, some species showed an increase in the number of
cells with enzyme activity under conditions of nutrient stress
(e.g. A. minutum), but these species were not present in Salt
Pond. As has been already discussed, high variability in the
presence of cells with activity could be due to high variability
in dinoflagellate nutrition; there are species that are strictly
autotrophic or heterotrophic as well as mixotrophic
(Schnepf & Elbrächter, 1992; Stoecker, 1999). Moreover,
during the 50-day-study, the populations might change
from one life stage to another (Anderson et al., 1983) and
this could result in changes in the enzyme activity. Further
work on the regulation of b-N-acetylglucosaminidase
FEMS Microbiol Ecol 64 (2008) 351–361
359
Chitinolytic activity in marine phytoplankton
activity by dissolved inorganic and organic nutrients and by
life history events are needed to understand the potentially
diverse role of this ectoenzyme in dinoflagellates.
Data from aquatic environments on bulk b-N-acetylglucosaminidase activity is limited, and complicated by methodological differences between studies. The comparison of
hydrolysis rate with freshwater activities is approximative,
because the substrate and also the sampling and incubation
conditions differed. If the bulk ( 4 20 mm) activities in Salt
Pond are back calculated, and bulk activities are estimated
for nonconcentrated Salt Pond samples, the average b-Nacetylglucosaminidase activity is c. 2 nmol L1 h1. This
value should be considered a minimum estimate as dissolved activity is excluded. In freshwater systems, the bulk
activity, determined using the substrate MUF-NAG, was
c. 4–9 nmol L1 h1 in a fish pond (Vrba et al., 1996),
0–142 nmol L1 h1 in European lakes and reservoirs (Vrba
et al., 2004), and 4.41–187 nmol L1 h1 in two acidified
lakes (Nedoma et al., 1994). Therefore, bulk activity detected in Salt Pond is roughly comparable to these freshwater studies and we can confirm through the ELF assays
that part of this activity is associated with eukaryotic
phytoplankton.
Summary
b-N-acetylglucosaminidase activity appears to be widespread across various eukaryotic marine phytoplankton
taxa. This was resolved with the use of an ELF substrate for
this enzyme which allowed for the detection of cell-specific
activity in individual cells. This approach, when combined
with more commonly applied bulk assays, shows promise
for identifying the activity associated with different groups,
and for distinguishing between the b-N-acetylglucosaminidase activity associated with heterotrophic bacteria and
phytoplankton in the field. Given the widespread presence
of b-N-acetylglucosaminidase activity, both in culture and
in field populations of eukaryotic marine phytoplankton,
these data suggest that a broad group of marine phytoplankton may be a relevant part of chitin-related DOM degradation and should be incorporated in our studies of chitin
cycling, and related DOM remineralization, in marine
systems. Future work in this area may identify the conditions which influence the presence or activity of this
important enzyme, and to what extent different phytoplankton groups use chitin, N-acetylglucosamine and its degradation products.
Acknowledgements
We thank Sheean Haley for her assistance in the laboratory
and for helpful comments on this manuscript. We also thank
Dave Kulis for his help with field sampling. This research
FEMS Microbiol Ecol 64 (2008) 351–361
was supported by a grant (R-83041501-0) from the US
Environmental Protection Agency to S.D. through the
ECOHAB Program, and a Fulbright-Masaryk Award to A.S.
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