Journal of
Plankton Research
plankt.oxfordjournals.org
J. Plankton Res. (2014) 36(1): 265– 270. First published online October 21, 2013 doi:10.1093/plankt/fbt106
SHORT COMMUNICATION
Incidence of dead copepods and factors
associated with non-predatory mortality
in the Rı́o de la Plata estuary
MARIANO MARTÍNEZ1,2, NOÉ ESPINOSA1,2 AND DANILO CALLIARI1,2*
1
OCEANOGRAFÍA Y ECOLOGÍA MARINA, FACULTAD DE CIENCIAS, UNIVERSIDAD DE LA REPÚBLICA, IGUÁ
4225, MONTEVIDEO CP 11400, URUGUAY AND 2ECOLOGÍA
FUNCIONAL DE SISTEMAS ACUÁTICOS, UDELAR, MONTEVIDEO, URUGUAY
*CORRESPONDING AUTHOR: [email protected]
Received May 30, 2013; accepted September 21, 2013
Corresponding editor: Roger Harris
We evaluated the incidence of dead copepods (IDC) in the Rı́o de la Plata estuary
utilizing a vital staining approach with neutral red. The dominant species was Acartia
tonsa. The overall IDC was on average 16.5% and it was higher in copepodite stage
IV through adult compared with earlier juvenile stages. Also, a negative relationship
between overall IDC and surface salinity was found.
KEYWORDS: Acartia tonsa; mortality; neutral red; estuary
I N T RO D U C T I O N
Copepods dominate metazoan plankton in marine
systems and play an important role in the transfer of
matter and energy to higher trophic levels (Calbet et al.,
2000). Much effort has gone into examining the growth
and fecundity of copepods but relatively little is known
about the rates and causes of mortality (Thor et al., 2008).
It is generally assumed that copepod mortality is
mainly due to predation (Tang et al., 2006). However, previous studies have shown that copepods can suffer from
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JOURNAL OF PLANKTON RESEARCH
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non-predatory mortality through environmental stresses
(Cervetto et al., 1999).
Due to methodological difficulties, the incidence of
non-predatory mortality is largely unknown. Motility has
been the main criterion for recognizing living plankters.
Dressel et al. (Dressel et al. 1972) developed a technique to
separate live from dead zooplankton based on the uptake
of the vital stain neutral red. This stain is only absorbed
by living cells, and therefore, following staining, organisms that were alive at the moment of collection would
appear red, whereas dead ones would appear unstained;
such a pattern remains after sample preservation. Other
stains, such as Evans blue and Aniline blue operate the
opposite way, staining dead cells, and have been utilized
with that same purpose (Crippen and Perrier, 1974;
Bickel et al., 2009), but these are only incorporated by carcasses of organisms whose time of death is between 2 h
and 2 – 3 days (Dubovskaya et al., 2003).
Neutral red is the most efficient among several
methods for live/dead determinations of planktonic
organisms (Zetsche and Meysan, 2012). Despite early
successful applications (Crippen and Perrier, 1974;
Fleming and Coughlan, 1978) neutral red vital stain has
been little used. Recently, Tang et al. (Tang et al., 2006)
and Elliott and Tang (Elliott and Tang, 2009, 2011)
refined the method to facilitate its application in the field,
demonstrating high percentages of dead copepods in
Chesapeake Bay. These authors found that staining efficiency decreases with the amount of seston and density of
plankton in the sample, that is less efficient at lower temperatures, and that it is not affected by salinity.
Estuaries are characterized by high temporal and
spatial environmental variability (e.g. salinity), which subjects organisms to persistent stressful conditions, especially
affecting juvenile stages (Attrill, 2002). Hence, determination of the incidence of dead copepods (IDC) in estuaries
is particularly relevant for an understanding of the sources
of non-predatory mortality.
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The Rı́o de la Plata (RdlP) estuary drains the second
largest basin of South America. It has an average depth
of 10 m and is characterized by a salt wedge regime and
a well-developed salinity front (Guerrero et al., 1997).
Despite its ecological relevance, little is known about the
zooplankton in this area (but see Berasategui et al., 2005);
studies about their vital rates are scarce (Calliari et al.,
2004) and there are no estimates of mortality for any
planktonic group. Here we assess the IDC in the RdlP
estuary and explore factors of non-predatory mortality.
We estimated stage-specific IDC for dominant species
and evaluated its relationship with salinity gradients. We
hypothesized that strong salt gradients, characteristic of
the RdlP estuary oligo-mesohaline region induce high
copepod mortality, and that juveniles experience higher
mortality than adults due to their higher susceptibility to
osmotic stress.
We sampled three stations ca. 3.7 km apart and 4 km
offshore of the northern bank of the oligo-mesohaline
region of the RdlP estuary during four cruises in September
and November 2009 (CR3, CR4), and September and
November 2010 (CR5, CR6). Temperature and salinity
profiles were obtained with an SBE 19V-plus CTD. Salinity
was measured according to the Practical Salinity Scale
and is reported without units. Plankton was always collected by horizontal sub-surface tows (1 – 2 m depth) due
to the shallowness of the water column (4 – 9 m, Table I).
Copepod density (ind m23) was estimated with a 47-cm
mouth-opening plankton net fitted with a 90-mm mesh
and a flowmeter. Samples were preserved in 4% formaldehyde for identification and counting. For vital staining,
zooplankton was collected by slow (0.5 m s21) and short
(2 min) horizontal tows using a plankton net fitted with a
120-mm mesh and non-filtering cod end. The net was
not rinsed to prevent collection of damaged organisms
adhered to the mesh; cod-end contents were immediately
transferred to a vessel, and incubated with neutral red
(1:1000 stock solution) for 15 min at a final concentration
Table I: Range of depth, surface temperature (SST), surface salinity (SSS), saline stratification coefficient
(SSC), horizontal saline gradient (HSG), Acartia tonsa population density and percentage of dead
individuals.
Physical environment
A. tonsa
Cruise code
Depth (m)
Surface layer
thickness (m)
SST (8C)
SSS
SSC (m21)
HSG (km21)
Population density
(ind m23)
% dead
individuals
CR3
CR4
CR5
CR6
4.5 –9.5
4.5 –8.5
8.5 –9.3
7.9 –9.2
1.7 –3.5
1.7 –4.5
1.1 –1.5
4.7 –6.2
13.5– 14.1
14.6– 15.3
16.1– 17.0
17.7– 18.1
4.0 –5.7
7.5 –16.4
2.0 –4.8
4.5 –6.9
8.0 –14.1
0.0 –3.3
8.1 –11.1
1.1 –7.1
0.02 –0.30
0.04 –1.75
0.06 –0.53
0.07 –0.28
1633 –6901
1470 –3908
709 –2579
1657
12.5 –13.0
6.2–13.2
16.5 –44.8
11.8 –22.8
Values represent the range observed for the three stations of each cruise.
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of 1:67 000, at in situ temperature and in darkness. The
sample was subsequently filtered through a 100-mm
sieve, rinsed with filtered seawater and stored at 2208C
until analysis (Elliott and Tang, 2009).
The presence of halocline/pycnocline, surface and bottom mixing layers was determined by visual analysis of
CTD data. Surface temperature and salinity were estimated as their respective average in the surface mixing
layer. A saline stratification coefficient (SSC, m21) was
calculated as the salinity difference between bottom and
surface layers, divided by the depth difference between
layers. A horizontal saline gradient (HSG, km21) was
estimated as the difference between surface salinity of two
consecutive stations divided by the distance between
them.
For vital staining analysis, samples were thawed using
cold in situ filtered water, acidified with 1M HCl (1:10
final concentration) to develop the stain’s colour and
observed under a stereomicroscope to determine the
state of the organisms (live/dead) and their developmental stages. Mortality was analysed for population stages
classified in two groups: small copepodites (copepodite stages
I through III, CI–CIII) and large copepodites (copepodite
stage IV through adult, CIV–CVI) of the dominant
species, usually only Acartia tonsa. Copepod molts were
identified but not considered for mortality estimations.
Preliminary tests with individuals of known vital status
(live/dead) indicated that organisms subjected to the
staining protocol could be classified into three colour categories: (i) bright red, (ii) light pink and (iii) white. Live
copepods corresponded to staining category 1, while
dead ones corresponded mostly to category 3 (92%) but a
minor fraction (8%) were classified into category 2.
The influence of salinity and temperature on staining
efficiency was evaluated using live plankton from the
same region of the RdlP estuary. Samples were diluted
immediately after collection in 20-L thermally insulated
buckets and taken to the laboratory where copepods
(A. tonsa) were sorted out and subjected to salinities of 2.5,
5, 10 and 15 (constant temperature ¼ 208C) and to temperatures of 108C, 158C and 208C (constant salinity ¼
2.5), incubated with neutral red for 15 min and further
processed as described for field samples. Each salinity/
temperature treatment consisted of 3 replicates of 10
living organisms and 10 freshly dead ones killed by immersion in seawater at 508C for 5 min. Conditions of salinity and temperature tested were representative of the
range found in situ. Salinity changes from in situ to experimental conditions were performed in steps of ca. 5 and
animals were held at each acclimatization condition (and
at each treatment prior staining) for 2 h (Calliari et al.,
2006, 2008). After the experiment organisms were classified into the categories mentioned above.
To assess differences in the in situ IDC between stage
groups small and large, the fraction of dead copepods of
each stage class in the stained sample was corrected by
the contribution of that same stage class in the population, as determined in quantitative samples. Corrected
IDC was estimated as the index “m”:
m¼
% dead individuals of stage ðiÞ in stained sample
% individuals of stage ðiÞ in the population
Index m for stage class i varies between 0 (no dead individuals in class i) and 100/Pi, where Pi is the percentage of
stage i in the population.
Corrected IDC for groups small and large were compared using Student’s t-test for dependent variables.
Pearson correlation analyses were conducted for overall
and group-specific IDC vs. surface salinity, surface temperature, SSC, HSG and copepod density. Normality
and homoscedasticity were verified using Shapiro– Wilk
and Levene’s test, respectively, and data were log or
squared-root transformed if needed.
Consistent with Elliott and Tang (Elliott and Tang,
2009), there was no effect of salinity on staining efficiency,
while temperature did have a significant effect. The
lowest efficiency was found at 108C, an unusual extreme
in the RdlP estuary (June– September) (Fig. 1).
Surface salinity and temperature were typical for the
study area, ranging from 2 to 16.4 and from 13.5 to
18.18C, respectively (Table I). SSC was highest in CR3
and CR5, while HSG was so in CR4 (Table I).
Acartia tonsa was the dominant species in every sample,
representing between 67 and 99% of total copepod
abundance and reaching the maximum density in CR3
(6901 ind m3). The overall IDC was on average 16.5%,
similar to that found by Elliott and Tang (Elliott and
Tang, 2011) in Chesapeake Bay (12 – 15%) under a
similar salinity range.
Higher mortality of juveniles was expected to be associated with strong salt gradients based on the results of
Cervetto et al. (Cervetto et al., 1999). However, in the
present study IDC of the group large was higher (t ¼ 3.306,
n ¼ 11, P ¼ 0.008) (Fig. 2a). Such a result suggests that
environmental stress would not be the only cause of nonpredatory death in copepods, and that physiology and
life history patterns (i.e. senescence) may have a relevant
role as well. Recently, Rodriguez-Graña et al. (RodriguezGraña et al., 2010) assessed the effect of ageing on different population parameters of A. tonsa, finding that
feeding and production rates decrease with age, and that
mortality rates increase with age of the organisms. Also,
the interaction of environmental stress and age, i.e.
higher susceptibility to environmental stress by senescent
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Fig. 1. Laboratory experiments: influence of salinity (left) and temperature (right) on staining efficiency. The thick line indicates the median,
the box represents the first and third quartile, and whiskers the 95% confidence interval.
individuals, could have contributed to a higher IDC in
the large group.
Considering IDC disaggregated by stations, a high
value was found in CR5 (44.8%). Tang et al. (Tang et al.,
2006) found up to 75% of dead copepods associated with
a saline front using the same vital staining. However, in
the present study IDC did not correlate with HSG.
Correlation analysis resulted in a single (negative) significant relationship between overall IDC and surface salinity (r 2 ¼ 0.521, P ¼ 0.018, n ¼ 10) (Fig. 2b) after
removing an outlier (following Moore and McCabe,
1999) in CR5. Highest mortality of A. tonsa at low salinities could be due to higher relative salinity changes
at lower salinities. Even though A. tonsa tolerates low
salinities (2 – 5), under these conditions it is subjected to a
strong physiological stress (Calliari et al., 2006, 2008).
Under natural conditions, the stress imposed by highly
diluted waters most likely adds to other factors not considered here (food quality, age, presence of contaminants)
which may contribute to increased mortality in oligohaline conditions. The pattern observed could also be a
consequence of the RdlP estuary circulation patterns, i.e.
accumulation of particles (including dead copepods) near
the turbidity front in low salinity areas.
The absence of correlation between IDC and other
variables could be due to the low number of samples analysed, or alternatively, it may indicate that non-predatory
mortality caused by gradients is episodic. It is reasonable
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FUNDING
This study was funded by Comisión Sectorial de Investigación Cientı́fica Grupos I þ D Programme, Universidad
de la República, and a Marie Curie Grant (European
Commission, International Incoming Fellowship, Framework Programme 6) to D.C.
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