Introduction
Many zooplankton and nekton organisms feed in the ocean's surface layer
during the night and migrate to beneath the photic zone during daytime,
mainly to avoid visual predation (Lampert, 1989). These so-called diel
vertical migrations (DVMs) mediate the active flux of particulate and
dissolved organic and inorganic matter from the surface layer to midwater
depths (Steinberg et al., 2000, 2002), as the zooplankton and nekton
organisms respire, excrete, defecate and die at depth. The DVM mediated
active flux is an important aspect of marine biogeochemical cycles and can be
as high as the passive flux via sinking particles (Putzeys, 2013). The active
flux to a certain depth depends on the migrating biomass to that depth and on
the respiration, excretion and defecation activity of the migrating
organisms, as well as their mortality rate at the respective depth. Here we
focus on the regulation of respiration and excretion rates via environmental
factors. Environmental temperature (Ikeda, 2014), oxygen (Ekau et al., 2010;
Seibel, 2011), and carbon dioxide levels (e.g. Rosa and Seibel, 2008; Maas et
al., 2012a) modulate the activity of migrating organisms, with temperature
being the best understood factor. In general, a 10 ∘C temperature
decrease results in an approximately 2-fold reduction of metabolic activity
(Ikeda, 2014). Likewise, hypoxia ultimately leads to reduced metabolic rates
(Ekau et al., 2010; Seibel, 2011), whereas elevated pCO2 can increase
or decrease metabolic rates (e.g. Rosa and Seibel, 2008; Thomsen and Melzner
2010, Kroeker et al., 2010; Maas et al., 2012a). The vertical gradients of
temperature, oxygen and carbon dioxide are very pronounced at the eastern
margins of tropical oceans. Here, migrating organisms encounter relatively
high temperatures, high oxygen and low carbon dioxide levels at the surface,
but relatively low temperatures, low oxygen and high carbon dioxide levels in
the oxygen minimum zone (OMZ) at midwater depth (Karstensen et al., 2008;
Paulmier et al., 2011).
OMZs in tropical oceans result from restrained mixing due to strong thermal
stratification and sluggish circulation that result in a reduced oxygen
supply and a comparatively high supply of particulate organic matter from the
surface layer that is respired at midwater depth (Brandt et al., 2015). OMZs
structure the pelagic habitat and influence the distribution and activity of
marine organisms, as many organisms cannot survive at low environmental
oxygen concentrations (e.g. Ekau et al., 2010). In the Eastern Tropical South
Pacific (ETSP), the upper boundary of the oxycline is the single most
critical factor structuring the habitat of most zooplankton organisms
(Semenova et al., 1982; Escribano et al., 2009). The ETSP OMZ features
severely hypoxic to anoxic conditions, with anoxia being detected at about 100 to 300 m depth and oxygen levels
between 0 and 5 µmol kg-1 (∼ 0 to about 0.4 kPa) at
about 30 to 100 m depth and 300 to 400 m depth (Thamdrup et al., 2012). In
contrast, oxygen concentrations in the Eastern Tropical North Atlantic (ETNA)
seldom fall below 40 µmol kg-1 (∼ 3.4 kPa at
12 ∘C, Karstensen et al., 2008), but transient severely hypoxic to
anoxic conditions have been observed in mesoscale eddies (Karstensen et al.,
2015). OMZs have expanded in the recent past and a further expansion, mainly
due to global warming, is expected (Stramma et al., 2008; Keeling et al.,
2010). An expansion of OMZs will have far-reaching consequences for marine
tropical ecosystems. For example it results in the compression of the habitat
of billfishes (Stramma et al., 2012), but also in the extension of areas
where fixed nitrogen is lost from the ocean (Kalvelage et al., 2011).
Many organisms have developed special adaptations like an enhanced oxygen
uptake capacity to thrive at particularly low oxygen levels of less than
5 kPa (∼ 40 µmol kg-1) oxygen (Childress and Seibel,
1998; Seibel, 2011). The abundance and biomass of zooplankton and nekton
permanently inhabiting extreme OMZs is rather low (e.g. Wishner et al., 1998;
Auel and Verheye, 2007; Escribano et al., 2009), but the migrating
zooplankton and nekton biomass can be very high even in these regions. Most
zooplankton and nekton organisms can regulate their oxygen uptake rate over a
wide range of oxygen concentrations, but this regulatory ability breaks down
at a certain critical oxygen concentration. The point at which the aerobic
metabolism can no longer be maintained independent of the environmental
oxygen partial pressure (pO2) is called the critical oxygen partial
pressure pcrit (e.g. Seibel, 2011). The pcrit is species
specific and varies with habitat oxygen concentrations (e.g. Childress and
Seibel, 1998; Richards, 2011) and temperature (Deutsch et al., 2015). Many
species inhabiting the OMZ have evolved significantly lower pcrit
values than non-OMZ inhabiting species (Childress and Seibel, 1998) and can
survive extended periods of time at oxygen levels below their pcrit
or even at anoxia (Childress, 1975; Kiko et al., 2015a). Migrations into
waters with oxygen levels below the pcrit should result in a
metabolic suppression that reduces the amount of oxygen respired and carbon
dioxide excreted. This reduction needs to be taken into account when
calculating the active flux of respiratory carbon into the OMZ and when
estimating the biological oxygen consumption within the OMZ.
The impact of hypoxia on other metabolic processes is not described as well
as that on respiration. Its impact on the excretion of nitrogenous compounds
is of particular interest, as pelagic primary productivity is primarily
dependent on dissolved inorganic nitrogen (e.g. Hauss et al., 2012; Meyer et
al., 2016). The active removal of fixed nitrogen from the euphotic zone to
midwater depths is therefore an important aspect of DVMs (Steinberg et al.,
2002). Furthermore, the release of ammonium in the OMZ by diel vertical
migrators could possibly fuel bacterial anaerobic ammonium oxidation
(anammox) – a process that removes fixed nitrogen from the ocean (Bianchi et
al., 2014). Several studies have found a reduction in ammonium excretion in
response to long-term (days to weeks) exposure to hypoxic and anoxic
conditions for benthic and pelagic crustaceans (e.g.; Hagerman et al., 1990;
Hagerman and Szaniawska, 1994; Rosas et al., 1999). Cass and Daly (2014) on
the other hand found both enhanced and reduced rates of nitrogen excretion in
response to short-term (few hours) exposure to mild hypoxia (initial O2
concentration 36 to 78 µmol O2 kg-1; initial pO2
2.7 to 6 kPa) in different calanoid copepod species and report that
excretion rates at severe hypoxia (initial O2 concentration 4 to
17 µmol O2 kg-1; initial pO2 0.3 to 1.3 kPa)
were too low to be measured. Svetlichny et al. (1998) showed for the Black
Sea calanoid copepod Calanus euxinus that short-term exposure to
mild hypoxia (26 µmol O2 kg-1; 1.9 kPa) did not affect
the ammonium excretion rate, whereas it resulted in a down-regulation of
respiration. Maas et al. (2012b) found no reduction of ammonium excretion in
three thecosomate pteropod species when animals were exposed to initial
oxygen concentrations of 31.5 ± 8 µmol O2 kg-1
(2.5 ± 0.6 kPa). Kiko et al. (2015a) observed a very strong
down-regulation of ammonium excretion in the squat lobster
Pleuroncodes monodon upon exposure to anoxia and calculated a
pcrit for ammonium excretion of 0.5 kPa (6.1 µmol
O2 kg-1 at 13 ∘C). In general, it still remains unclear
how nitrogen excretion is regulated in response to low environmental oxygen
concentrations in diel vertical migrators.
It is also not clear how the increase in pCO2 that coincides with a
decrease in pO2 impacts the metabolic activity of zooplankton and
nekton organisms and if interactive effects exist. Previous studies to
analyse the impact of OMZ conditions on zooplankton metabolic activity often
let the animals respire the oxygen in the test bottle (e.g. Childress, 1975;
Donnelly and Torres, 1988), which would result in elevated pCO2 levels
consistent with the rise in pCO2 levels in the OMZ. However, this
approach could also lead to the build-up of potentially harmful amounts of
metabolic endproducts. In other studies, pure nitrogen or nitrogen and oxygen
mixes were used to adjust oxygen levels (e.g. Svetlichny et al., 1998;
Trübenbach et al., 2013; Cass and Daly, 2014). The latter approach leads
to the removal of carbon dioxide from the incubation water and therefore to a
pCO2 reduction and a pH increase, non-representative of OMZ
conditions (Melzner et al., 2013). In other studies, the pCO2 was
adjusted to OMZ-conditions, but not the pO2 (e.g. Maas et al., 2012a),
or both pO2 and pCO2 were adjusted, but interactive effects
were not tested (Kiko et al., 2015a). It is therefore necessary to develop
procedures to test metabolic activity at realistic pO2 and pCO2
levels.
We here established a method to mimic the OMZ conditions (temperature,
pO2 and pCO2) of the ETNA and ETSP using premixed
N2 / O2 / CO2 gas mixes and tested the hypothesis that
metabolic activity in copepods and euphausiids is changed under these
incubation conditions compared to the incubation conditions realized when
only N2 / O2 gas mixes are used. Furthermore, we tested the
hypothesis that exposure to OMZ conditions not only reduces the amount of
oxygen respired, but also the amount of ammonium excreted. To test these
hypotheses, we measured respiration and ammonium excretion rates of migrating
and non-migrating copepods from the ETNA (Pleuromamma
abdominalis and Undinula vulgaris,
respectively) and krill species from the ETSP OMZ (Euphausia mucronata) and the ETNA OMZ (Euphausia gibboides) at different
oxygen levels, carbon dioxide levels and temperatures to characterize their
metabolic response. Calanoid copepods from the Pleuromamma genus are
ubiquitous members of the migrating zooplankton community of the tropical
Atlantic (Steinberg et al., 2000; Auel and Verheye, 2007), whereas
Undinula vulgaris is a ubiquitous non-migrating epipelagic calanoid
copepod of the tropical oceans (Chahsarvar-Archard and Razouls, 1982; Razouls
et al., 2015). Euphausia gibboides is found regularly at low
latitudes in the Atlantic and Pacific (Siegel, 2015), and E. mucronata is the dominant euphausiid in the ETSP (Antezana, 2009, 2010).
Both euphausiid species conduct diel vertical migrations into the OMZ. Our
work should help to better parameterize ammonium excretion and respiration
rates of crustacean zooplankton and nekton in OMZ regions and aims to provide
a base for revising model formulations of DVM-mediated export in OMZ regions.
Material and methods
Animal collection and maintenance
Sampling was conducted during RV Maria S. Merian cruise MSM22 (ETNA;
24 October to 23 November 2012) and RV Meteor cruises M93 (ETSP;
6 February to 4 March 2013) and M97 (ETNA; 25 May to 28 June 2013; Fig. 1).
Zooplankton was collected using a Hydrobios Multinet Midi (0.25 m2
mouth opening, 200 µm mesh size, 5 nets), a Hydrobios Multinet Maxi
(0.5 m2 mouth opening, 330 µm mesh size, 9 nets), a WP-2 net
(0.26 m2 mouth opening, 200 µm mesh size), a MOCNESS
(1 m2 mouth opening, 2 mm mesh size) or a CalCOFI-Net (0.78 m2
mouth opening, 500 µm mesh size). All specimens used for
experiments were caught in the upper 400 m of the water column and only
animals appearing unharmed and fit were used for experiments. Specimens were
sorted, identified and transferred into aquaria with filtered,
well-oxygenated seawater immediately after the catch and maintained for 1 to
13 h prior to physiological experiments at the respective experimental
temperature. Only adult euphausiids and adult female copepods were used for
the experiments. OMZ temperatures during MSM22 and M97 ranged from 5.5 to
13.6 ∘C at 300 to 600 m depth and from 13 to 29 ∘C in the
upper 100 m. Temperature at 200 to 300 m depth during M93 ranged from 10.2
to 13.3 ∘C. Maintenance and physiological experiments were therefore
conducted in darkness in temperature-controlled incubators at 11, 13 or
23 ∘C (±1∘). Animals were not fed before or during
experiments.
Sampling locations of specimens for respiration and excretion
measurements. Circles: Pleuromamma abdominalis and Undinula vulgaris during cruise MSM22 (October/November 2012); Squares:
Euphausia mucronata during cruise M93 (February 2013); Diamonds:
Euphausia gibboides during cruise M97 (May 2013).
Stomach fullness and colouration of the mid gut gland were routinely
categorized in E. gibboides. Possible scores of the mid gut gland
colouration were 0–5, indicating transparent through green to dark brown
colouration (Morris et al., 1983).
Incubation conditions
Respiration and ammonium excretion rate measurements (both in
µmol h-1 gDW-1) at varying oxygen concentrations were
conducted simultaneously in 12 to 60 mL gas-tight glass bottles. These were
equipped with oxygen microsensors (∅ 3 mm, PreSens Precision
Sensing GmbH, Regensburg, Germany) attached to the inner wall of the bottles
to monitor oxygen concentrations non-invasively. Read-out of oxygen
concentrations was conducted using multi-channel fiber optic oxygen
transmitters (Oxy-4 and Oxy-10 mini, PreSens Precision Sensing GmbH,
Regensburg, Germany) that were connected via optical fibers to the outside of
the bottles directly above the oxygen microsensor spots. Calibration of the
oxygen microsensors was conducted at the beginning of each cruise or when a
different incubation temperature was set with a Na2SO3-solution
(0 % oxygen) and aerated seawater (100 % air saturation) at the
respective measurement temperature. Oxygen concentration was calculated from
air saturation according to the PreSens manual. All other oxygen unit
conversions were conducted using the R-package AquaEnv (Hofmann et al., 2010)
and R scripts obtained from Andreas F. Hofmann. Measurements were started at
pre-adjusted oxygen and carbon dioxide levels. For this, seawater stocks with
adjusted pO2 and pCO2 were prepared by equilibrating 3 to 4 L
of filtered (0.2 µm Whatman GFF filter) and UV-sterilized (Aqua
Cristal UV C 5 Watt, JBL GmbH and Co. KG, Neuhofen, Germany) water with
premixed gases (certified gas mixtures from Air Liquide) for 4 h at the
respective experimental temperature. pCO2 levels were chosen to mimic
the environmental pCO2 in the ETSP OMZ or the ETNA OMZ. pCO2
levels for the respective area were calculated from data published by the
World Ocean Circulation Experiment (WOCE 2002; ETSP: data from the upper
150 m between 14.74 to 16.38∘ S and 75.25 to 76.93∘ W;
ETNA: data from the upper 400 m between 4.5∘ S to 11∘ N
and 7 to 26∘ W) using CO2sys_v2.1 (Pierrot et al., 2006).
Furthermore, gas mixes with different levels of oxygen, but without CO2
were used to test the effects of this experimental manipulation. The detailed
composition of the premixed gases used is described in Table 1. Antibiotics
(25 mg L-1 ampicillin and 25 mg L-1 streptomycin) were added to
the stocks after equilibration to inhibit microbial activity.
Volumetric composition of used premixed gases.
50 % air sat.
33 % air sat.
10 % air sat.
Nitrogen
89.58
93.02
97.85
ETNA CO2-plus
Oxygen
10.36
6.91
2.07
Carbon dioxide
0.06
0.07
0.08
Nitrogen
89.53
92.96
97.77
ETSP CO2-plus
Oxygen
10.36
6.91
2.07
Carbon dioxide
0.11
0.13
0.16
Nitrogen
89.64
93.09
97.93
CO2-minus
Oxygen
10.36
6.91
2.07
Carbon dioxide
0.00
0.00
0.00
The salinity and pH of the prepared water were measured immediately on board
with a handheld multiparameter metre Multi 350i equipped with a Sentix 41 pH
and a ConOx conductivity probe (WTW). Calibration of the pH-probe was
conducted daily prior to the measurements using 7.000 and 10.012 pH IUPAC
standard buffers (Radiometer analytical) and the conductivity probe was
calibrated at the beginning of each cruise using 0.01 mol L-1 KCl. 250
mL of the prepared water was collected in gas-tight glass bottles and fixed
with 100 µL saturated mercury chloride solution for later
measurements of total alkalinity (AT) and dissolved inorganic
carbon (CT) in the home laboratory. CT was measured
using an AIRICA system (Marianda, Kiel, Germany) via a LI-COR 7000 infrared
CO2 / H2O analyser. AT was measured in duplicates
via potentiometric titration with an automated titrator (Titroline 7000, SI
Analytics, Germany) using 50 mL of sample and 0.05 M HCl. CT
was measured for samples from cruise MSM22, whereas AT was
measured for samples from cruises M93 and M97, due to a breakdown of the
CT measurement system. AT and CT were
measured against certified reference material provided by Andrew Dickson of
Scripps Institution of Oceanography (http://andrew.ucsd.edu/co2qc/).
The pCO2 established in the different incubation trials was calculated
with CO2sys_v2.1, using experimental temperature, water salinity,
pHNBS and CT or AT as input.
Excretion and respiration measurement
Experimental runs to simultaneously measure the ammonium excretion and
respiration rates were conducted with 11 to 15 trial incubations (1 or 2
animals per incubation bottle and three different treatment levels) and three
animal-free control incubations (experimental
treatment) per run. During each run,
experimental treatments comprised 100 % air saturation as well as one
reduced air saturation level with and without CO2. A typical run for
example comprised three replicates plus one animal-free control at 100 %
air saturation, four replicates plus one animal-free control at 50 % air
saturation plus CO2 and four replicates plus one animal-free control at
50 % air saturation minus CO2. To start the incubations, the
incubation bottles were filled with the respective prepared water and the
animals were added immediately. This handling took a maximum of 5 min for
each animal and about 30 min for all replicates. Subsequently, the
respective incubation bottle was immediately transferred to the incubator.
Oxygen concentrations in the incubation bottles were recorded every 5 min
using the fiber-optic microsensor system and the recording of data for the
determination of respiration rates was started immediately after all animals
were transferred. Respiration rates were calculated from the slope of oxygen
decrease over selected time intervals. Chosen time intervals were 20 to
105 min long. No respiration rate was calculated for the first 20 to 60 min
after animal transfer to avoid the impact of enhanced activity of the animal
or changes in the bottle water temperature during initial handling on the
respiration rates and oxygen readings. Respiration rates were obtained over a
maximum incubation time of 16 h and slopes were linear at normoxia to mild
hypoxia. Respiration rates in animal-free control bottles were used to
correct for microbial activity. These rates were < 2 % of animal
respiration rates at normoxia.
Animals were incubated for 2 to 10 h for the measurement of ammonium
excretion rates. Ammonium concentration was determined fluorometrically
(Holmes et al., 1999). Ammonium excretion was calculated as the concentration
difference between incubation and animal-free control bottles. Some specimens
died during the respiration and excretion rate measurements, as indicated by
a cessation of respiration. No excretion rate measurements were conducted in
this case, but the oxygen level at which the animal died was noted.
Euphausia mucronata survived exposure to anoxia, enabling the
measurement of ammonium excretion rates under severely hypoxic to anoxic
conditions. These measurements were started after 2–5 h when animals had
respired all oxygen from the incubation bottles. Thereafter, a 1 mL water
sample was taken from the 12–13 mL bottles used to determine the ammonium
concentration at the onset of the trial. The withdrawn water was replaced
with 0 % oxygen water prepared using pure nitrogen gas. Oxygen levels
slightly increased during this procedure, but never rose above
∼ 3 % air saturation. This small amount of oxygen was in most cases
respired after about 30 min, but in some cases anoxia was not reached until
the end of the trial. After an incubation period of 2.5 h at severe hypoxia
to anoxia, a second sample for the ammonium concentration measurement was
taken. The ammonium excretion rate was calculated as the difference between
the first and second measurement and the oxygen concentration was calculated
as the mean of the initial and end oxygen concentrations. All individuals
used in the described experiments were subsequently frozen at
-80 ∘C, dried at
50 ∘C for 72 h and weighed to determine their dry weight. Oxygen
consumption and ammonium excretion rates (R,
µmol h-1 gDW-1) were standardized to an average dry
weight (DW in mg) of 0.1 g applying a scaling coefficient b of -0.25
(Moloney and Field, 1989) as
Rstd=R×0.1weight-0.25.
The rates presented should be considered routine metabolic rates, as activity
was not monitored and animals were not fed (Prosser, 1961).
Statistical analysis
General linear models (GLM) with pO2 as a continuous variable and
pCO2 (two levels) and temperature (for all except E. mucronata, two levels)
as a categorical predictor were used on log-transformed respiration and excretion
rate data for each species separately to explore the overall effect of
experimental conditions on the metabolic response.
Pairwise t-tests were employed to compare the respiration and excretion rates
of the two pCO2-treatments maintained under similar temperature and
pO2 conditions. For these tests, the mean respiration rate obtained at
a given experimental condition (starting pO2 value and experimental
temperature) for a replicate was determined. E.g. if an experimental run with
an animal was started at 50 % air saturation, stopped at 20 %
air saturation and lasted 8 h, four determinations of the respiration rate
were available for this replicate and the mean of these respiration rates was
determined. To analyse the effects of different oxygen levels on the
respiratory activity, the four determinations of the respiration rates
obtained at different oxygen levels were used separately for subsequent
analysis.
Where possible, metabolic rates were modelled as a function of pO2
using nonlinear regressions with the python module lmfit. If hypoxia was
found to have lethal effects at a given temperature, the metabolic data were
transformed prior to fitting the power function by subtracting the lowest
observed lethal pO2 from the abscissa data. A power function with a
y-intercept of zero
Rstd,O2=a×pO2b
was fitted to the standardized (according to Eq. 1) and transformed
respiration rate data as the respiration rate at 0 kPa oxygen or that of
dead individuals is zero by definition. A power function with y intercept
Rstd,NH4=a×pO2b+c
was fitted to the standardized and transformed ammonium excretion rate data.
To calculate the pcrit, the standardized and transformed
respiration or excretion rates were normalized by dividing each respiration
or excretion rate by the highest observed respiration or excretion rate,
respectively. Power functions as described above were fitted to these
standardized, transformed and normalized rates. The respiration or excretion
pcrit was then calculated according to Marshall et al. (2013) as
pcrit=0.065a×b11-b,
where a and b are the respective factors obtained from fitting Eq. (2) or (3)
to the standardized and normalized rates.
The fitted functions and the calculated pcrit were back-transformed
to the original scaling of the abscissa.
Results
Impact of pCO2-levels on respiration
and excretion
We prepared incubation water using both O2 / N2-mixes
(CO2-minus) and O2 / N2 / CO2-mixes (CO2-plus) to test
if differences in pH and pCO2 in the incubation water lead to
significant differences in the metabolic activity of copepods and euphausiids
from the ETNA and ETSP. O2 / N2 / CO2-mixes representative of the
respective regions were used. The use of O2 / N2-mixes led to an
artificial reduction of the pCO2 and an artificial increase in pH
compared to untreated seawater (pO2 ∼ 21 kPa; Fig. 2).
Differences to the respective simulated OMZ conditions were even larger and
increased with decreasing oxygen levels (Fig. 2). The use of
O2 / N2 / CO2-mixes resulted in pH and pCO2 levels that
were very close to the environmental target conditions for the ETNA OMZ,
whereas pCO2 levels were slightly above those of the ETSP OMZ.
pHNBS and pCO2 in incubation water (black symbols),
compared to environmental levels (grey symbols) for the ETNA OMZ and the ETSP
OMZ. Black circles: CO2-plus treatment. Open diamonds: CO2-minus
treatment. Grey circles: environmental pH or pCO2 data, pCO2
calculated from AT and CT. Grey triangles in F:
pCO2 calculated from pH and AT.
Weight-specific respiration rates of calanoid copepods and
euphausiids at different temperatures, air saturation and CO2-levels.
p = CO2-plus, m = CO2-minus. Red barplot colour
indicates a statistically significant difference (t test, p<0.05) between CO2-plus and CO2-minus treatments. Each row contains
data for only one species, with low temperatures on the left side of the row
and high temperatures on the right side and air saturation increasing from
left to right.
Weight-specific ammonium excretion rates of calanoid copepods and
euphausiids at different temperatures, air saturation and CO2-levels.
p = CO2-plus, m = CO2-minus. Red barplot colour
indicates a statistically significant difference (t test, p<0.05) between CO2-plus and CO2-minus treatments. Each row contains
data for only one species, with low temperatures on the left side of the row
and high temperatures on the right side and air saturation increasing from
left to right.
In the general linear model (GLM) with the mean pO2 as continuous and
pCO2 and – if applicable – temperature as categorical predictors,
applied to each species separately, no significant overall effects of the
CO2-level on the respiration or excretion rates were detected (p>0.1), while temperature and pO2 were significant
(univariate p<0.0001 for respiration, p<0.05 for
excretion, all species) predictors of metabolic activity. Respiration whole
model adjusted r2 was 0.63, 0.36, 0.41 and 0.37 for E. gibboides, E. mucronata, Pleuromamma abdominalis and
Undinula vulgaris, respectively and ammonium excretion whole model
adjusted r2 was 0.30, 0.31, 0.84 and 0.77 for E. gibboides,
E. mucronata, P. abdominalis and U. vulgaris,
respectively.
In the pairwise comparison, respiration and ammonium excretion rates of
animals incubated in CO2-plus waters were not found to be significantly
different from those incubated in CO2-minus waters at most oxygen and
CO2 levels analysed (t tests, p value > 0.05; Figs. 3
and 4). However, in Euphausia mucronata incubated at 33 % air
saturation at 13 ∘C, the CO2-plus treatment resulted in a
significant 1.35-fold increase (p<0.05) in respiration. No effect
was observed in this species at 10 % air saturation and no consistent
effects were observed in any other species. Likewise, the CO2-plus
treatment resulted in a significant 1.6-fold increase (p<0.05) in
ammonium excretion in P. abdominalis at 33 % air saturation and
11 ∘C, but no effects were observed in this species at any other
condition tested or in any other species. To estimate the sensitivity of our
analysis we artificially increased the respiration rates at CO2-plus or
CO2-minus conditions stepwise by a factor of 0.1 to determine the
fold changes at which more than 50 % of the tests become significant.
This was the case at a 1.2-fold increase for the respiration rate tests (7 of
14 tests significant) and a 1.5-fold increase for the excretion rate tests (6
of 11 tests significant). As no consistent significant effects of our
CO2-treatment were found, we combined data from pCO2-plus and
pCO2-minus incubations to further characterize the effects of
temperature and oxygen levels on metabolic activity.
Temperature and oxygen-dependence of respiration and hypoxia
tolerance
Respiration rates at normoxia (trial start conditions 100 %
air saturation) were always significantly lower at 11 or 13 ∘C than
at 23 ∘C in Undinula vulgaris, Pleuromamma abdominalis and Euphausia gibboides (t test, p<0.05). The Q10 temperature coefficient of respiration at normoxia
(pO2 > 15 kPa, Temp. = 11–23 ∘C) was 1.4
for U. vulgaris, and 2.0 for P. abdominalis and
E. gibboides (Table 3). Standardized individual respiration rates
and fitted power functions are shown in the upper panels of Figs. 5 and 6.
Respiratory pcrit of E. gibboides and P.
abdominalis were 2.4 (at 13 ∘C) and 0.6 kPa (at
11 ∘C), respectively (Figs. 5 and 6, Table 3). As no data on
respiratory activity below 1.9 kPa were obtained for P.
abdominalis, the pcrit likely lies between the calculated
pcrit of 0.6 and 1.9 kPa. In the case of E. gibboides the
respiratory pcrit was almost identical with the mean lethal
pO2 of 2.5 kPa (SD = 1.4, n = 20) at 13 ∘C. The
mean lethal pO2 of U. vulgaris was 2.7 kPa (SD = 0.3,
n = 6) at 11 ∘C. No mortality was observed for P.
abdominalis down to 1.9 kPa at 11 ∘C. E. mucronata survived several hours at anoxia at 13 ∘C, but survival
time at anoxia was not systematically determined. The respiratory
pcrit of E. mucronata was found to be 0.6 kPa at
13 ∘C. Data from Teal and Carey (1967) supplement our observations
on the respiratory rates of E. mucronata. From their Fig. 2, an
approximate pcrit of 2 to 4 kPa at 20 ∘C can be deduced.
In all species that we tested, the respiratory pcrit was higher at
23 ∘C than at 11 or 13 ∘C (Table 3). At 23 ∘C, the
mean lethal pO2 was 6.6 kPa in U. vulgaris (SD = 0.7,
n = 6), 6.3 in P. abdominalis (SD = 1.0, n=2) and 7.5
in E. gibboides (SD = 1.9, n= 20). Respiration rates
calculated according to Ikeda (2014) for 0.1 g copepods or euphausiids
coincided well with the standardized respiration rates observed at normoxia
in all species (Figs. 5, 6).
Weight-specific respiration and ammonium excretion rates of
Undinula vulgaris and Pleuromamma abdominalis at different
oxygen partial pressures and temperatures (CO2-plus and CO2-minus
treatments combined). Magenta symbols = 23 ∘C, Blue
symbols = 11 ∘C. Transparent dots represent individual
measurements. Solid curves indicate the power function fits to the data.
Regression coefficients are given in Table 3. Solid vertical lines indicate
the respective pcrit. Horizontal error bars in the upper panels
indicate the mean (±SD) lethal pO2 at the respective temperature.
A power function could not be fitted to the ammonium excretion data for
U. vulgaris at 23 ∘C, therefore the mean (±SD)
excretion rates for the three pre-set oxygen levels (100 % air
saturation, 50 and 33 %) are plotted with vertical errorbars. Respiration
and excretion rates were standardized to a mean dry mass of 0.1 g. Error
bars right beside the plots indicate the respiration or ammonium excretion
rate (±SE) calculated according to Ikeda (2014) for calanoid copepods
(0.1 gDW) at the respective temperature.
Weight-specific respiration and ammonium excretion rates of
Euphausia gibboides and Euphausia mucronata at different
oxygen partial pressures and temperatures (CO2-plus and CO2-minus
treatments combined). Magenta symbols = 23 ∘C, Green
symbols = 13 ∘C. Transparent dots represent single measurements.
Solid curves indicate the power function fits to the data. Regression
coefficients are given in Table 3. Solid vertical lines indicate the
respective pcrit for respiration and ammonium excretion. Horizontal
error bars indicate the mean (±SD) lethal pO2 at the given
temperature for E. gibboides. A power function could not be fitted
to the ammonium excretion data for E. gibboides, therefore the mean
(±SD) excretion rates for the three pre-set oxygen levels (100 % air
saturation, 50 and 33 %) are plotted with vertical errorbars. Respiration
and excretion rates were standardized to a mean dry mass of 0.1 g. Error
bars right beside the plots indicate the respiration or ammonium excretion
rate (±SE) calculated according to Ikeda (2014) for euphausiids
(0.1 gDW) at the respective temperature.
Ammonium excretion rates
The Q10 temperature coefficient of ammonium excretion at normoxia (trial
start conditions 100 % air saturation) was 1.6 for Undinula vulgaris, and 2.3 for both Pleuromamma abdominalis and
Euphausia gibboides. It was possible to fit power functions to most
ammonium excretion data (Figs. 5 and 6, Table 3) except for U. vulgaris at 23 ∘C and for E. gibboides at 13 and
23 ∘C. For the latter species and experimental conditions students
t-tests grouped by initial oxygen concentrations revealed no statistically
significant changes in excretion rates with decreasing oxygen levels,
compared to the excretion rates at normoxia. The ammonium excretion
pcrit was very similar when compared to the respiratory
pcrit in all species observed and ammonium excretion at anoxia was
drastically down-regulated in E. mucronata. Mean (±SD)
standardized ammonium excretion at severe hypoxia
(pO2 < 0.5 kPa) was significantly reduced by a factor of
5.9 compared to ammonium excretion (0.65 ± 0.33 vs.
3.82 ± 2.54 µmol h-1 gDW-1) under normoxia
(15–22 kPa pO2, t test, p<0.01) in E. mucronata.
Ammonium excretion rates calculated according to Ikeda (2014) for 0.1 g
copepods or euphausiids coincided well with the excretion rates observed at
normoxia in almost all species (Figs. 5, 6). Only the excretion rates
observed in E. gibboides were lower than predicted. Stomachs of
E. gibboides were mostly half-full and midgut colouration ranged
between 1 and 2 in this species.
Discussion
Empirical models exist to predict copepod (Ikeda, 2014) and euphausiid
(Tremblay et al., 2014; Ikeda, 2014) respiration and excretion rates, but
these models do not include pO2 or pCO2 as environmental factors. We developed
here an approach to determine copepod and euphausiid respiration
and excretion rates at temperatures, and oxygen and carbon dioxide levels
consistent with those found in the ETNA and ETSP OMZ. We furthermore tested
whether respiration and excretion rates are altered when the oxygen, but not
the carbon dioxide level is experimentally adjusted to represent OMZ
conditions compared to the scenario when both levels are adjusted according
to OMZ conditions.
Recommended volumetric composition for premixed gases for the ETSP
OMZ.
50 % air sat.
33 % air sat.
10 % air sat
Nitrogen
89.56
93.00
97.81
ETSP CO2-plus
Oxygen
10.36
6.91
2.07
Carbon dioxide
0.08
0.09
0.11
Lethal pO2, parameter estimates (a, b) of fitted power
functions, pcrit and Q10 values for respiration and excretion
rate data.
Species
Target
mean lethal
minimum
aresp
bresp
pcrit, resp
aNH4-excr
bNH4-excr
pcritNH4-excr
Q10, resp
Q10,NH4-excr
Temp.
pO2(kPa),
lethal pO2
(kPa)
(kPa)
(∘C)
(SD, n)
(kPa)
U. vulgaris
11
2.7 (0.3, 6)
2.3
15.48
0.20
2.4
2.29
0.13
2.9
1.4
1.6
U. vulgaris
23
6.6 (0.7, 6)
5.5
27.55
0.17
6.2
NA
NA
NA
P.abdominalis
11
NA
NA
9.62
0.33
0.6
1.51
0.30
1.8
2.0
2.3
P.abdominalis
23
6.3 (1.0, 2)
5.3
31.31
0.25
6.5
6.37
0.13
6.5
E. gibboides
13
2.5 (1.4, 20)
1.1
19.70
0.22
2.4
NA
NA
NA
2.0
2.3
E. gibboides
23
7.5 (1.9, 20)
4.5
27.12
0.33
6.2
NA
NA
NA
E. mucronata
13
NA
NA
10.00
0.39
0.6
1.27
0.36
0.73
NA
NA
E. mucronata
20
NA
NA
NA
NA
2 to 4*
NA
NA
NA
NA
NA
* Approximate pcrit deduced from Fig. 2 in Teal
and Carey (1967). NA = not applicable.
Impact of pCO2-levels on respiration
and excretion
As expected, the use of CO2-minus gas mixes resulted in a removal of
CO2 from the incubation water, mirrored in a pH increase and a
pCO2 decrease. The use of CO2-plus gas mixes allowed a more
realistic simulation of OMZ conditions. pH, AT and
CT data were available for the Peruvian OMZ (WOCE, 2002) and we
calculated the CO2-plus gas mix composition based on the available pH
and CT data. pCO2 calculated from AT and
CT is lower and more realistic, as these two parameters can be
estimated more reliably. We therefore now suggest the use of the gas mixes
listed in Table 2 to experimentally establish Peruvian OMZ pO2 and
pCO2 levels.
Realistic adjustment of the carbonate system did not result in consistent,
statistically significant changes in the respiration or ammonium excretion
rate in tropical Atlantic copepod or euphausiid species, or the Pacific
Euphausia mucronata, compared to artificially lowered
pCO2-levels. In some experiments (e.g. respiration rate of U. vulgaris at 11 ∘C and 10 % air saturation) the number of
replicates was low, but also in cases where many replicates were available,
as well as with the GLM no significant effects were found. The approximate
detection level of our approach was a 1.2-fold difference in respiration and
a 1.5-fold difference in ammonia excretion. We did not acclimatize the
animals to the respective test conditions, as we aimed to mimic DVM changes
in temperature, pO2 and pCO2 experienced by the animals when
migrating from the OMZ to the surface layer and back. These migrations are
conducted within 1 to 5 h (Fischer and Visbeck, 1993; Heywood, 1996) and the
animals should therefore be able to cope with fast changes in environmental
conditions. Previously, copepods were found to be relatively robust if it
comes to acute changes in pCO2 (Isari et al., 2015; Thor and Dupont,
2015; Thor and Oliva, 2015). Food quality and quantity, life history (Thor
and Dupont, 2015; Thor and Oliva, 2015) and other environmental constraints
(e.g. temperature, pO2) seem to be more important than pCO2 in
determining metabolic rates. Thor and Dupont (2015) for example found that
respiration rates of Pseudocalanus acuspes copepods reared in the
laboratory for two generations at elevated CO2-levels (0.09 kPa) had
1.14-fold higher respiration rates than copepods reared at 0.04 kPa
pCO2. Respiration rates of copepods reared at 0.15 kPa were
significantly reduced by a factor of 0.84 compared to those of copepods
reared at 0.04 kPapCO2. However, if animals reared at 0.09 kPa were
tested at 0.04 kPa or vice versa, no significant changes in respiration
rates were found. The same holds true for tests of animals raised at 0.04 and
0.15 kPa. Isari et al. (2015) found no statistically significant differences
in respiration rates in Acartia grani and Oithona davisae
upon acute exposure to 0.12 kPa pCO2, compared to 0.04 kPa. Results
are however difficult to assess as the number of replicates was low (n=2),
and mortality in the batch incubations seemingly was not zero. Thor and Oliva
(2015) found both increases and decreases in respiration rates after exposure
to elevated pCO2 for 10 days in a Skagerrak population of P. acuspes, but not in a Svalbard population. Food was a better predictor of
respiration rate than pH in both populations. Until now, only one study
assessed metabolic responses of Euphausiids to pCO2-level changes
(Saba et al., 2012). A statistically significant 3-fold increase in DOC
excretion in response to an elevated pCO2 (0.07 vs. 0.04 kPa; 24 h
incubations) was found for E. superba, whereas ammonium, phosphate
and urea excretion were not significantly impacted. The increase in DOC
excretion might be related to an observed increased feeding activity at
elevated pCO2, which currently remains unexplained (Saba et al.,
2012).
The fact that Pleuromamma abdominalis, E. gibboides and
E. mucronata are naturally exposed to daily varying
pCO2-levels when migrating into and out of the OMZ might also explain
why no consistent changes in respiration or excretion rates under
CO2-plus vs. CO2-minus conditions were found in these species.
Migrating organisms that are regularly exposed to pCO2-changes may be
less sensitive than those that live at stable conditions. Rosa and
Seibel (2008) found that in the cephalopod (Dosidicus gigas)
routine metabolism was not impacted by exposure to a pCO2 of 0.1 kPa
at low temperatures characteristic for the ETSP OMZ. Maas et al. (2012a)
found no significant differences in respiration rates when pteropods
migrating into the OMZ were exposed to OMZ CO2-levels (0.1 kPa)
compared to surface CO2-levels (0.04 kPa) at 20 ∘C. Acute
CO2 effects on metabolic activity in copepods and other DVM organisms
therefore seem to be small and might also be masked by the response to
concomitant changes in O2-levels the animals experience when migrating
into and out of the OMZ.
Effects of temperature and oxygen on metabolic activity
That temperature strongly affects metabolic activity is well established and
our results are consistent with the general observation that zooplankton
metabolic activity doubles with a 10 ∘C increase in temperature
within the thermal window of the species (Ikeda, 2014). The Q10 of
respiration and ammonium excretion was relatively close to 2 in
Euphausia gibboides and Pleuromamma abdominalis. The
Q10 was below 2 in Undinula vulgaris, but this might be related
to the fact that this surface dwelling species is seldom exposed to
11 ∘C and the Q10 therefore might not be representative for the
normal thermal range of this species. An increase in temperature furthermore
impaired hypoxia tolerance as indicated by a higher lethal pO2,
respiration pcrit and ammonium excretion pcrit in all
species tested. That elevated temperatures impair hypoxia tolerance has been
found for numerous other marine species and quantitative estimates of the
pcrit can help to understand distribution patterns of marine
organisms (e.g. Deutsch et al., 2015). Consistent with previous studies (e.g.
Childress and Seibel, 1998), respiratory pcrit values of E. mucronata from the ETSP were found to be lower than in species from weak-
(E. gibboides, P. abdominalis and U. vulgaris)
or non-OMZ regions (Childress and Seibel, 1998). No large differences in
respiratory pcrit could be observed when comparing the tropical
Atlantic copepod and krill species. In general, our findings support the
hypothesis that the critical oxygen partial pressure evolved to largely match
the minimum oxygen level to which a species is regularly exposed (Seibel,
2011; Richards, 2011).
It seems reasonable to also transfer this concept to the impact of oxygen
levels on ammonium excretion rates. A reduction of ammonium excretion under
severely hypoxic or anoxic conditions was observed in E. mucronata
and is consistent with similar observations from the squat lobster
Pleuroncodes monodon (Kiko et al., 2015a) and from several calanoid
copepods (Cass and Daily, 2014). It follows that both respiration and
ammonium excretion are drastically reduced when crustacean zooplankton
organisms are exposed to severe hypoxia or anoxia. The characterization of
metabolic rates across the entire tolerated oxygen level spectrum (including,
if possible, anoxia) is key to properly estimate effects of hypoxia and
anoxia on species distribution and activity, as well as related
biogeochemical fluxes. Furthermore, the determination of respiration and
excretion rates over a wider temperature range and at higher resolution could
be helpful to better understand differences in performance between migratory
and non-migratory species. The inclusion of further factors (e.g. time of day
and feeding status) could also lead to a more precise predictive model of
zooplankton and nekton respiration and excretion rates.
Implications for the calculation of biogeochemical fluxes of
oxygen, carbon and nitrogen
Several studies have assessed the active DVM-mediated fluxes and the passive
particle-mediated fluxes of carbon and nitrogen in regions that mostly do not
feature severe hypoxia (pO2 < 1 kPa) or anoxia at midwater
depths (Longhurst et al., 1990; Zhang and Dam, 1997; Hidaka et al., 2001;
Steinberg et al., 2002; Davison et al., 2013). Excretion rates used in these
studies to calculate the active flux were obtained at mildly hypoxic
(pO2 >1 kPa) to normoxic conditions (e.g. Donnelly and
Torres, 1988; Dagg et al., 1980; Steinberg et al., 2002). For OMZs
demonstrating only mild hypoxia, such as the ETNA OMZ, this approach seems
reasonable. The respiration and ammonium excretion pcrit of
Pleuromamma abdominalis were found to be 0.6 and 1.8 kPa at
11 ∘C, respectively. The respiratory pcrit of
Euphausia gibboides was found to be 2.4 kPa at 13 ∘C and
no significant reduction of ammonium excretion was observed at a mean
pO2 of 5.1 kPa for E. gibboides at this temperature. P.
abdominalis and E. gibboides normally do not encounter
extremely low oxygen levels in their natural habitat. Oxygen concentrations
in the ETNA seldom fall below 40 µmol kg-1(∼ 3.4 kPa
at 12 ∘C, Karstensen et al., 2008). It follows that zooplankton
respiration and excretion rates for biogeochemical flux calculations in the
ETNA can be calculated from published empirical models of zooplankton
respiration and excretion (Ikeda, 2014), if oxygen levels above
∼ 2.4 kPa are encountered. However, food conditions should also be
taken into account. Excretion rates of E. gibboides at normoxia were
lower than predicted via the empirical model by Ikeda (2014), possibly due to
low food availability as indicated by low midgut colouration scores.
Estimates of DVM-mediated fluxes to midwater depth with drastically reduced
oxygen levels in the Pacific (e.g. Longhurst et al., 1990; Escribano et al.,
2009; Bianchi et al., 2014) are likely too high. E. mucronata
migrates to the core of the ETSP OMZ that features oxygen concentrations
below 4 µmol O2 kg-1 (∼ 0.34 kPa) (Antezana
2009). Later work by Thamdrup et al. (2012) showed that the core of the ETSP
OMZ is often anoxic. Antezana (2002) tested the dependence of respiration on
oxygen concentration at 13 ∘C, but only tested oxygen levels
> 1.8 kPa (21 µmol O2 kg-1) and therefore
did not characterize the response of E. mucronata to the ETSP OMZ
core conditions. A reduction in respiration activity at oxygen levels of 1.8
to 3.5 kPa (21 to 43 µmol O2 kg-1) in comparison to
measurements at normoxia was not observed (Antezana, 2002). We here show that
reduction in respiration and ammonium excretion sets in at about 1 kPa
(∼ 12 µmol O2 kg-1) in E. mucronata.
Specimens migrating to the OMZ core therefore are exposed to oxygen
concentrations below their pcrit for respiration and ammonium
excretion. We obtained similar results for the squat lobster
Pleuroncodes monodon that also seems to conduct regular migrations
to the OMZ core (Kiko et al., 2015a) and Cass and Daly (2014) report a strong
reduction of nitrogen excretion in several copepod species at oxygen levels
of 4 to 17 µmol O2 kg-1 (0.3 to 1.3 kPa). It follows
that calculations of DVM-mediated respiratory carbon dioxide and ammonium
release into the anoxic core of the Pacific OMZ need to be adjusted. We
observed here a 5.3-fold reduction of ammonium excretion in E. mucronata and found a 4-fold reduction in P. monodon (Kiko et
al., 2015a). Generalizations that DVM mediated ammonium export is 20 % of
the passive export flux (Bianchi et al., 2014) seem invalid and it seems
unlikely that DVM mediated ammonium supply to anoxic OMZs can support anammox
to a large extent. Parameterizations that account for changes in respiration
and ammonium excretion with decreasing oxygen levels should be applied in
biogeochemical modelling studies to calculate DVM-mediated impacts on
biogeochemical cycles in a consistent model framework.
OMZs are thought to expand due to climate change, as warming reduces the
solubility of oxygen in seawater and enhances stratification (Stramma et al.,
2008). During the last decades, it has been observed that the vertical extent
and the area covered by tropical oceanic OMZs expanded in the Eastern North
Pacific (Bograd et al., 2008) as well as in the Indian Ocean, the ETSP and
the ETNA (Stramma et al., 2008). Modelling studies predict a further decrease
in the global oxygen inventory (Bopp et al., 2013; Cocco et al., 2013).
However, the detailed extension of OMZs seems to be difficult to model and
Cocco et al. (2013) come to the conclusion that “projections of the
evolution of low O2 regions will vary among models and be affected by
large uncertainties”. Median deviations of the water volumes with oxygen
levels below 5 and 50 µmol oxygen kg-1 predicted for the year
1990 by the 17 models investigated (Cocco et al., 2013; Bopp et al., 2013)
from the volumes observed in 1990 (Bianchi et al., 2012) are 6.7 (range: 1.0
to 21.1) and 1.9 (range: 1.1 to 4.8), respectively. To some extent problems
to predict the observed OMZ extensions might be related to an unrealistic
representation of oxygen-dependent processes, including the regionally
varying dependency of DVM-mediated export processes on the physiological
capacities of the migrating organisms to cope with low oxygen levels. We
expect that a decrease in oxygen levels below approximately 2.4 kPa
(∼ 30 µmol O2 kg-1, at 11 to 13 ∘C) in
the ETNA will cause avoidance of these regions and will therefore result in a
reduction of DVM mediated export. Expansion of the anoxic core of the ETSP
should also result in a reduction of DVM-mediated fluxes, not via exclusion
of anoxia-tolerant migrators, but via repression of their metabolic activity.
Both effects would weaken the biological pump and would therefore affect the
oceanic CO2 uptake capacity, possibly enhancing global warming.