Copepods Boost the Production but Reduce the Carbon Export Efficiency by Diatoms

Abstract

The fraction of net primary production that is exported from the euphotic zone as sinking particulate organic carbon (POC) varies notably through time and from region to region. Phytoplankton containing biominerals, such as silicified diatoms have long been associated with high export fluxes. However, recent reviews point out that the magnitude of export is not controlled by diatoms alone, but determined by the whole plankton community structure. The combined effect of phytoplankton community composition and zooplankton abundance on export flux dynamics, were explored using a set of 12 large outdoor mesocosms. All mesocosms received a daily addition of minor amounts of nitrate and phosphate, while only 6 mesocosms received silicic acid (dSi). This resulted in a dominance of diatoms and dinoflagellate in the +Si mesocosms and a dominance of dinoflagellate in the −Si mesocosms. Simultaneously, half of the mesocosms had decreased mesozooplankton populations whereas the other half were supplemented with additional zooplankton. In all mesocosms, POC fluxes were positively correlated to Si/C ratios measured in the surface community and additions of dSi globally increased the export fluxes in all treatments highlighting the role of diatoms in C export. The presence of additional copepods resulted in higher standing stocks of POC, most probably through trophic cascades. However it only resulted in higher export fluxes for the −Si mesocosms. In the +Si with copepod addition (+Si +Cops) export was dominated by large diatoms with higher Si/C ratios in sinking material than in standing stocks. During non-bloom situations, the grazing activity of copepods decrease the export efficiency in diatom dominated systems by changing the structure of the phytoplankton community and/or preventing their aggregation. However, in flagellate-dominated system, the copepods increased phytoplankton growth, aggregation and fecal pellet production, with overall higher net export not always visible in term of export efficiency.

Full text

ORIGINAL RESEARCH
published: 22 March 2018
doi: 10.3389/fmars.2018.00082
Frontiers in Marine Science | www.frontiersin.org 1March 2018 | Volume 5 | Article 82
Edited by:
Eric ’Pieter Achterberg,
GEOMAR Helmholtz Centre for Ocean
Research Kiel, Germany
Reviewed by:
Jan Taucher,
GEOMAR Helmholtz Centre for Ocean
Research Kiel, Germany
Javier Arístegui,
University of Las Palmas de Gran
Canaria, Spain
*Correspondence:
Brivaëla Moriceau
moriceau@univ-brest.fr
Specialty section:
This article was submitted to
Marine Biogeochemistry,
a section of the journal
Frontiers in Marine Science
Received: 08 August 2017
Accepted: 26 February 2018
Published: 22 March 2018
Citation:
Moriceau B, Iversen MH, Gallinari M,
Evertsen A-JO, Le Goff M, Beker B,
Boutorh J, Corvaisier R, Coffineau N,
Donval A, Giering SLC, Koski M,
Lambert C, Lampitt RS, Le Mercier A,
Masson A, Stibor H, Stockenreiter M
and De La Rocha CL (2018)
Copepods Boost the Production but
Reduce the Carbon Export Efficiency
by Diatoms. Front. Mar. Sci. 5:82.
doi: 10.3389/fmars.2018.00082
Copepods Boost the Production but
Reduce the Carbon Export Efficiency
by Diatoms
Brivaëla Moriceau 1
*, Morten H. Iversen 2, Morgane Gallinari 1, Antti-Jussi O. Evertsen 3,
Manon Le Goff 1, Beatriz Beker 1, Julia Boutorh1, Rudolph Corvaisier 1, Nathalie Coffineau 1,
Anne Donval 1, Sarah L. C. Giering 4, Marja Koski 5, Christophe Lambert 1,
Richard S. Lampitt 4, Alain Le Mercier 1, Annick Masson 1, Herwig Stibor 6,
Maria Stockenreiter 6and Christina L. De La Rocha 1
1Laboratoire des Sciences de l’environnement Marin (LEMAR) UMR6539 CNRS/UBO/IFREMER/IRD, Université de Bretagne
Occidentale, Institut Universitaire Européen de la Mer (IUEM), Technopole Brest-Iroise, Plouzané, France, 2Alfred Wegener
Institute for Polar and Marine Research, MARUM, University of Bremen, Bremen, Germany, 3Department of Biology,
Norwegian University of Science and Technology, Trondheim, Norway, 4National Oceanography Centre Southampton,
Natural Environment Research Council, University of Southampton, Southampton, United Kingdom, 5Institute for Aquatic
Resources (DTU Aqua), Technical University of Denmark, Charlottenlund, Denmark, 6Biology II, Aquatic Ecology,
Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany
The fraction of net primary production that is exported from the euphotic zone as
sinking particulate organic carbon (POC) varies notably through time and from region to
region. Phytoplankton containing biominerals, such as silicified diatoms have long been
associated with high export fluxes. However, recent reviews point out that the magnitude
of export is not controlled by diatoms alone, but determined by the whole plankton
community structure. The combined effect of phytoplankton community composition
and zooplankton abundance on export flux dynamics, were explored using a set of 12
large outdoor mesocosms. All mesocosms received a daily addition of minor amounts of
nitrate and phosphate, while only 6 mesocosms received silicic acid (dSi). This resulted
in a dominance of diatoms and dinoflagellate in the +Si mesocosms and a dominance
of dinoflagellate in the −Si mesocosms. Simultaneously, half of the mesocosms had
decreased mesozooplankton populations whereas the other half were supplemented
with additional zooplankton. In all mesocosms, POC fluxes were positively correlated to
Si/C ratios measured in the surface community and additions of dSi globally increased
the export fluxes in all treatments highlighting the role of diatoms in C export. The
presence of additional copepods resulted in higher standing stocks of POC, most
probably through trophic cascades. However it only resulted in higher export fluxes for the
−Si mesocosms. In the +Si with copepod addition (+Si +Cops) export was dominated
by large diatoms with higher Si/C ratios in sinking material than in standing stocks. During
non-bloom situations, the grazing activity of copepods decrease the export efficiency in
diatom dominated systems by changing the structure of the phytoplankton community
and/or preventing their aggregation. However, in flagellate-dominated system, the
copepods increased phytoplankton growth, aggregation and fecal pellet production, with
overall higher net export not always visible in term of export efficiency.
Keywords: biogenic silica, POC, marine snow, zooplankton, mesocosm, Bay of Hopavågen, plankton community,
biological pump

Moriceau et al. Diatom and Copepod Control on Export
INTRODUCTION
The export of particulate organic carbon (POC) from the surface
ocean, in terms of the overall amount or as the fraction of local
net primary production, varies seasonally as well as regionally
(Lutz et al., 2002; Boyd and Trull, 2007; Honjo et al., 2008;
Buesseler and Boyd, 2009; Lam et al., 2011; Henson et al., 2012;
Siegel et al., 2016). Numerous factors intervene in this variability:
turbulence, stratification, and mixed layer depth; phytoplankton
community composition; the rates, timing, and extent of
seasonality of primary production; meso- and microzooplankton
abundance and feeding strategies; the aggregation of particulate
organic matter (POM) into large, rapidly sinking particles of
marine snow; and the occurrence of ballast particles like biogenic
silica, calcium carbonate, and dust. The actions and interactions
of these factors determine the ocean food web, which either
recycles most of the organic matter in the surface ocean (resulting
in only minor export to depth) or is “leaky” (exporting a large
portion of the net primary production to depth). It is necessary
to understand food web interactions in order to predict the
biological pump’s variability and its ability to sequester CO2in
a future ocean with warmer temperatures, higher CO2, more
acidity, and differing nutrient inputs and ratios compared to the
present ocean (Bopp, 2005; Passow and Carlson, 2012; Alvain
et al., 2013; Bopp et al., 2013).
Relationships between food webs, ballast minerals and fluxes
have been investigated in various physical regimes. On the
smaller scale of such investigations are microcosm studies of
sinking particles in rolling tanks (Shanks and Trent, 1980;
Passow and De La Rocha, 2006) and flow through systems
(Ploug et al., 2008; Long et al., 2015) that allow controlled
examination of selected processes and interactions. At the
other extreme are regional and global scale studies based on
models, remote sensing, and observational data from time-series,
cameras, autonomous platforms, and sediment traps (Klaas and
Archer, 2002; Honjo et al., 2008; Klaas et al., 2008; Lee et al., 2009;
Lam et al., 2011; Assmy et al., 2013; Quéguiner, 2013; Giering
et al., 2014; Sanders et al., 2014; Guidi et al., 2016). However,
whereas small scale laboratory investigations allow full control
over environmental variables and mechanistic investigations,
they often lack natural community composition. Field studies
allow identification of larger scale patterns and correlations with
environmental variables but no clear identification of causal
relationships owing to complex confounding factors. Mesocosms
offer a middle ground, where some of the control of small
scale laboratory experiments is combined with parts of the
complexity of environmental variables of field observations.
Mesocosms enclose part of an in situ water column allowing
manipulations of target parameters. Mesocosms are large enough
to host a reasonably complex food web (e.g., including micro-
and mesozooplankton) (Wassmann et al., 1996; Svensen et al.,
2001; Sommer et al., 2004; Stibor et al., 2004; Olsen et al., 2006;
Stange et al., 2017) while still allow controlled manipulations of
parameters such as nutrients, ballast minerals, turbulence, and
phytoplankton and zooplankton community composition.
A small number of mesocosm experiments have been used to
study phytoplankton community interactions and POC export.
Early work noted a strong link between the addition of silicic
acid (in addition to nitrate and phosphate) and POC export
fluxes (Wassmann et al., 1996). It was hypothesized that the
addition of silicic acid promoted the growth of diatoms, which
increased export of POC (Engel et al., 2002; Kemp et al., 2006;
Kemp and Villareal, 2013; Rynearson et al., 2013; Lasbleiz et al.,
2014). However, later studies showed that artificial mixing of
upper water layers also initiated aggregate formation even in
the absence of diatoms, suggesting that diatoms (and indirectly
silicic acid) were not the sole trigger of high POC fluxes (Svensen
et al., 2001, 2002). Several factors could results in high POC
export, including phytoplankton aggregation of both diatom and
non-diatom phytoplankton and zooplankton grazing, suggesting
that the whole plankton community structure - more than just
presence of diatoms–is important to determine export fluxes
(Gehlen et al., 2006; Guidi et al., 2016).
Generally carbon export follows the seasonality of primary
production but is even more dependent on the fraction of slow-
sinking versus fast-sinking aggregates, with higher export for fast
sinking aggregates (Moriceau et al., 2007; Henson et al., 2015).
However, the influence of seasonality and plankton community
composition on global export efficiency (proportion of primary
production that is transported below the mixed layer depth) is
still poorly understood. Except for recent studies on the impact
of acidification or elevated CO2concentrations (Paul et al., 2015;
Bach et al., 2016; Spilling et al., 2016; Gazeau et al., 2017) most
mesocosms studies have focused on the processes that lead to
export during phytoplankton bloom conditions, even though
non-blooming periods can potentially be important for global
export fluxes, and can be periods of efficient export of carbon and
bSiO2(Fujii and Chai, 2005; Morris et al., 2007; Lam et al., 2011).
Efficient export during non-bloom conditions has been linked to
zooplankton abundance, which can repackage small particles into
dense, fast-sinking particles (Lalande et al., 2016).
We designed a mesocosm study to investigate the link between
plankton composition and export flux during non-blooming
conditions. We explicitly tested the impact of zooplankton
abundance on export fluxes for two different phytoplankton
populations.
MATERIALS AND METHODS
Study Area
The experiment was conducted in 2012, between August 2 and
24 in the Bay of Hopavågen (63◦36′N, 9◦33′E), a tidally-driven,
semi-enclosed marine lagoon on the west coast of Norway, 20 km
west of the outlet of the Trondheimsfjord (Figure 1). This semi-
enclosed marine lagoon has a maximum depth of 32 m, a volume
of roughly 6.7 ×106m3, and exchanges roughly 14% of its water
daily with the ocean through a narrow inlet (van Marion, 1996).
Nutrient concentrations in the lagoon at the time of the
experiment were extremely low (<1µM for silicic acid, <0.3 µM
for ammonium, and <0.1 µM for nitrate, nitrite, and phosphate).
Concentrations of chlorophyll a (Chl) in the upper 10 m of
the lagoon at the time of the experiment ranged from 0.5 to
2.4 mg m−3, corresponding well to the typical average summer
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Moriceau et al. Diatom and Copepod Control on Export
FIGURE 1 | The Bay of Hopavågen.
concentrations for this lagoon (between 1–3 mg m−3,Olsen et al.,
2006).
Mesozooplankton commonly found in the Bay of Hopavågen
include the ctenophore Bolinopsis sp., calanoid copepods (such
as Temora sp., Centropages sp., and Pseudocalanus sp.) at an
average summer concentration of 20 ind L−1, the cyclopoid
copepod Oithona sp., and the appendicularian Okipleura sp.
(van Marion, 1996; Stibor et al., 2004; Vadstein et al., 2004).
The phytoplankton community in the lagoon consists of
diatoms (e.g., Rhizosolenia sp., Skeletonema sp., Thalassiosira sp.,
Nitzschia sp., and Pseudonitzschia sp.), autotrophic picoplankton,
dinoflagellates (e.g., Gymnodinium sp., Prorocentrum sp.), and
nanoflagellates (Sommer et al., 2005).
Mesocosms
Each mesocosm consisted of a 10 m deep polyethylene tube, with
a diameter of approximately 1 m, a volume of roughly 9 m3, and
a sealed, conical bottom. The twelve mesocosms were filled on
August 2 by lowering the entire mesocosm bag to a depth of
10 m and then raising the top gently back up to the surface. The
filled mesocosms were secured to a raft that was anchored in the
deepest part of the Bay of Hopavågen.
The light conditions in each mesocosm were similar, with light
intensities of 15–20% of surface light at 1 m depth (generally
around 100 µmol m−2s−1) and ∼1% at ∼6 m depth (Figure 2).
These conditions were similar to the light intensities observed
in the Bay of Hopavågen (around the mesocosms) during the
experiment (∼40% ∼300 µmol m−2s−1at 1 m and 8% at 6 m).
To avoid disturbances of formed aggregates within the
mesocosms and to avoid interfering with sinking fluxes, the
mesocosms were not mixed during the experiment. However,
some small-scale mixing and turbulence might have occurred
within the mesocosms due to waves and tidal currents within
the Bay of Hopavågen during the study. According to the Chl
profiles, the upper 2 m of the mesocosm bags were well-mixed
(Figure 2). Turbulence was not measured during the experiment,
but previous work in similar types of mesocosms suggested that
the energy dissipation rates in the mesocosms would have been
on the order of 10−9–10−8m2s−3, corresponding to a wind
velocity of 3–6 m s−1(Svensen et al., 2001).
In the Bay of Hopavågen, nutrients are resupplied daily by
the natural water inflow (Sommer et al., 2004). We mimicked
the natural nutrient input by manually adding nutrient to
the enclosed mesocosms, using nitrogen concentrations that
compensated for the loss of N caused by sedimentation and
Redfield ratios for phosphorus and silicon additions as described
in previous studies (Sommer et al., 2005; Olsen et al., 2006). Such
nutrients additions maintained the low natural phytoplankton
concentrations yet avoided accumulation of unrealistically high
concentrations of phytoplankton biomass (Børsheim et al., 2005).
Twelve mesocosms were set up in total, allowing the
investigation of four different treatments in triplicate. The
treatments were (1) silicate addition and decreased copepod
abundance (+Si −Cops), (2) silicate addition and increased
copepod abundance (+Si +Cops), (3) no silicate addition and
decreased copepod abundance (−Si −Cops), (4) no silicate
addition and increased copepod abundance (−Si +Cops).
Nutrients were added via an 8-m long tube. The tube was lowered
slowly to a depth of 8 m in each mesocosm and then fully
filled with a nutrient solution calculated to add the required
concentration of nutrients to each mesocosm. The tube was then
slowly lifted out to minimize disturbances of the water in the
mesocosms. Through the process of displacement, this allowed
the nutrients to distribute evenly throughout the water column
of the mesocosms (Olsen et al., 2007). Nutrients uptake are lower
in the dark (Dortch and Maske, 1982; Litchman et al., 2004) and
samplings were done in the morning. Nutrients were added in
the evening to avoid contamination of the morning sampling,
and began the day after the bags were filled (on the evening of
August 3) and 4.5 days before the first day of sampling. Previous
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Moriceau et al. Diatom and Copepod Control on Export
FIGURE 2 | Typical physical conditions within the mesocosms as exemplified by bag 3 (+Si −Cops) on August 23–24 (experiment Days 16–17).
study evidenced that such a time lag allows the phytoplankton
communities to differentiate depending on nutrient additions
(Gismervik et al., 2002; Sommer et al., 2004; Larsen et al.,
2015).
Following Stibor et al. (2004), mesozooplankton
concentrations were reduced in the −Cops treatments by
repeated vertical hauls with a 150-µm plankton net (Sommer
et al., 2004; Stibor et al., 2004). We refer to these mesocosm as
‘+Si −Cops’ and ‘−Si −Cops’, respectively. In the evening of
August 7 (5 days after the filling of the bags and 1 day before
the first sampling day), we increased the copepod abundance
in the +Cops treatments by adding copepods (4 copepods per
liter), mainly Centropages sp. and Oithona sp., collected from the
lagoon using a 150-µm plankton net.
Sample Collection and Analysis
Day 1 of the sampling was on the morning of August 8, 5.5 days
after filling the bag and 4.5 days after the first nutrient addition.
The sampling period lasted 17 days in total. On Days 1, 3, 6, 10,
13 and 16 of the sampling period, depth-integrated samples of
the upper 4.5 m of the mesocosms were collected between 7:00
and 8:00 in the morning. Sampling was done by repeated and
slow deployments of a 1 m long integrated water sampler. We
collected a total of 12-20 L from each mesocosm (0.1–0.2% of
initial water volume), depending on the sampling strategy of the
day. Water samples were placed in opaque 20 L LDPE carboys
(Nalgene), which were subsampled for total and fractionated
particulate organic carbon (POC) and nitrogen (PON), biogenic
silica (bSiO2), pigments, nutrients, phytoplankton cell counts,
and microscopic taxonomy of phytoplankton.
Sinking particles were collected with sediment traps from 8 m
depth three times during the course of the experiment, from
Day 3 to Day 8, from Day 9 to Day 12, and from Day 14 to
Day 17. This was done with cylindrical sediment traps with an
aspect ratio (height to width) of 6 in mesocosms in two of each
treatment triplicate. In the four remaining mesocosms (one of
each treatment triplicate), we deployed gel traps (McDonnell
and Buesseler, 2010) at 8 m to collect and preser ve the size
and structure of the sinking particles. The gel traps were only
deployed for 2 days in order to avoid individual particles landing
on top of each other in the gel. Gel traps were deployed on Day 3,
8, and 14.
Phytoplankton community compositions were determined
for one of each treatment triplicates (four mesosocosms in
total) four times during the sampling period. Water samples
were preserved with Lugol’s iodine (1% final concentration)
and taxonomically identified to species level using an inverted
microscope (Utermöhl, 1958).
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Moriceau et al. Diatom and Copepod Control on Export
Samples for particulate organic carbon (POC) and for
particulate organic nitrogen (PON) were filtered onto
precombusted (450◦C, 5 h) 25-mm GF/F filters (0.7-µm
nominal pore size, Whatman). Every second sampling day,
POC/PON samples were size fractionated before filtration using
different screens. Size fractions were 0.7–2.7, 2.7–20, 20–44,
44–100, and >100 µm. Filters were rinsed with MilliQ water
to remove salts and dried overnight at 60◦C. Inorganic carbon
was removed from the filters by fuming with HCl before analysis
with a Flash 1112 Series elemental CN analyzer (ThermoQuest).
Samples for Chl a were filtered onto GF/F filters (0.7-µm
nominal pore size, Whatman) and immediately frozen in liquid
nitrogen. The filters were stored at −80◦C until analysis by HPLC
(Shimadzu LC-10A HPLC system with LC Solution software;
Shimadzu). The HPLC system was calibrated with pigment
standards (DHI Water and Environment).
Samples for biogenic silica (bSiO2) were filtered onto 0.4-µm
polycarbonate filters (Millipore), dried overnight at 60◦C and
stored at room temperature until digestion and analysis. bSiO2
in the samples was dissolved in 0.2 M NaOH at 100◦C for 60 min
(Ragueneau et al., 2005) and neutralized with 1 M HCl for silicic
acid analysis by colorimetry. Particulate bSiO2concentrations
were corrected for lithogenic contribution following a second
digestion of the particulate matter to yield the Si:Al ratio of
the lithogenic silica (Ragueneau et al., 2005). Aluminum was
determined via inductively coupled plasma optical emissions
spectroscopy (ICP-OES).
Samples for nitrate, nitrite, silicic acid, phosphate and
ammonium were filtered through 0.4-µm polycarbonate filters
(Millipore) and analyzed using a Bran+Luebbe AAIII auto-
analyzer. Concentrations of ammonium in water samples were
measured manually on a spectrophotometer (Shimatzu UV 1700)
following Koroleff (1969).
Statistical Analysis
To test and differentiate the effect of time from the effect of the
different treatments: +Si vs. −Si and +Cops vs. −Cops, a two-
way or three-way analysis of variance (ANOVA) with a multiple
comparison procedure (Holm-Sidak method) were applied to our
data set (Sigmaplot 12, Systat Software, Inc.,) except for sediment
traps data for which only two replicates were done. The overall
significance level was chosen as p<0.05.
RESULTS
Nutrient Concentrations and Uptake
At the beginning of the sampling period, phosphate
concentrations were lower in the +Si mesocosms than in
the −Si mesocosms (0.02 ±0.02 µM vs. 0.06 ±0.03 µM).
Phosphate concentrations in the mesocosms increased during
the first 10 days of the experiment. After Day 10, however,
concentrations of phosphate decreased in all but the +Si +Cops
mesocosms (Figure 3A). The total net phosphate uptake during
the sampling period (Figure 4A,Table 1) in the −Si −Cops
mesocosms (0.11 ±0.02 µM) was lower than in the other three
treatments (+Si −Cops: 0.17 ±0.04 µM; +Si +Cops: 0.16 ±
0.04 µM; −Si +Cops: 0.18 ±0.01 µM).
Concentrations of silicic acid (dSi) in the −Si treatments
remained close to zero throughout the experiment (Figure 3B),
suggesting no measureable net dSi uptake in these mesocosms.
In the +Si mesocosms, there was no dSi uptake between Days 6
and 10, with net dSi uptake between Day 1 and 6 and between
Day 10 and 16 (Figure 4C). The total net dSi utilization in the
mesocosms during the sampling period was 1.4 ±1.0 µM (+Si
−Cops), 2.5 ±0.9 µM (+Si +Cops), 0.0 ±0.1 µM (−Si −Cops),
and −0.1 ±0.1 µM (−Si +Cops) (Table 1).
Continuously increasing cumulative net utilization of DIN
in the +Si mesocosms resulted in lower concentrations
compared to −Si mesocosms (Figures 3F,4E,Table 1). In
addition to the daily external input of nitrate of 0.22 µM,
we observed DIN production likely caused by ammonium
regeneration within the mesocosms. This was evident from
the mid-experiment peak in ammonium concentrations in all
mesocosms (Figure 3E). Following Day 10, however, ammonium
concentrations decreased, except in +Si +Cops, were the
decrease started at Day 13, suggesting that uptake exceeded
regeneration in all mesocosms in the final days of the experiment.
During the same period, nitrate, nitrite and overall DIN
concentrations declined (Figures 3C,D), suggesting increased
rates of DIN uptake rather than a decrease in the rates of
ammonium regeneration (n.b. over the 17 day duration of the
experiment, we would not expect to see notable amounts of
nitrate being regenerated).
In the +Si mesocosms, net removal rates of DIN were, on
average, 2–3 times faster than net removal rates of dSi (Table 1).
Since dSi removal rates were undetectable in the −Si mesocosms,
we could not calculate a net uptake ratio of DIN to dSi for
these mesocosms. DIN to phosphate uptake ratios remained
reasonably close to Redfield values of 16:1 in most of the
mesocosms (Table 1).
Standing Stocks and Phytoplankton
Composition
Integrated standing stocks of POC (upper 4.5 m) decreased
during the first days of the experiments and stabilized after
Day 3 for +Cops mesocosms and after Day 6 for −Cops
mesocosms (Figure 5A). The mean POC concentrations over the
experimental period were 18.5 ±6.4 µmol POC L−1in the +Si
−Cops, 24.7 ±7.7 µmol POC L−1for the +Si +Cops, 14.4 ±
4.9 µmol POC L−1for the −Si −Cops and 21.0 ±4.1 µmol
POC L−1for the −Si +Cops treatments, with values ranging
from 10 to 37 µmol POC L−1. Average POC concentrations
were similar to those measured in the lagoon at the end of
the experiment (19.5 µmol POC L−1;Figure 5A) and to those
obtain at similar nutrient additions in Børsheim et al. (2005).
Standing stocks of POC in the upper 4.5 m of the mesocosms
differed more between treatments than between the replicates for
each individual treatments or for each replicate over time (two
way ANOVA α=0.01; p<0.001). POC standing stocks were
higher in +Si mesocosms than in the other treatments at the
beginning of the sampling period. Differences in POC standing
stocks decreased over time. POC standing stocks were similar for
+Cops and −Cops treatments until Day 3, but started to differ
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Moriceau et al. Diatom and Copepod Control on Export
FIGURE 3 | Concentrations in the upper 4.5 m of the nutrients (A) phosphate, (B) silicate, (C) nitrate, (D) nitrite, (E) ammonium, and (F) total DIN. Data points and
error bars represent averages and standard deviations for the three mesocosms of each treatment.
FIGURE 4 | Cumulative net uptake in the different mesocosm treatments of (A) phosphate, (B) nitrate, (C) silicate, and (E) total DIN. (D) show the cumulative net
regeneration of ammonium. Data points and error bars represent averages and standard deviations for the three mesocosms of each treatment.
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Moriceau et al. Diatom and Copepod Control on Export
TABLE 1 | Total net nutrient removal during the experiment, ratios between removal, and average number of species during the duration of the experiment.
Mesocosms Total net removal Ratios of total net removal Average number of species
PO4 DIN Si(OH)4 DIN/Si DIN/P total Diatoms Dinoflagellates
+Si −Cops 0,17 2,8 1,4 2,0 16,6 13 4 8
+Si +Cops 0,16 3,8 2,5 1,5 23,9 19 4 12
−Si −Cops 0,11 2,0 0 - 18,5 11 2 8
−Si +Cops 0,18 2,7 −0,1 - 15,1 16 2 12
after Day 6, with more POC present in the +Cops mesocosms.
Over the whole sampling period, POC concentrations were
significantly higher in the six mesocosms that had received a
supplement of copepods compared to those where copepods had
been removed (Three way ANOVA; p<0.001). We also observed
significantly higher POC concentrations in +Si −Cops compared
to −Si −Cops and in +Si +Cops compared to −Si +Cops (Three
way ANOVA; p=0.006).
On the last sampling day (Day 16), the +Si +Cops mesocosms
reached an average standing stock of POC of 25.3 ±0.9 µmol
POC L−1and the −Si +Cops mesocosms an average of 23.6 ±
2.4 µmol POC L−1compared to 17.6 ±2.6 µmol POC L−1and
15.6 ±0.7 µmol POC L−1in the +Si +Cops and −Si −Cops
mesocosms, respectively.
Concentrations of total PON were also relatively stable during
the experiment with significant differences between +Cops
and −Cops mesocosms. However, unlike POC standing stocks,
differences between +Si +Cops and −Si +Cops or +Si −Cops
and −Si −Cops treatments were not significant (Figure 5B). C
to N ratios (C/N) of the organic matter in the upper 4.5 m of
the +Si mesocosms decreased from 6.7 ±0.8 mol mol−1on Day
1 to 4.4 ±0.5 mol mol−1on Day 6 and remained close to that
for the rest of the experiment. The C/N of the −Si mesocosms
averaged 4.4 ±0.3 mol mol−1during the experiment. The C/N
ratios of the standing stocks were very similar between treatments
with an average for the entire experiment of 4.7 ±0.8 mol mol−1
(Figure 5C).
Concentrations of Chl a ranged from 0.5 to a maximum of
4.7 µg L−1(Figure 5D). Chl a concentrations were generally
within the range for non-bloom conditions, in accordance with
the nutrient addition (Børsheim et al., 2005). Standing stocks of
Chl a decreased rapidly in the +Si mesocosms, from 3.0 ±0.5 and
4.2 ±0.4 µg/L on Day 1 for the +Cops and −Cops respectively,
to 0.6 ±0.2 and 0.7 ±0.2 µg/L on Day 6. After Day 6, Chl a
increased until the end of the experiment to reach 2.3 ±0.4 µg/L
in +Si +Cops and 1.1 ±0.6 µg/L in the +Si −Cops. Except
for the first day of the sampling period, Chl a concentrations
were higher in the +Cops mesocosms (three way ANOVA; p<
0.001; Figure 5D). The −Si −Cops mesocosms also experienced
a decrease from Day 1 to Day 6 (1.9 ±0.2 µg/L to 0.5 ±0.1
µg/L), before increasing to 0.9 ±0.4 µg/L at the end. The −Si
+Cops mesocosms Chl a concentrations was less variable. Chl
a concentrations slightly decreased from 2.2 µg/L on Day 1 to
1.4 ±0.4 µg/L at the end. The final average concentrations of
Chl a in the −Si +Cops mesocosms were lower than those in the
+Si +Cops (1.4 ±0.4 µg L−1vs. 2.3 ±0.4 µg/L, respectively, on
Day 16), but they were still ∼30% greater than the average final
concentrations in the +Si −Cops and −Si −Cops (1.1 ±0.6 and
0.9 ±0.4 µg L−1, respectively).
Integrated concentrations of bSiO2in the upper 4.5 m of the
−Si mesocosms, decreased to 0 and 0.1 µmol/L for −Cops and
+Cops at Day 6 respectively. At the beginning of the sampling
period, bSiO2concentrations were 0.8 ±0.1 µmol/L and 0.7 ±
0.1 µmol/L for the −Cops and +Cops respectively. In the +Si
mesocosms, bSiO2concentrations generally increased from Day
1 to Day 3 and then declined until Day 10 before increasing again
until the end of the experiment (Figure 5E). At the end of the
experiment, the bSiO2integrated concentrations in the upper
4.5 m of the +Si +Cops mesocosms, averaged 1.6 ±0.6 µmol
L−1, which was roughly 3-fold higher than that measured in the
+Si −Cops mesocosms (0.5 ±0.6 µmol L−1). The molar Si to
POC ratios (Si/C) of standing stocks mirrored the changes in
bSiO2concentrations rather than reflecting overall patterns in
the POC standing stocks (Figure 5F). Outside the mesocosms,
Si/C ratios were around 0.015, which is similar to the lowest ratios
reported for North Atlantic Waters (Ragueneau et al., 2002). The
+Si mesocosms had Si/C ratios of 0.04–0.10, similar to global
ocean average (0.04–0.25, Ragueneau et al., 2002). The Si/N ratios
in the nutrient stocks of +Si treatments were higher than 1, the
average Si/N utilization in the +Si mesocosms was around 1 with
fluctuations from 0.6 and 2.1.
POC standing stocks and Chl a concentrations were both
high at the beginning of the experiments especially in the +Si
mesocosms. Chl a estimated from CTD measurements 2 days
before the beginning of the sampling period gave an average
value of 2 mg/L. The daily uptake rate estimated from the
total nutrient additions were also lower than the first uptake
calculated during the experiment (0.2 vs. 0.3 µM/days for nitrate,
0.004 µM/days vs. 0.02 µM/days for phosphate and 0.3 vs. 0.3
µM/days for silicate), suggesting that no phytoplankton bloom
had developed before the sampling period. Throughout the entire
experiment, and in all treatments, 30–50% of the total biomass
was in the 2.7–20 µm size fractions, while the other four size-
fractions (0.7–2.7 µm, 20–44 µm, 44–100 µm, and >100 µm)
each had between 10 and 20% of the total biomass.
Taxonomic analysis confirmed that differing nutrient
additions induced changes in phytoplankton population, with
diatoms and flagellates dominating the +Si treatments, and
flagellates alone dominating the −Si treatments (Figure 6). We
identified up to 43 phytoplankton species in the +Si +Cops
mesocosms with an average of 19 over the entire sampling
period (Table 1). By contrast, a maximum of 23 species were
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Moriceau et al. Diatom and Copepod Control on Export
FIGURE 5 | Composition of standing stocks of biomass in the upper 4.5 m of the four treatments: (A) POC, (B) PON, (C) C/N, (D) chlorophyll a, (E) bSiO2, and (F)
Si/C. Data points and error bars represent averages and standard deviations for the three mesocosms of each treatment. Gray lines represent concentrations in the
Bay outside mesocosms as measured at the end of the experiment (Day 16).
found in the +Si −Cops, with an average of 13. For the −Si
mesocosms, the maximum number of species in +Cops and
−Cops treatments was 24 and 17, respectively, with an average
total amount of species of 16 and 11 for −Si +Cops and −Si
−Cops treatments, respectively. In the +Si +Cops treatments,
diatom concentrations decreased from Day 3 to Day 10, after
which their abundance increased (Figure 6). At Day 13, diatoms
concentrations were much lower in the +Si mesocosms. This
result was not mirrored by standing stock measurement of
bSiO2concentrations or by total cell concentrations according
to cytometry. This sudden change of diatom concentrations
may therefore potentially be due to analytical problems with
phytoplankton enumeration at this sampling day.
In the +Si treatments, 97% of the diatom populations were
constituted by small species (cell volume <2500 µm3) until
Day 6. Skeletonema marinoii (diameter ∼10 µm) dominate all
treatments until Day 3 and disappeared from −Si mesocosms
thereafter. From Day 13 the diatom population of the +Si
−Cops treatments switched from small S. marinoii to medium
sized-diatoms, with Cylindrotheca closterium (50 to 125 µm
length) forming most of the population in +Si −Cops while
Leptocylindrus danicus constituted more than 90% of the +Si
+Cops diatom population. The +Si treatments also developed
a large population of dinoflagellates, with the Gymnodinium sp.
dominating the dinoflagellates population, except for the +Si
−Cops after Day 6 when Scripsiella contribute for more than 50%
to the dinoflagellates population.
Dinoflagellates dominated the −Si treatments from Day 3
until the end of the experiments, with Gymnodinium sp. and
Scrippsiella (trochoida and sweenea) making up 60 to 95% of
the total dinoflagellates community in terms of cell abundance.
Gymnodinium sp. dominate the community before Day 6 and
Scrippsiella after.
Sinking Fluxes
Gel traps showed that sinking particles mostly consisted of fecal
pellets and large aggregates such as marine snow. During the
first trap deployment the +Si −Cops mesocosms particle fluxes
were characterized by a higher contribution of marine snow
and fewer fecal pellets than the +Si +Cops treatments. The
flux composition between these two treatments became more
similar toward the end of the study. At the end of the study,
particle fluxes in the +Si treatments where characterized by more
marine snow aggregates than the −Si that had a much higher
contribution by fecal pellets (Figure 7). Sinking fluxes of POC,
PON, and bSiO2decreased over time (Figure 7), not mirroring
standing stocks in the upper 4.5 m of the mesocosms (Figure 5).
The total POC exported during the experiment was 1.47 ±0.30 g
of C m−2in the +Si −Cops, 1.05 ±0.28 g of C m−2in the +Si
+Cops, 0.78 ±0.10 g of C m−2in the −Si −Cops and 0.86 ±
0.24 g of C m−2in the −Si +Cops (Figure 8). The C/N ratio
of the material sinking into sediment traps was higher than the
C/N ratios found in the suspended particles with more variability
between traps than between treatments (C/N in standing stocks
=4.5 ±0.6; C/N in sinking particles =7.6 ±2.0). The difference
between C/N in sediment traps and standing stocks was very
pronounced in the −Si −Cops at the end of the experiment,
when flux C/N ratios value reached an average of 10.6 ±5.
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Moriceau et al. Diatom and Copepod Control on Export
FIGURE 6 | Phytoplankton community structure in the different mesocosm treatments, in percentage, total cell numbers and biovolume.
The ratios of bSiO2to POC of sinking particles (Si/C) were
twice those measured in suspended particles (Figure 8). This
difference increased with elevated copepod abudances. Despite
the lack of measurable bSiO2in the suspended particles after day
6, Si/C ratios for the sinking material were still 0.02 in the −Si
mesocosms and some diatoms were observed in the sediment
traps. The POC fluxes were positively correlated to the Si/C ratios
of the standing stocks (Figure 9). Only the sinking material of the
traps set up at day 8 and 16 were analyzed for the phytoplankton
taxonomy. Small diatoms formed 96 to 100% of the diatom
fluxes collected in all traps except at the end of the experiment.
In the +Si −Cops the medium sized (<125 µm) Cylindrotheca
closterium formed 60% of the diatom cell numbers found in the
sediment traps. The contribution of Proboscia alata increased in
the +Si +Cops to achieve half of the sinking at the end.
DISCUSSION
Diatoms with their ballasted frustule (Armstrong et al., 2002;
François et al., 2002) have long been recognized to be efficient
for downward transport of matter (Smetacek, 1985; Nelson et al.,
1995; Sarmiento et al., 2004). Recent studies are challenging this
belief highlighting rather potential links between export and the
structure of the whole plankton community (Henson et al., 2012;
Lima et al., 2014; Guidi et al., 2016). Additionally a combination
of poorly understood processes, such as gravitational settling
as aggregates or fecal pellets, physical transport of particulate
and dissolved organic matter, or zooplankton disaggregation
or migration may also contribute substantially to downward
transport of matter (Sanders et al., 2014; Turner, 2015; Siegel
et al., 2016).
Impact of Phytoplankton Community
Structure
Daily addition of small amount of dSi triggered the growth
of diatoms in the +Si mesocosms and globally increased POC
fluxes (Figure 8), with however, 50 to 100-fold less carbon
exported per unit dSi in our non-blooming situation compared
to similar bloom simulating experiment (Wassmann et al., 1996).
All treatments showed strong positive correlations between the
POC fluxes and the Si to POC ratios of suspended material
in the upper 5 m of the mesocoms (Figure 9) suggesting
that the magnitude of POC export was first driven by the
presence of diatoms. This is supported by the high POC
flux in the +Si −Cops at Day 6 (Figure 7) and by the high
export efficiency in the +Si −Cops mesocosms, in terms
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Moriceau et al. Diatom and Copepod Control on Export
FIGURE 7 | Composition of the particle fluxes in the four treatments, measured from images of gel traps and POC fluxes in sediment traps.
of the amount of POC produced reaching the sediment
traps (Figure 10). Globally, POC flux intensities follow the
aggregate contribution to the flux, with downward trend when
the aggregate contribution to fluxes decrease (Figure 7). On
the +Si mesocosms the aggregate contribution was slightly
lower than the average aggregate contributions in the flux of
the −Si mesocosms at Day 6 and 11, when Gymnodinium
sp. dominate the phytoplankton population. However, when
aggregate contribution to the flux is similarly high such as
seen at Day 6 for +Si −Cops and −Si −Cops, the flux
is almost twice as high in the diatom dominated treatment
compared to −Si treatments. Aggregation capacity is not
restricted to diatom species (Cataletto et al., 1996), but
diatom aggregates transport more carbon than non-ballasted
aggregates due to higher density and sinking rates (Long
et al., 2015). However, even for non-diatom species, decreased
aggregation involved lower fluxes as seen at the end of the
experiment in −Si mesocosms in association to the growth of
Scrippsiela sp..
Aggregate contribution to the fluxes stay around 82% in
the +Si −Cops, where the aggregating species Skeletonema
marinoii, and Pseudonitzschia sp and Cylindroteca closterium
prevailed (Cataletto et al., 1996). In the +Si +Cops the
aggregating S. marinoii constituted most of the diatom
population at the beginning of the sampling period. The
non-aggregating Leptocylindrus danicus (Cataletto et al., 1996)
that dominate diatom population at the end were not
visible in the sediment traps where the aggregates still
formed 70% of the particles collected. Instead the large
non-aggregating Proboscia alata and the small aggregating
S. marinoii were majoritary in the sediment traps confirming
that size may be also an important factor, together with
ballast and aggregation capacity for an efficient carbon
export.
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Moriceau et al. Diatom and Copepod Control on Export
FIGURE 8 | Flux of material into the sediment traps and its elemental composition averaged for each mesocosm treatment. (A) POC, (B) PON, (C) bSiO2,(D) the
C/N of the sinking flux, and (E) the Si/C of the sinking flux.
The Importance of Zooplankton
Abundance to POC Flux
The global role of zooplankton for POC export is difficult
to quantify since zooplankton indirectly influence export by
changing the structure of the phytoplankton community, from
small to larger species as seen in the previous paragraph and in
other study (Quéguiner, 2013). Moreover, zooplankton activity
may both (1) increase POC fluxes by converting slow-sinking
single cells into fast-sinking fecal pellets and (2) reduce the flux by
fragmenting large aggregates into small particle with slow sinking
velocities and high degradation in the surface ocean (Iversen
and Poulsen, 2007; Giering et al., 2014; Sanders et al., 2014).
In our mesocosm study, higher copepod concentrations resulted
in higher standing stock of phytoplankton (Figure 4) and in
higher phytoplankton diversity (Table 1). The flagellate diversity
appeared to be mainly driven by the copepod presence. Diatom
diversity was more influenced by the silicic acid addition but the
dominant species change depending on the copepod abundances
(Table 1). Higher phosphate and nitrate uptake in the +Cops
treatments compared to the −Cops treatments (Figure 4),
suggest that the phytoplankton community benefitted from the
presence of mesozooplankton. Such a counter-intuitive influence
of meso-zooplankton addition on phytoplankton growth has
already been observed in other studies (Sommer et al., 2001,
2004, 2005). The silicate uptake also increased with copepods
addition (Figure 4). This could be explained by the change
of diatoms community toward more silicified species (Assmy
et al., 2013; Quéguiner, 2013), or by the increase of diatom
silicification in the presence of grazers (Pondaven et al., 2007).
Grazers feeding activity may also increase the remineralization of
the dead diatom frustules (Schultes et al., 2010). Such an effect
would benefit diatom growth but couldn’t be seen in our study
because we only measured the net uptake. Moreover, the trophic
connections are much more complex than only copepods and
prey. Additionally, trophic cascades can explain the increased
phytoplankton biomass in the +Cops mesocosms (Gismervik
et al., 2002; Sommer et al., 2004; Stibor et al., 2004). Copepods
may partially feed on microzooplankton. The grazing pressure
of micro-zooplankton on phytoplankton would then be reduced
as observed in previous mesocosm experiments (Gismervik
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Moriceau et al. Diatom and Copepod Control on Export
FIGURE 9 | POC fluxes versus Si/C ratios in the standing stocks in the upper
4.5 m.
FIGURE 10 | Export efficiency: proportion of the POC produced in the surface
layer, collected in the sediment traps versus time for the four treatments +Si
−Cops, +Si +Cops, −Si −Cops and −Si −Cops.
et al., 2002; Stibor et al., 2004). In this case, an increase of the
copepods abundance mean a decrease of the grazing pressure on
phytoplankton, and an increase of the phytoplankton net growth.
In terms of export fluxes, we observed that elevated copepod
concentrations decreased the POC flux in the +Si while the
opposite was observed for −Si treatments (Figure 8). The highest
difference can be seen for +Si at Day 6 where copepods strongly
impact the flux composition decreasing aggregate contribution
to the flux (from 92 to 68% for −Cops and +Cops, respectively,
Figure 7). This could have resulted from two mechanisms, a
mechanic breakage of the aggregates (Alldredge et al., 1990;
Dilling and Alldredge, 2000) or by changing diatom community
because diatoms are not equal in terms of aggregation capacity
(Cataletto et al., 1996). However until Day 6 the aggregating
species Skeletonema marinoii dominate the two treatments
suggesting at this time that physical breakage of aggregates
by zooplankton feeding and swimming activities must have
prevailed. But copepods activities also influenced the diatom
community: in the +Si +Cops, the biovolume of the diatom
population increased toward the end (Figure 6) with larger
species progressively dominating the diatom community (from
S. marinoii to L. danicus). We also observed an impact of copepod
activity on the species composition of the sinking material.
Similarly to observations done in the Kerguelen area, preferential
feeding by copepods resulted in an increased proportion of
heavily silicified diatoms in the sinking flux (Quéguiner, 2013)
even when they are not so well represented in the surface water
populations. Diatom composition of the export fluxes in +Si
−Cops reflected that of the suspended diatom community, but
most of the material recovered in the +Si +Cops traps were
constituted by the large and heavily silicified Proboscia alata and
aggregates of S. marinoii.
Reversely in the −Si mesocosms, higher copepod
concentrations were associated with increased POC fluxes,
with a stronger contribution of aggregates at Day 11 and of intact
fecal pellets toward the end of the mesocosm study (Figure 7).
While the intensity of the POC fluxes generally follow the
aggregate contribution to the flux, the slight decrease of the
flux intensity at the end in the −Si +Cops mesocosm traps
confirm that fecal pellets production can compensate for the
disaggregation process in a non-diatom situation.
Overall, our data suggests that zooplankton decreased the
efficiency of the system to export POC in diatom dominated
ecosystem (Figure 10). For dinoflagellates dominated systems,
the net export is increasing due to the positive effect of
copepod activity on phytoplankton growth. However, except at
Day 6, the efficiency of the system in terms of how much of
POC production was exported was not affected by copepod
abundance (Figure 10). Instead of copepods simply ingesting
and repackaging phytoplankton into fecal pellets in proportions
similar to the community composition in the mixed layer,
copepods actively shape the phytoplankton community structure
in the surface layer and in the export fluxes by selective
grazing which determines which phytoplankton species are being
retained in the upper ocean, and which ones are being exported.
CONCLUSIONS
In our large scale experimental non-bloom conditions, POC
fluxes were positively correlated to Si/C in all mesocosms,
highlighting the global importance of diatoms for POC
export. The addition of dSi increased POC fluxes, confirming
the results by Wassmann et al. (1996). Lightly silicified
diatoms, which dominated water column in most mesocosms
during our study, drive the export through aggregation when
copepods concentration is decreased. With elevated copepod
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Moriceau et al. Diatom and Copepod Control on Export
concentrations, net phytoplankton growth globally increased,
probably due to trophic cascade effect. Grazing pressure also
structure the phytoplankton community, with a shift from small
to medium or large diatoms in the water column and in
the export fluxes. Simultaneously aggregates are mechanically
broken by copepod activity which is not compensated by the
increase density of the diatoms escaping the grazing. These
two opposite effects in diatom dominated ecosystem resulted
in a net decrease of the export efficiency. Reversely by favoring
phytoplankton growth and formation of fast sinking fecal pellets,
copepods increase the net export by non-diatom species, but do
not clearly change the export efficiency.
AUTHOR CONTRIBUTIONS
BM and CD contributed to the conception of the experiment, to
find the fundings, to the acquisition, analysis, and interpretation
of data and wrote the manuscript. MI and MG contributed to
the conception of the experiment, to the acquisition, analysis,
and interpretation of data and writting of the manuscript. ML,
RL, HS, MS, and A-JE, contributed to the conception of the
experiment, to the acquisition and analysis of data and revised
the manuscript. BB, JB, RC, NC, AD, SG, MK, CL, AL, and AM
contributed to the acquisition and analysis of data and revised the
manuscript.
ACKNOWLEDGMENTS
Immeasurable thanks are owed to everyone whose technical
support and advice made this experiment possible: A. Neyts,
HS, RC, AL, E. Achterberg, O. Vadstein, Y. Olsen, and M. St.
John. This research was supported by the European Community’s
7th Framework Programme’s Integrating Activity HYDRALAB
IV (No. 261520) and Integrating Project EURO-BASIN (No.
264933). Thank you to the two reviewers that greatly help to
improve this manuscript.
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Conflict of Interest Statement: The authors declare that the research was
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The reviewer JT and handling Editor declared their shared affiliation.
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