FEMS Microbiology Letters, 363, 2016, fnv216
doi: 10.1093/femsle/fnv216
Advance Access Publication Date: 14 November 2015
Research Letter
R E S E A R C H L E T T E R – Environmental Microbiology
Colonization and release processes of viruses and
prokaryotes on artificial marine macroaggregates
Yvan Bettarel1,∗ , Chiaki Motegi2 , Markus G. Weinbauer3,4 and Xavier Mari3,4,5
1
IRD, UMR 9190 MARBEC, 34095 Montpellier, France, 2 Takuvik Joint International Laboratory, University Laval,
Québec G1V 0A6, Canada, 3 CNRS, UMR 7093, LOV, Observatoire Océanologique, 06230 Villefranche-sur-Mer,
France, 4 Sorbonne Universités, UPMC, Université Paris 06, UMR 7093, LOV, Observatoire Océanologique,
06230 Villefranche-sur-Mer, France and 5 Aix Marseille Université, CNRS/INSU, Université de Toulon, IRD,
Mediterranean Institute of Oceanography (MIO) UM 110, 13288, Marseille, France
∗
Corresponding author: Université de Montpellier 2 - CC 093, Montpellier, 34095 cedex 5, France. Tel: +33 6 07 18 95 57; E-mail: [email protected]
One sentence summary: By taking advantage of a novel approach to create artificial macroaggregates, we have examined the small-scale movements of
viruses and bacteria between marine snow particles and the surrounding water.
Editor: Andrew Millard
ABSTRACT
Marine organic aggregates are sites of high of viral accumulation; however, still little is known about their colonization
processes and interactions with their local bacterial hosts. By taking advantage of a novel approach (paramagnetic
functionalized microsphere method) to create and incubate artificial macroaggregates, we examined the small-scale
movements of viruses and bacteria between such marine snow particles and the surrounding water. The examination of
the codynamics of both free-living and attached viral and bacterial abundance, over 12 hours of incubation in virus-free
water, suggests that aggregates are rather comparable to viral factories than to viral traps where a significant part of the
virions production might be locally diverted to the water column. Also, the near-zero proportion of lysogenized cells
measured in aggregates after mitomycin-C induction seems to indicate that lysogeny is not a prominent viral reproduction
pathway in organic aggregates where most viruses might rather be virulent. Finally, we hypothesize that, contrary to
bacteria, which can use both strong attachment and detachment from aggregates (two-way motion of bacteria), the
adsorption of planktonic viruses appears to be numerically negligible compared to their massive export from the aggregates
into the water column (one-way motion of viruses).
Keywords: viruses; bacteria; aggregates; seawater; ecology
INTRODUCTION
Viruses are biological entities of tremendous importance in
aquatic biomes where they fulfill a number of crucial ecological
functions (Fuhrman 1999; Suttle 2007). Prokaryotes are viruses’
main target in the water column of both marine and freshwater systems, and we now know that such phages can methodically control their host abundance and diversity (Weinbauer
and Rassoulzadegan 2004; Rohwer and Thurber 2009). Although
the main life traits of aquatic viruses have been factually de-
rived from their free-living existence in the plankton, previous
reports have also revealed the large occurrence of viral particles attached to organic and inorganic surfaces including aggregates and suspended matters (Mari, Kerros and Weinbauer
2007; Weinbauer et al. 2009). These adsorptive processes can
typically mobilize a substantial fraction of the pool of aquatic
viruses (Bongiorni et al. 2007; Luef, Neu and Peduzzi 2009; Cattaneo et al. 2010) which, on one side, may reduce the lytic pressure
in the water column, and on the other side may promote viral
Received: 15 October 2015; Accepted: 3 November 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Letters, 2016, Vol. 363, No. 1
Table 1. Mean (±SD) nutrient and Chlorophyll a concentrations, viral and bacterial abundances measured in both Bay and Lagoon (LAG) waters
and their related aggregates (immediately after their generation).
BAY
LAG
[NO3 +NO2 ]
μM
[SRP]
μM
[NH4 ]
μM
[Chla]
Mg L−1
[Virus]agg
106 mL−1
[Bacteria]agg
106 cells mL−1
0.40 ± 0.03
0.12 ± 0.01
0.13 ± 0.01
0.02 ± 0.00
1.61
0.04
0.71
0.40
9.51 ± 0.72
8.70 ± 0.49
4.04 ± 0.43
2.60 ± 0.36
infection and gene exchanges in these miniature, condensed
‘snow’ microcosms (Azam and Long 2001; Riemann and Grossart
2008; Weinbauer et al. 2009). The ecological implications of such
abundant attached forms are now presumed to be considerable with substantial effects on the food web structure and biogeochemical cycles (Grossart et al. 2003; Peduzzi and Luef 2008;
Kernegger, Zweimüller and Peduzzi 2009), but more investigations are required to gain a greater understanding in their colonization mechanisms.
Previous reports have shown that viral abundances are highly
variable on aggregates and generally range from 105 to 1011
viruses mL−1 (Weinbauer et al. 2009) and prevail over that of their
planktonic counterparts (Peduzzi and Weinbauer 1993; Luef
et al. 2007). The main factors cited to explain this variability are
the quality, size and age of aggregates as well as the exposure
time of viruses to particles (Bongiorni et al. 2007; Mari, Kerros and
Weinbauer 2007; Kernegger, Zweimüller and Peduzzi 2009; Luef,
Neu and Peduzzi 2009; Luef et al. 2009). Several chemical and microbiological reasons have also been evoked to justify their high
abundances: first, aggregates and their high nutritive value represent ‘hot spots’ for bacterial growth (Heissenberger, Leppard
and Herndl 1996; Grossart et al. 2003; Kiørboe et al. 2003; Blom
et al. 2010) which may stimulate viral activity (as viral replication
is dictated by their host physiological state) (Maurice et al. 2011;
Payet and Suttle 2013; Palesse et al. 2014), and second because
aggregates can act as chemical trap with adhesive properties
on both bacteria and viruses (Mari, Kerros and Weinbauer 2007;
Luef, Neu and Peduzzi 2009). Conversely, rapid mechanisms
of dissolution and aggregation of marine snow can also dynamically reduce or increase the abundance of bacteria (Simon
et al. 2002; Grossart et al. 2003; Kiørboe et al. 2003) although such
movements are not yet fully understood for viral communities.
Also, the intrinsic nature and origin of aggregated viruses
still remain intriguous as they either can be produced in the
water column and be later adsorbed on the aggregates during
their formation (planktonic origin), or conversely they could be
produced within the aggregates after local infections of the resident prokaryotes (sessile origin). In other words, it remains
to be elucidated whether aggregates are actually viral factories or viral scavengers (Weinbauer et al. 2009). There are also
large uncertainties on whether viruses use lytic or lysogenic
strategies to multiply within marine snow, and more generally on their effective control on bacterial community abundance and composition. Yet this information is crucial for the
understanding of the nature of viral movements in aquatic
biomes.
In this study, we took advantage of a recent innovative approach aiming at generating aggregate analogs (Mari et al. 2012)
to examine viral and bacterial trajectories between artificial aggregates and the surrounding water. More specifically, we investigated the short-term dynamics of their respective abundance
to tentatively understand the specific colonization mechanisms
of both entities, and how they replicate. One important objective
was to determine whether lysogeny is an important reproduc-
tion pathway for aggregated phages, or conversely if they rather
use lytic strategies for their expansion.
MATERIAL AND METHODS
Concentration of marine organic aggregates
components
Aggregates were generated from subsurface seawater collected
in the southwest lagoon of New Caledonia, in June 2009. Samples were taken at two different sites (see Table 1): one nearshore
eutrophic station ‘Bay’ (22◦ 15 48 S, 166◦ 26 19 E) and one offshore
oligotrophic station ‘Lagoon’ (22◦ 19 12 S, 166◦ 17 10 E) (Table 1). After sampling, seawater was kept in 20-L polycarbonate carboys,
during the transport to the laboratory (within 1 h) and was immediately processed for aggregates generation. In the laboratory,
seawater was prefiltered through a 10-μm nylon mesh using a
147 mm diameter filtration unit to remove large particles. The
filtrate was then processed with a 30 kDa Biomax filter (Pellicon, Millipore). The ultrafiltrate was kept for rinsing procedures,
and the concentrate containing the 30 kDa to 10 μm size fraction was collected into sterile 250-mL tissue culture flasks (concentration factor of 100) used to produce the aggregates. The
30 kDa–10 μm size fraction concentrated by ultrafiltration contained naturally occurring living and non-living particulate and
colloidal organic material, such as exopolymeric substances,
bacteria and viruses. The present method relies on the ability
of a specific fraction of exopolymeric substances found in seawater, the transparent exopolymeric particles (TEP) to stick to
other particles and form mixed aggregates. The principle of this
methodological approach is, first, to form small magnetizable
mixed aggregates of TEP, bacteria, viruses and paramagnetic carboxylated microspheres, and second, to collect and cluster these
magnetizable small aggregates into a single macroaggregate using a magnetic nucleus (Mari et al. 2012) (see whole procedure
in the supplemental information). Each newly formed aggregate
was finally transferred into a 2-mL centrifugation vial filled with
1.8 mL of 0.02 μm-filtered seawater of the same origin (Bay or
Lagoon) and incubated for 3, 6, 9 and 12 hours, at room temperature (23◦ C). Finally, a total of 40 vials containing duplicate
macroaggregates (one aggregate per vial) + ultrafiltered seawater, for each time point, were generated for both stations. At
each time point, both duplicate aggregates and surrounding water were sampled for measurement of viral and bacterial abundances, and of the proportion of lysogenized bacteria (Fig. 1).
Extraction conditions and enumeration of viruses
and bacteria
The number of viruses and prokaryotes attached to the newly
formed macroaggregates were determined after a detachment procedure, following the recommendations by Lunau et
al. (2005). Each aggregate was fixed prior to detachment by
adding glutaraldehyde into the solution (1% final concentration).
Bettarel et al.
Motion of viruses and bacteria between aggregates and
surrounding water
3
Lysogenic production of viruses in aggregates
+ mitomycin-C
sampling of
surrounding water
sampling of
surrounding water
sampling of
aggregate
T0h
sampling of
aggregate
T0h +12h
mitomycin-C
T3h+12h
T3h
Hook
String of
nylon
T6h
T6h+12h
Virus-free
seawater
T9h+12h
T9h
mitomycin-C
T12h+12h
T12h
magnet
(a)
(b)
2-mL
centrifuge
vial
(c)
(d)
Figure 1. Experimental design for the assessment of viral and bacterial colonization and lysogenic production within marine aggregates. Two milliliters vials were
incubated with one artificial macroaggregate and virus-free seawater. Vials were sampled every 3 hours (one set of duplicate vials per incubation time point) for the
measurement of viral and bacterial abundance within aggregates (a) and surrounding water (b). At each time point, the FLC in duplicate aggregates was also estimated
by comparing viral and bacterial counts in mitomycin-C treated (c) and untreated control samples (d), after 12 hours of incubation.
After fixation, 1.8 mL of solution was removed from each tube
(the aggregate being left undisturbed in 0.2 mL at the bottom
of the tube) and was replaced by 1.8 mL of a solution of ultrafiltered seawater and 10% methanol (final concentration). The
tubes filled with the macroaggregates and the 10% methanol solution were placed into a heating sonicator, and were sonicated
at 40 kHz for 5 min at 35◦ C. This procedure allows solubilizing the polysaccharidic matrix and detaching the particles (i.e.
bacteria and viruses). Once the sonication completed, the tubes
were disposed inside a magnetic particle concentrator (MPC) designed to hold six microcentrifuge tubes (Dynal MPC-S; Invitrogen Dynal) in order to separate the magnetic microspheres from
the organisms left suspended in the solution. The abundances
of bacteria and viruses (in both dissolved aggregates and surrounding water) were determined by standard techniques using epifluorescence microscopy (Patel et al. 2007). The number of
virus-like particles and bacteria contained in duplicate samples
of 300 μL were determined after particle retention of the particles on 0.02-μm pore-size Anodisc membrane filters (Whatman,
Maidstone, UK) and staining with SYBR Gold (Molecular Probes
Europe, Leiden, the Netherlands). On each filter, 300–600 bacteria and viruses were counted under a Leitz Laborlux D epifluorescence microscope with blue excitation, in 20 fields.
Viral colonization of macroaggregates and lysogenic
infections
At each time point (T0h , T3h , T6h , T9h and T12h ), duplicate
vials were sampled for the measurement of viral and bac-
terial abundance in both aggregates and surrounding water.
This procedure was used to evaluate the movements of both
types of particles between aggregates and water, and to ultimately estimate their respective adsorption and release rates.
The fraction of lysogenic cells (FLC) was also determined in
both aggregates and water samples by using the method of
Jiang and Paul (1996) to initiate prophage induction in virally infected bacteria. At each time point, mitomycine-C (1
μg mL−1 final concentration, Sigma Chemical Co, No M-0503)
was added to duplicate vials. Duplicate untreated samples
served as the control (Fig. 1). All samples were formalin fixed
after being incubated for 12 h in the dark, at room temperature. Viral and bacterial counts were performed in both water and aggregates, following the procedure described above.
For each sample, prophage induction was calculated as the difference in viral abundance between the mitomycin-C treated
(Vm) and control incubations (Vc). The FLC was calculated
as
FLC (%) = 100 [(Vm–Vc) / (BS × BAt0 )]
where BS = burst size (virus bacteria−1 ) (we used a BS of
24, which represents the mean value obtained from a variety of marine ecosystems, as calculated by Wommack and
Colwell (2000) and Parada, Herndl and Weinbauer 2006) and
BAto = bacterial abundance at the start of the experiment,
i.e. before adding mitomycin-C (Weinbauer, Brettar and Höfle
2003).
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FEMS Microbiology Letters, 2016, Vol. 363, No. 1
Figure 2. Dynamics of the abundance (mean values of duplicate incubations) of attached and free-living bacteria and viruses of the aggregates from station Bay (a and
b) and Lagoon (c and d), and in the surrounding water. Error bars correspond to standard deviations.
Statistical analyses
Data were log transformed to satisfy the requirements of normality and homogeneity of variance necessary for parametric
analyses. Differences between the means (± standard deviations) were tested using a t-test. Simple relationships between
original data sets were tested using Pearson correlation analysis. All statistical analyses were performed using SIGMASTAT
software.
RESULTS AND DISCUSSION
Viral and bacterial dynamics in aggregates
While the mean abundances of viruses were not significantly
different between the two types of aggregates (mBAYay = 9.5 ±
0.7 × 106 virus mL−1 , mLAG = 8.7 ± 0.5 × 106 virus mL−1 , t-test, P
< 0.05), that of bacteria exhibited significant differences (mBAY =
4.0 ± 0.4 × 106 cells mL−1 , mLAG = 2.6 ± 0.4 × 106 cells mL−1 , t-test,
P < 0.05) (Table 1). Such abundances were within the range of values for natural snow particles, reported in previous studies (i.e.
105 –1011 viruses mL−1 ; 106 –108 bacteria mL−1 ), however, at the
lower end of the spectrum (Simon et al. 2002; Weinbauer et al.
2009). Although the ‘magnetic microspheres’ approach does not
enable to produce particles of high abundances of viruses and
bacteria, they still remain good analogs of natural ones, easy to
manipulate and are robust to disruption. The resulting macroag-
gregates can be used to study the colonization by bacteria or
metazoans and the release of microorganisms from the aggregate to the surrounding environment during aging experiments.
The fact that the proposed protocol allows immobilization of the
newly formed macroaggregates in a non-turbulent system allows close and precise sampling near the aggregate, and thus,
precise determination of the variations of microorganisms’ concentration or community composition in the vicinity of the aggregate. The main cons of this method are that it does not allow
producing a model aggregate perfectly mimicking naturally occurring aggregates (as none of the other existing model aggregates does). The main difference between these model aggregates and naturally occurring ones is that they possess a central core, which obviously modifies their porosity, which, in turn,
may modify the water flow within aggregates (see Mari et al.
2012).
The total abundance of both free-living and attached viruses
and bacteria generally increased over the 12-h experiment,
regardless of the origin of the aggregates (Bay or Lagoon)
(Fig. 2). This net production of both viruses and bacteria could
be explained by low bacterial losses in the absence of predators
(i.e. flagellate or ciliate grazers, after prefiltration, and protection from UV in laboratory conditions), which also allows the
achievement of viral lytic cycles and the subsequent release of
viruses that would otherwise not occur with the grazing of infected bacteria (or even viruses, see Bettarel et al. 2005). However,
Bettarel et al.
Table 2. Pearson coefficients of correlation (r) between viral (VIR) and
bacterial abundances (BAC) measured in surrounding water (wat)
and in aggregates (agg) obtained from stations ‘BAY’ and ‘LAG’. Numbers in bold indicate significant correlations, P < 0.05, n = 10.
BAY
VIRwat vs VIRagg
BACagg vs VIRagg
BACwat vs VIRwat
BACwat vs BACagg
r = 0.880
r = 0.983
r = −0.942
r = −0.717
LAG
VIRwat vs VIRagg
BACagg vs VIRagg
BACwat vs VIRwat
BACwat vs BACagg
r = 0.916
r = 0.934
r = 0.105
r = 0.184
Water
VIRBAY vs VIRLAG
BACBAY vs BACLAG
r = 0.491
r = 0.911
Aggregate
VIRBAY vs VIRLAG
BACBAY BACLAG
r = 0.879
r = 0.754
the detailed short-term dynamics of viruses and bacteria were
clearly divergent, and these differences were also pronounced
between water and aggregates (Fig. 2).
First, at T0h , for both Bay and Lagoon samples, we observed an
immediate increase in bacterial abundance (and of viruses to a
lower extent) in the surrounding water that was initially virus
free (Fig. 2a–d). Such a presumed detachment may suggest that
rapid exchanges can occur between aggregates and water, as reported by other investigators (Kiørboe et al., 2002, 2003; Grossart
et al. 2003). The short-term dynamics of viral abundance then
followed a similar pattern in both compartments (Fig. 2a and c),
characterized by a positive and significant correlation between
attached viruses and their free-living counterparts (rBAY = 0.88,
rLAG = 0.92, P < 0.05, n = 10) (see Table 2).
Within the first 3 hours, viral abundance increased, on average, by 5.1 and 2.9 in both water and aggregates, respectively.
Intuitively, one might envisage that the resuspension of the aggregates in the virus-free water may have triggered induction
of lysogens, and therefore could explain the rapid and abrupt
enhancement in viral abundance. However, for both Bay and
Lagoon samples, lysogeny remained very low, almost insignificant (FLCBAY = 0.78 ± 0.97%; FLCLAG = 0.10 ± 0.19%) (Fig. 3),
which invalidates the hypothesis that aggregates are hotspots of
lysogeny. Such low proportions of lysogenized cells are not surprising as this replication pathway is usually seen as a refuge
strategy favored when host abundance is low, and environmental conditions unfavorable for host growth (low-nutrient, lowtemperature habitats) (Weinbauer 2004; Paul 2008). Conversely,
aggregates typically represent highly nutritive biotopes for bacteria (Kiørboe and Jackson 2001; Grossart et al. 2003). In addition,
the incubation temperature in the laboratory (23◦ C) was comparable to the in situ seawater temperature in the Lagoon of New
Caledonia, which also represent favorable conditions for bacterial growth. For all these reasons, aggregates do not seem to
provide an ideal environment for promoting lysogeny, although
further experiments with natural aggregates are necessary to
confirm this statement.
Finally, if lysogeny is not seemingly responsible for the increase in viral abundance, then the aggregated viruses might
rather mutliply by using lytic cycles. Previous reports of very
short burst time in virally infected marine bacteria revealed that
the lytic cycle can be completed within 20 and 30 min in culture
conditions (Middelboe 2000). The high confinment and proxim-
5
ity between viruses and bacteria within aggregates, the experimental thermal conditions (average temp: 23◦ C) and the high
DOC contents of aggregates form conditions that are close to
those found in cultures. Although viral lytic activity was not
measured in this study, it is then very likely that aggregates
might represent active spots of viral lytic replication, as also
suspected by Mari, Kerros and Weinbauer (2007). The synchronized dynamics of free-living and attached viruses raise a number of questions; the most fundamental questions are: Where
are the actual sites of viral production? Are they produced locally within aggregates and released in the water after cell burst?
Or conversely, are they originated from the surrounding water
and were then lately adsorbed onto aggregates? To tentatively
elucidate these questions, one should pay particular attention
into the dynamics of their bacterial hosts. Indeed, in both Bay
and Lagoon samples, the viral dynamics strictly followed that
of bacteria in aggregates (rBAY = 0.93; rLAG = 0.98, P < 0.05, n =
10) but not in the surrounding water (Fig. 2, Table 2), suggesting that attached viruses might be produced locally within the
aggregates, rather than in the water column. Our hypothesis is
supported by the relatively high fraction of visibly infected bacterial cells measured in aggregates by using transmission electron microscopy by Proctor and Fuhrman (1991) (up to 3.7% of
the total abundance), and which is usually in the highest range
of what is commonly found in the oceanic water column (i.e. 1–
4%) (Wommack and Colwell 2000; Weinbauer 2004; Suttle 2007).
Finally, we postulate that aggregates can be seen as active viral factories, rather than scavengers, and therefore, viral lysis is
probably a major factor of prokaryotic losses in marine snow. If
viruses are mainly produced within aggregates, then the matching patterns between attached and free-living viruses might imply that there could be a massive release of viruses from the
aggregate into the surrounding water fraction. This seems to indicate a rather unidirectional (one-way) motion of viruses from
aggregate to water, and adsorption processes seem to be relatively insignificant compared to viral losses from the aggregate
(Fig. 4).
Unlike viruses, the abundance of free-living bacteria greatly
decreased within the first 3 hours, and conversely increase in
an almost symetrical way in aggregates (Fig. 2, Table 2). This
may indicate a substantial colonization of planktonic bacteria
on aggregates, together with cellular growth inside the particles.
Such a cellular tropism of free-living bacteria towards organic
aggregates is relatively well known, and has been demonstrated
on several occasions (Grossart, Riemann and Azam 2001; Simon
et al. 2002; Kiørboe et al. 2003). First, the colonization of aggregates might represent a defense strategy for bacteria, trying to
escape predation by protists (Simek et al. 2001; Blom et al. 2010).
We also know that aggregates can provide ecological niches of
high nutritive value for prokaryotes where cell growth is typically higher than that of planktonic cells (Grossart et al. 2007).
For example, this chemotactism has been previously described
on numerous occasions where dissolved organic substances released from natural aggregates were shown to enhance the colonization rate of chemosensing bacteria by a factor of 2 to 5
(Kiørboe and Jackson 2001).
Then bacterial abundance kept following an opposite trend
between aggregates and surrounding water, although this was
more pronounced for Bay samples. In other words, when bacterial abundance decreased in water, it increased in aggregates
and vice versa, suggesting that both compartments feed each
other successively on a rotating basis. Such dynamics may imply
that bacteria can go both ways between the two compartments.
Then, unlike viruses, bacteria appeared to exhibit a rather
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FEMS Microbiology Letters, 2016, Vol. 363, No. 1
Figure 3. Dynamics of viral abundance (mean values of duplicate incubations) in both types of artificial aggregates: BAY (a) and LAGOON (b), with and without (control)
addition of Mitomycin-C (mito. C). FLC, Frequency of lysogenic cells. Error bars correspond to standard deviations.
two-way (bidirectional) motion between aggregate and surrounding water. The reason for such trajectories still remain unclear but it is likely that the chemical and antipredatory attraction of bacteria towards aggregates, could be compensated by
the release of bacteria when the size of aggregates become too
large and reached a threshold beyond which some parts of aggregates might simply dislocate. Further studies will be needed
to examine viral and bacterial ‘trajectories’ during longer periods of aggregate life.
Finally, the results obtained in this small-scale study support the idea that aggregates represent highly attractive habitats for bacterial cells which may locally enjoy explosive growth,
but also promoting viral lytic infections. Herein, we hypothesize
that such ’viral factories’ may generate a continuous release of
newly produced virions into the surrounding water, potentially
enhancing the mortality rates of planktonic bacteria and thus
influencing the cycling of organic matter in the oceanic water
column.
Bettarel et al.
7
Figure 4. Conceptual outline for viral and bacterial motions between aggregates and surrounding water.
SUPPLEMENTARY DATA
Supplementary data are available at FEMSLE online.
ACKNOWLEDGEMENTS
This work was supported by the French National Research
Agency (MAORY project, contract: ANR-07-BLAN-0116) and the
French Research Institute for Development (IRD).
Conflict of interest. None declared.
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