Harilanto Felana Andrianjakarivony

Christelle Desnues

Philippe Cecchi

Yvan Bettarel
email: yvan.bettarel@ird.fr

Viral life strategies in a heavily anthropized tropical lagoon

Keywords: virus, bacteria, lagoon, eutrophication, lysogeny, ammonium

Ecological traits of aquatic microorganisms have been poorly investigated in tropical latitudes, especially in lagoons, which are often subjected to strong anthropogenic influence, conducive to microbial development. In this study, we examined the abundance of both viral and bacterial communities, as well as their interactions (lytic and lysogenic infections) in the water and sediment of seven main stations of the Ebrié Lagoon (Ivory Coast) with contrasting levels of eutrophication. The highest bacterial and viral concentrations in both planktonic and benthic samples were found in the most eutrophicated stations, where viral lytic infections also exhibited their highest values. Conversely, the highest fractions of inducible lysogens were measured in the most oligotrophic stations, suggesting that these two main viral life strategies are mutually exclusive in this lagoon. Our findings also revealed the importance that nutrients (especially ammonium) play as drivers of the interactions between viruses and their bacterial hosts in tropical lagoons.

Introduction

Over the last two decades, much attention has been given to viruses and their multiple ecological and biogeochemical roles in aquatic ecosystems [START_REF] Suttle | Viruses in the sea[END_REF]. Besides being the most abundant and ubiquitous biological entities [START_REF] Bergh | High abundance of viruses found in aquatic environments[END_REF], viruses also play a fundamental role in shaping the structure and diversity of aquatic microbial food webs, both in water and sediment [START_REF] Suttle | Marine viruses-major players in the global ecosystem[END_REF]. It is now widely accepted that aquatic viral communities consist mostly of phages [START_REF] Batinovic | Bacteriophages in natural and artificial environments[END_REF], which can control the abundance, activity, and diversity of their bacterial hosts [START_REF] Weinbauer | Are viruses driving microbial diversification and diversity?[END_REF]). The dynamics of aquatic viral and bacterial abundance (BA) are most often closely linked at spatial or temporal scales [START_REF] Mei | Virus production and life strategies in aquatic sediments[END_REF][START_REF] Bettarel | Virus-bacterium interactions in water and sediment of west African inland aquatic systems[END_REF], 2011a[START_REF] Sotomayor-Garcia | Assessing viral abundance and community composition in four contrasting regions of the southern ocean[END_REF]. Marine phages use two major mechanisms for replication: lysogenic and lytic infections [START_REF] Williamson | Interaction of the HSIC virus with its host: lysogeny or pseudolysogeny?[END_REF]. Lytic strategies result in host lysis and production of new progeny viruses, which are directly released in the ambient water. Conversely, in lysogenic infections, the viral DNA is integrated into the host's genome and replicates together with it in a dormant state while waiting for favorable conditions or events (in particular UV, temperature, and pollutants) to trigger prophage activation, virion production, and host lysis.

Numerous studies have examined virus-bacteria interactions to unravel the factors that influence the occurrence of lytic and lysogenic infections in aquatic systems [START_REF] Weinbauer | Lysogeny and prophage induction in coastal and offshore bacterial communities[END_REF][START_REF] Hewson | Virus-like particle distribution and abundance in sediments and overlying waters along eutrophication gradients in two subtropical estuaries[END_REF],P a r u c he ta l . 2019, [START_REF] Sotomayor-Garcia | Assessing viral abundance and community composition in four contrasting regions of the southern ocean[END_REF]. Phage life strategies have been shown to be governed by physicochemical factors such as salinity (Bettarel et al. 2011b), temperature (Abdulrahman Ashy and Agustí 2020), and solar radiation [START_REF] Weinbauer | Lysogeny and prophage induction in coastal and offshore bacterial communities[END_REF], Bettarel et al. 2011b). Lysogeny is generally favored in environments where bacterial hosts are less abundant and/or live in conditions unfavorable to their growth, whereas lytic infections are usually high in productive ecosystems [START_REF] Bongiorni | Viral production, decay rates, and life strategies along a trophic gradient in the north Adriatic Sea[END_REF][START_REF] Laybourn-Parry | Viral dynamics and patterns of lysogeny in saline Antarctic lakes[END_REF], where bacteria are abundant and physiologically active, sustaining the rapid proliferation of phage parasites according to the "Kill-the-Winner" paradigm [START_REF] Thingstad | Theoretical models for the control of bacterial growth rate, abundance, diversity and carbon demand[END_REF]Lignell 1997,Winteretal. 2010). However, this relationship is undoubtedly more complex and is increasingly contested: e.g. by the recent theory of "Piggybackthe-Winner," which postulates that lysogeny could be a dominant strategy in environments where bacterial hosts are present in high concentrations [START_REF] Knowles | Lytic to temperate switching of viral communities[END_REF]).

Yet, most of the research on viral replication cycles in aquatic environments has so far been conducted in temperate or polar latitudes. Only a handful of studies have explored the occurrence of phage lifecycles in tropical waters, where microbial processes are somewhat different due to the high level of solar radiation and high temperatures [START_REF] Peduzzi | Bacteria and viruses in the water column of tropical freshwater reservoirs[END_REF][START_REF] Bettarel | Virus-bacterium interactions in water and sediment of west African inland aquatic systems[END_REF][START_REF] Tsai | Seasonal variations in virioplankton and picoplankton in semi-enclosed and open coastal waters[END_REF][START_REF] Ram | Bacterial-viral interactions in the sea surface microlayer of a black carbon-dominated tropical coastal ecosystem (Halong Bay, Vietnam)[END_REF],C a b r a le ta l . 2017,C h e ne ta l . 2019). Moreover, most of the aquatic ecosystems that have been "virally" investigated are restricted to coastal, estuarine, riverine, or lacustrine waters, with lagoons remaining poorly studied. Lagoon ecosystems are subject to strong hydrological and physicochemical variations, linked to significant exchanges between fresh water (rainfall and fluvial inputs) and marine water [START_REF] Mahapatro | Coastal lagoon: present status and future challenges[END_REF]. They are also often bordered by large urban areas, due to the economic resources and services they provide to local populations. These characteristics, added to their restricted water exchange with the sea, long water residence times, and massive amounts of allochthonous inputs (natural or anthropogenic), make lagoons among the most vulnerable and stressed aquatic ecosystems on Earth [START_REF] Pérez-Ruzafa | Coastal lagoons: environmental variability, ecosystem complexity, and goods and services uniformity[END_REF].

The case of the Ebrié Lagoon in Ivory Coast, one of the largest lagoons in West Africa, is emblematic [START_REF] Adingra | Organic and bacterial pollution in the Ebrié lagoon, Côte d'Ivoire[END_REF]. The lagoon is closely linked to the culture, history, and economic development of the country. It has been affected by the rapid population growth of the capital city of Abidjan, accompanied by intensifying industrial, domestic, and agricultural activities. This has resulted in a high level of eutrophication in Ebrié's waters over the last two decades [START_REF] Scheren | Integrated water pollution assessment of the Ebrié lagoon, Ivory Coast, West Africa[END_REF][START_REF] Yao | Détermination de certains paramètres de pollution dans les baies d'une lagune tropicale : la lagune Ebrié (Côte d'Ivoire)[END_REF]). The lagoon serves as a receptacle of huge quantities of solid and liquid wastes, often untreated, from bays and rivers around Abidjan [START_REF] Kouassi | Land-based sources of pollution and environmental quality of the Ebrié lagoon waters[END_REF][START_REF] Scheren | Integrated water pollution assessment of the Ebrié lagoon, Ivory Coast, West Africa[END_REF]). These pollutants can interact with environmental factors (chemical and physical), and consequently disrupt microbiological processes in the water column and sediment.

The objective of this study was to investigate the distribution of both viral and bacterial communities and their interactions (lysogenic versus lytic phage lifecycles) in this highly anthropized ecosystem: to our knowledge the first study to examine this in the Ebrié Lagoon. To this end, we sampled different stations around the lagoon with contrasting sources and levels of pollution.

Materials and methods

Study site and sample collection

Samples were collected between 21 and 28 May 2019 in the Ebrié Lagoon, which is surrounded by the city of Abidjan (which has a population of almost 5 million), Ivory Coast's main city. A total of seven different stations were sampled around the lagoon, which had contrasting sources and levels of anthropogenic influence: (S1, 05 • 11' 15.8' N, 03 • 41' 20.8' W) Mondoukou is the furthest station from the city center, and the least impacted by anthropogenic activities ;(S2,05 • 19' 41.1' N,03 • 59' 26.4'

Sampling strategy

At each station, both sediment and water samples were sampled. Water samples were collected between a depth of 40 and 50 cm with 1.5 l sterile plastic Nalgene bottles. Sediment samples were collected with a grab sampler (Van Veen). The top layer of the sediment cores (∼ 2 cm) was retrieved and placed in sterile zipped plastic bags (Whirl-pak). All samples were kept cool in an icebox during transportation to the laboratory, where they were analyzed within 3 h after collection.

Measurements of physicochemical parameters

During the field sampling, temperature, pH, dissolved oxygen (O 2 ) concentration, and conductivity of the water samples were determined by using a multiparameter probe (Noetek Ponsel Odeon) that was calibrated just prior to the field campaign. The salinity was calculated from the conductivity and temperature value pairs. Water samples for the measurement of suspended matter (SM) were filtered on preweighed (1/10 mg) Whatman GF/F glass fiber filters (diameter 47 mm; porosity 0.7 µm) previously dried (105 • C). After filtration, the filters were dried again (105 • C, 2 h) and then reweighed. The SM concentrations were obtained by relating the difference in mass to the filtered volume. Filtrates were frozen (-15 • C) immediately after filtration and remained frozen until nutrient analyses were performed. Dissolved inorganic nitrogen (ammonium (NH 4 )a n dn i t r a t e( N O 3 )) and dissolved reactive phosphorus (PO 4 ) concentrations were determined by spectrophotometry following the conventional protocols recommended by [START_REF] Aminot | The determination of total dissolved free primary amines in seawater: critical factors, optimized procedure and artefact correction[END_REF]. Water samples dedicated to phytoplankton biomass measurements were filtered through Whatman GF/F filters (diameter 25 mm; porosity 0.7 µm). Filters were later grounded in 90% acetone and extracted for 24 h in the dark at 4 • C. Chlorophyll a (Chl a) concentrations were measured by spectrophotometry (Perkin-Elmer LS50b) and calculated following [START_REF] Neveux | Spectrofluorometric assay of chlorophylls and phaeopigments using the least squares approximation technique[END_REF].

Enumeration of planktonic and benthic viruses and bacteria

At each station (n = 7), triplicate water and sediment samples (n wat = 21; n sed = 21) were distributed into cryotubes (4.5 ml) and fixed with 0.02 µm filtered formaldehyde (final concentration 2% v/v), then flash-frozen in liquid nitrogen and stored at -80 • C prior to enumeration of viruses and bacteria. The abundance of planktonic viruses and bacteria was determined from 300 µlo f formaldehyde-fixed water samples after retention of particles on 0.02 μm pore-size membranes (Anodisc) and staining with SYBR Gold, as previously described [START_REF] Patel | Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR green I[END_REF]). Viruses and bacteria were counted with an Olympus AX70 epifluorescence microscope in 20 fields at x100 magnification under blue light excitation (488 nm). Benthic viruses and bacteria were extracted following the recommendation of [START_REF] Danovaro | Separation of free virus particles from sediments in aquatic systems[END_REF]. This involved weighing 1 g of formaldehyde-fixed sediment samples and then resuspending them with 9 ml of distilled water filtered at 0.02 μm. The sediment suspension was homogenized and a pyrophosphate solution was added (5 mM final concentration v/v). The suspension was then incubated for 15 min in the dark. The samples were treated with an ultrasonic processor (Bioblock) at 45 Khz amplitude for 3 min (3 cycles of 1 min followed by 30 s of manual shaking) and then centrifuged at 800 g for 15 min. The supernatant was collected, and a volume of 300 µl was used for the enumeration of viruses and bacteria as described above.

Fraction of lysogenic bacteria

At each station, the fraction of lysogenic bacteria (FLC) was obtained from triplicates of water and sediment (n wat = 21; n sed = 21) by prophage induction using mitomycin-C [START_REF] Weinbauer | Lysogeny and virus-induced mortality of bacterioplankton in surface, deep, and anoxic marine waters[END_REF]. Mitomycin-C (final concentration 1 μgm l -1 , Sigma Aldrich) was added to 3 ml of samples (treated samples). Untreated samples (control incubations) and treated samples with mitomycin C were incubated for 24 h in the dark at in situ temperature and were then fixed with 0.02 µm filtered formaldehyde (final concentration 2% v/v). Viral abundance (VA) in untreated samples and treated samples with mitomycin C was determined using SYBR Gold and epifluorescence microscopy as described above. Phage induction was estimated as the difference between the VA in mitomycin Ctreated samples (Vm) and the VA in control samples (Vc). The FLC was calculated as: FLC (%) = [(Vm -Vc)/BS × BA t0 )] × 100, where BA t0 represents the BA before the addition of mitomycin C, and BS represents the burst size (number of virus particles per bacterium). The chosen value for BS was 24, as this is an average value from previous aquatic environment studies [START_REF] Wommack | Virioplankton: viruses in aquatic ecosystems[END_REF]Colwell 2000, Parada et al. 2006).

Viral lytic production

Viral decay, which is the decrease in viral concentration over time, was recorded after inhibition of new viral lytic production (VP) by the addition of potassium cyanide (KCN; final concentration, 5 mM) to 4.5 ml of each triplicate of water and sediment samples (n wat = 21; n sed = 21; [START_REF] Fischer | Benthic and pelagic viral decay experiments: a model-based analysis and its applicability[END_REF]. All incubations for decay experiments were performed at in situ temperature for 24 h, in the dark. Incubations were stopped after the addition of 0.02 µm filtered formaldehyde (final concentration 2% v/v). The sediment samples were further treated to desorb viruses, based on extraction with a pyrophosphate solution followed by sonication (see details in the above section Enumeration of planktonic and benthic viruses and bacteria) VA was determined in KCN-treated and KCNuntreated water and sediment samples using SYBR Gold and epifluorescence microscopy (see above). The difference between the abundance of viruses with and without KCN allows the estimation of VP [START_REF] Heldal | Production and decay of viruses in aquatic environments[END_REF].

Statistical analyses

The ordination and hierarchical clustering of the seven stations (S1-S7) by bi-plot graph [using a principal component analysis (PCA)] based on physicochemical parameters, and the correlation between all datasets (using a nonparametric Pearson's correlation test), were performed with the software PAST "PAlaeontological STatistics" [START_REF] Dieumegard | logiciel statistique naturaliste[END_REF]. Using R software, viral and bacterial abundances, VBR, the fraction of lysogenic bacteria, and the VP rate were compared between the resulting clusters with the nonparametric Kruskal-Wallis and Anova test. Statistical significance was assumed when P ≤ .05.

Results and discussion

To assess the level of anthropogenic influence in the seven sampling stations of the Ebrié Lagoon, the ordination and clustering of the stations were performed based on the dissolved oxygen rate (O 2 ), nutrient concentration (NH 4 and NO 3 ), SM, and Chl a concentration, with the two principal components PC_1 and PC_2 (Figs 1 and 2; Table 1). A total of three different clusters were clearly observed, following a clear level of eutrophication: "LOW" (in sites S1 and S5) exhibited a high dissolved oxygen rate, low nutrient concentration, low SM, and low Chl a concentration; "MEDIUM" (in sites S2 and S3) was moderately oxygenated, with high NO 3 concentration, low SM, and low Chl a concentration; and "HIGH" (in sites S4, S6, and S7), which were the least oxygenated sites with the highest NH 4 concentration, SM, and Chl a concentration (Fig. 1).

The bacterial and viral abundances (benthic and planktonic) obtained in this study were within the same range (10 7 -10 9 cells and VLPs ml -1 as those usually reported in tropical waters, where high temperatures stimulate bacterial growth, promoting the production of their phage parasites [START_REF] Ram | Bacterial-viral interactions in the sea surface microlayer of a black carbon-dominated tropical coastal ecosystem (Halong Bay, Vietnam)[END_REF][START_REF] Bettarel | Virus-bacterium interactions in water and sediment of west African inland aquatic systems[END_REF][START_REF] Parikka | Deciphering the virus-toprokaryote ratio (VPR): insights into virus-host relationships in a variety of ecosystems: deciphering the virus-to-prokaryote ratio[END_REF][START_REF] Yucel-Gier | Evaluation of the relationship of picoplankton and viruses to environmental variables in a lagoon system (Çakalburnu Lagoon, Turkey)[END_REF];s e e Fig. 2). On average, viral and bacterial concentrations were significantly higher in the benthic than planktonic samples (Wilcoxon test, P ≤ .01). Marine sediments, by providing virus and bacteria with excellent preservation and reproduction conditions [START_REF] Mei | Virus production and life strategies in aquatic sediments[END_REF][START_REF] Bettarel | Virus-bacterium interactions in water and sediment of west African inland aquatic systems[END_REF][START_REF] Rastelli | Drivers of bacterial α-a n dβ-diversity patterns and functioning in subsurface hadal sediments[END_REF], have been identified as microbial hotspots harboring highly diversified and abundant prokaryotic and viral communities [START_REF] Hewson | Virus-like particle distribution and abundance in sediments and overlying waters along eutrophication gradients in two subtropical estuaries[END_REF][START_REF] Filippini | Viral abundance and genome size distribution in the sediment and water column of marine and freshwater ecosystems: virus genome size distribution in aquatic systems[END_REF][START_REF] Danovaro | Viriobenthos in freshwater and marine sediments: a review[END_REF][START_REF] Quero | Patterns of benthic bacterial diversity in coastal areas contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)[END_REF]. Since sediment represents a confined compartment with reduced distance between bacteria and phages, the probability of contact between them is typically high [START_REF] Danovaro | Viriobenthos in freshwater and marine sediments: a review[END_REF], and this may affect the viral lysis rate and enhance viral proliferation and accumulation. VBR, which is sometimes used a proxy of virus-bacteria interactions, did not vary significantly between the planktonic and benthic compartments, nor between the three clusters (Fig. 2E andF). The VBR ranged between 3.4 and 6.3 in the water samples (mean wat = 4.4 ± 1.0) and between 3.6 and 6.9 in the sediment (mean sed = 4.9 ± 1.2). Similar values were found in a study [START_REF] Bettarel | Virus-bacterium interactions in water and sediment of west African inland aquatic systems[END_REF]. However, contrary to our results, the authors observed that VBR was significantly higher in the water column than in the sediment. The existence of a relationship between RBV and the trophic level of aquatic ecosystems also remains unclear. Indeed, [START_REF] Maranger | Viral abundance in aquatic systems: a comparison between marine and fresh waters[END_REF] found no correlation between the VBR and the trophic status of 22 lakes in Quebec lakes. Conversely, [START_REF] Bettarel | Viral activity in two contrasting lake ecosystems[END_REF] proposed that VBR increase together with the lake productivity. Finally, the lack of clear pattern and the relatively high variability in the VBR values may be explained by the burst size and infection rate of bacteria cells, which are also highly variable among ecosystems [START_REF] Wommack | Virioplankton: viruses in aquatic ecosystems[END_REF]Colwell 2000, Hewson et al. 2001). Due to contradictory results, trends in VBR have led to misinterpretation [START_REF] Parikka | Deciphering the virus-toprokaryote ratio (VPR): insights into virus-host relationships in a variety of ecosystems: deciphering the virus-to-prokaryote ratio[END_REF]) and according to our result, VBR values were not satisfying to explain interactions between viral and bacterial communities and their link with the levels of eutrophication.

One of the study's main findings was that bacterial and VA, as well as phage lytic production, were all significantly higher in the most eutrophicated cluster (HIGH) than in the two other less productive clusters (MEDIUM and LOW), regardless of the compartment (water and sediment; see Figs 2 and3). Interestingly, all these parameters were positively and significantly correlated with NH 4 and Chl a concentrations (Pearson correlation, P ≤ .05, Fig. 4). Nutrient-rich environments typically act as fertilizers promoting phytoplankton growth, which contributes greatly to the utilizable dissolved organic material required to supply bacterial growth [START_REF] Kritzberg | Bacterial growth on allochthonous carbon in humic and nutrient-enriched lakes: results from whole-lake 13 C addition experiments[END_REF][START_REF] Pringault | Impacts of chemical contamination on bacterio-phytoplankton coupling[END_REF]. Furthermore, inorganic nutrients are essential for the growth of bacterial phage hosts [START_REF] Wilson | Lysogenic and lytic viral production in marine microbial communities[END_REF]Mann 1997, Freese et al. 2007); we know that the ability of viruses to replicate is directly influenced by the physiological state of their host, with lytic viral infections favored by metabolically active hosts [START_REF] Weinbauer | Ecology of prokaryotic viruses[END_REF][START_REF] Parada | Viral burst size of heterotrophic prokaryotes in aquatic systems[END_REF]). Given the large demand for nitrogen and phosphorus during viral replication processes [START_REF] Jover | The elemental composition of virus particles: implications for marine biogeochemical cycles[END_REF], it is not surprising to observe the highest levels of VP and concentration in the stations with the most eutrophication.

Conversely, the fraction of lysogenic cells (FLC) was significantly lower in the most eutrophicated cluster (Fig. 3). Such results are in line with previous findings showing that lysogeny is a dominant reproduction pathway in low productive environments, where the abundance of potential hosts is reduced [START_REF] Williamson | Interaction of the HSIC virus with its host: lysogeny or pseudolysogeny?[END_REF][START_REF] Weinbauer | Lysogeny and virus-induced mortality of bacterioplankton in surface, deep, and anoxic marine waters[END_REF],Paul2008, Bettarel et al. 2011b[START_REF] Maurice | Seasonal variations of phage life strategies and bacterial physiological states in three northern temperate lakes[END_REF],Brumetal. 2016). However, since the "Piggyback-the-Winner" hypothesis was put forward [START_REF] Knowles | Lytic to temperate switching of viral communities[END_REF], recent findings have strongly questioned this view. That model, developed essentially from data obtained on animal microbiomes, suggests that lysogeny could also be a relevant strategy at high bacterial host density, by providing many ecological benefits to infected bacterial communities, in particular by allowing them to escape the lytic pressure of phages and conferring superinfection exclusion, and thus continue to fulfill their functions (defence and nutrition) for the host [START_REF] Silveira | Piggyback-the-Winner in host-associated microbial communities[END_REF][START_REF] Howard-Varona | Lysogeny in nature: mechanisms, impact and ecology of temperate phages[END_REF][START_REF] Silveira | The landscape of lysogeny across microbial community density, diversity and energetics[END_REF].

Finally, our results indicated that the specific environmental context of the study site (i.e. a tropical lagoon with high temperatures and high eutrophication) seemed favorable to the development of virulent rather than temperate viruses. Overall, given the numerous but relatively contradictory data reported in the literature, our study suggests that the conditions favoring lysogeny or lytic strategies remain highly ambiguous and would benefit from further investigation, especially along larger latitudinal and eutrophication level.

Figure 1 .

 1 Figure 1. Ordination of the seven stations (S1-S7) by bi-plot graph using the PCA based on physicochemical parameters.

  W) is a confined site near the French Embassy receiving hospital, agriculture, and urban discharge; (S3, 05 • 18' 26.0' N, 04 • 02' 18.0' W) Yopougon Beach is a site with significant fish landing activity and a large marketplace; (S4, 05 • 18' 31.1' N, 04 • 04' 08.3'W) Pougon Bay is a confined embayment receiving large amounts of untreated urban sewage; (S5, 05 • 17' 15.0' N, 04 • 01' 48.6' W) Boulay Island is a densely populated fishing village without sanitation facilities; (S6, 05 • 15' 58.3' N, 03 • 58' 01.1' W) Bietri slaughterhouse is a site that receives animal feces (particularly sheep and cattle) before slaughter; and (S7, 05 • 15' 37.0' N, 03 • 58' 28.8' W) Bietri Neck is historically Ébrié Lagoon's most confined site, receiving huge quantities of urban discharge[START_REF] Lanusse | La contamination microbienne d'une lagune tropicale (Lagune Ebrié,côte d'Ivoire): influences de l'hydroclimat[END_REF][START_REF] Adingra | Organic and bacterial pollution in the Ebrié lagoon, Côte d'Ivoire[END_REF][START_REF] Kouassi | Caractéristiques hydrochimiques et microbiologiques des eaux de la lagune ebrié (Côte d\'Ivoire). Partie I : variabilité saisonnière des paramètres hydrochimiques[END_REF]).

Figure 2 .

 2 Figure 2. Comparison of the different microbiological traits [(A) and (B): BA, (C) and (D): VA, and (E) and (F): VBR) between the three clusters (i.e. Low, Medium, and High) in sediment and water samples. All the three colored boxplots display the median values (horizontal lines) and quartiles (boxes edges) values in the three clusters. The boxplot whiskers indicate the minimum and maximum values. Each dot in the boxplots represent the value of the microbial traits measured for individual stations and lozenge represents the mean value of the microbial traits for each cluster. Significant differences between the three clusters are represented by * * (P ≤ .01) and * * * (P ≤ .001). Virus-like particles (VLPs).

Figure 3 .

 3 Figure 3. Distribution of the two viral replication cycles [(A) and (B): lytic infections, (C) and (D): lysogenic infections] in the three clusters (i.e. Low, Medium, and High) in sediment and water samples. Each dot in the boxplots represents the value of the fraction of lysogenized cells (FLC) and of VP measured at each station and lozenge represents the mean value of FLC and VP for each cluster. Significant differences between the three clusters are represented by * * (P ≤ .01) and * * * (P ≤ .001). Virus-like particles (VLPs).

Figure 4 .

 4 Figure 4. Pearson correlations between environmental parameters and microbial traits in water (wat) and sediment (sed) samples. Temperature (T), dissolved oxygen (O 2 ), pH, salinity, Chl a,NO 3 ,NH 4 , phosphate (PO 4 ), SM, BA, VA, VBR, fraction of lysogenic cells (FLC), and Viral Production (VP). Red and blue circles indicate a positive and negative value, respectively. Significant P-values for Pearson's correlation coefficients are represented by * (P ≤ .05), * * (P ≤ .01), and * * * (P ≤ .001).

Table 1 .

 1 Physicochemical values measured in the water samples of the seven sampling stations (S1-S7). Temperature (T), dissolved oxygen (O 2 ), Chl a,NO 3 ,NH 4 , phosphate (PO 4 ), and SM.

	Station	Cluster	T ( • C)	O 2 (%)	pH	Salinity	Chl a (µgl -1 )N O 3 (µM)	NH 4 (µM)	PO 4 (µM)	SM (mg l -1 )
	S1	LOW	31.4	78.0	7.5	14.1	3.5	0.4	4.4	0.1	16.9
	S5		29.2	72.0	7.3	15.9	11.5	5.4	2.2	0.8	27.9
	S2	MEDIUM	30.2	58.2	7.2	8.4	17.7	18.4	8.6	1.4	16.9
	S3		29.4	52.2	7.1	13.8	5.7	19.1	22.4	3.5	26.4
	S4	HIGH	29.9	23.0	6.9	13.2	43.6	0.0	50.0	1.5	42.6
	S6		29.3	35.5	7.2	12.5	29.7	6.6	51.7	4.0	31.3
	S7		29.7	34.1	7.2	16.3	27.0	5.3	36.3	5.1	32.0
	conducted in tropical freshwater sites located in the basin of the
	Senegal River

Acknowledgments

The authors thank the Centre de Recherches Océanologiques (CRO) of Abidjan for its collaboration, and access to its microbiology laboratory. Thanks to Tom Jamonneau for his contribution and technical assistance during sampling. Tender thoughts go to our dear friend and colleague, Christelle Desnues who has just tragically passed away, who was the inspiration and one of the supervisors of this work. We will not forget your kindness, your advice, and your generosity. We are aslo grateful to the boat captain Kouadio Kouakou, who diligently carried out the sampling in the Ebrie Lagoon and who also recently passed away.

Conflict of interest. None declared.


Daniel Claves
email: daniel.claves@univ-bpclermont.fr

Jérôme Rossignol

Fluorine addition to single-wall carbon nanotubes revisited

Contrarily to the widely-held opinion, the addition pattern issuing from the high temperature fluorination of single wall carbon nanotubes does not consist of a distribution of exo-addends on the outer surface of a tube. Instead, fluorotubes possess a varied bonding scheme consisting of graphite fluoride-like, fluorofullerene-like and internally "addended" sections.

Introduction:

The set idea of an exo-addition pattern, following fluorine addition to a single-walled carbon nanotube (SWNT), has intuitively prevailed hitherto [1][2][3][4][5][6], guided by the initial view using scanning tunneling microscopy (STM) [1] on the outer profile of a fluorotube (F-SWNT) and probably also influenced by antecedent findings about the parent, exofunctionalised, fluorofullerene molecules [7][8][9][10][11]. Yet, attention should be drawn on the facts that STM does not allow observation of inner regions, which might well bias our empirical apprehension of such systems, and that fullerene molecules are closed cages which only permit external "addending", while this is usually not the case for tubes, open at tips most of the time. Consequently, a more rigorous state of mind on the question is necessary and in parallel, seems also dictated by the most recent experimental characterisations performed on F-SWNTs. Hence, the existence within such entities of a mixture of up to three different C-F bond types, differing by their respective strength, has been mentioned by several authors [12][13][14][15][16] but without reliable explanation regarding their origin up to now. Though it has sometimes been argued [12][13][14] that the former differentiation between the fluorine chemisorption modes present in single-walled fluorotubes has to be related to the emergence of an enhanced partial ionic character at the level of some bonds, the irrelevance of such a bonding concept for fluorocarbons is now established both experimentally and theoretically [17][18][19][START_REF] Claves | Carbon Nanotubes : New Research[END_REF].

The present brief overview brings to light that, contrarily to a widespread opinion, F-SWNTs exhibit too important disparities, of unknown origin, in their bonding properties for a selectively exo-decoration of their surface with fluorine addends to be inferred. Therein, a new model of organisation is proposed.

Results:

A sample of bundled SWNTs has been fluorinated at 300 °C for two hours, up to the final nominal composition C2F, following a procedure already described [16]. After fluorination, the TEM micrograph on figure 1a seemingly shows local swelling in the sidewall of the scarce individualised tubes observable, that may witness the already depicted [1] sequenced character of fluorine addition along the main axis. Yet, no advanced conclusion regarding the atomic scale architecture can be drawn from such a direct observation. The corresponding F1s XPS spectrum is made of an apparently single symmetric feature centred on 687.5 eV, not excluding the presence of unresolved components, however. On the other hand, the IR spectrum (fig. 1b) spreads over a wide wave number range and is therefore consistent with the presence of several C-F bond types, which a priori contrasts with the conceptual view of a simply exo-spatial distribution of fluorine.

Accordingly, the less sophisticated way to tentatively account for an again evidenced divergence in the fluorine chemisorption modes present in fluorotubes consists in anticipating a multiple location of the fluorine addends. Given that grafting to a tubular sidewall can be made following two distinct ways only, i.e. either from the inner or from the outer side, the simple mixed bonding scheme illustrated on figure 2 forces then itself upon us and would well account for up to three energetically different categories of C-F bonds, in compliance with earlier experimental statements [12][13][14][15][16]. Indeed, from the energetic standpoint, one can intuitively postulate stability in the respective order: group I> II> III, so that II and III appear as metastable subsystems that may be at the origin of progressively softened covalent bonds.

As a matter of fact, fluorine atoms of group I abide by the well-known architecture [17] of the highly stable (CF)n compound (poly-monocarbon fluoride), while fluorofullerene-like motives of group II are destabilised by local strain at the level of the carbon frame plus probable steric hindrance between nearest neighbours in eclipsed position, if 1-2 addition is respected. The group III endo-addends possess a still more reduced stability, owing to the concave deformation that they should generate at the convex carbon surface and also to their probable mutual steric repulsion.

The pertinence of this model finds a proper justification throughout different experimental and theoretical facts. First, the structure of fluorinated multiwall carbon nanotubes was claimed to be made of a stacking of rolled (CF)n-like sheets [START_REF] Hamwi | [END_REF]22], in which addends regularly alternate on both sides of a corrugated carbon sheet, so that at least partial filiation is highly likely to occur when dealing with single-walled homologues. In parallel, the scarce computational investigations [5,6], which considered such an a priori exotic succession of exo/endo-fluorine addends on the sidewall of an F-SWNT, concluded in the enhanced or competing thermodynamic stability of this latter functionalisation pattern with respect to its equivalent but purely exo counterpart. More importantly, the 19 F NMR spectrum of F-SWNTs synthesised at high temperature was described [23] as being made of a broad hump centred on -175 ppm, which is very close to the -180 ppm chemical shift recorded for the (CF)n compound [17]. All these points suggests almost unambiguously that (CF)n-like sections should constitute a notable, if not dominant, part of the fluorine addition pattern within F-SWNTs, contrarily to the more intuitive than really rigorous former interpretations made.

Moreover, the present IR spectroscopy data strictly confirm this latter assumption. Indeed, the spectrum displayed on figure 1 distinctly shows a major absorption band at 1205 cm -1 , approaching the well-known feature at 1215-1220 cm -1 that characterises the C-F stretching vibration in the (CF)n compound. The small frequency downshift simply occurs as a manifestation of the curvature effect on bond strength.

In the second place, IR spectroscopy also fully confirms the parallel existence of fluorine atoms belonging to the second group here envisaged. Indeed, a broad but distinct absorption feature is clearly apparent over the 1100-1170 cm -1 zone, which is the typical main absorption range of the exo-C-F moieties present in the structurally parent fluorofullerene molecules [7][8][9][10][11], taken as reference. Note also the presence of a significant shoulder near 1250 cm -1 which perfectly matches to the lateral bands of the global fluorofullerenes' signature in the IR region [7][8][9][10][11]. The coexistence of a fluorofullerene-like subsystem in the overall addition pattern of F-SWNTs is therefore highly likely, as well. In this framework, the earlier interpretation of fluorine addition to SWNTs appears partly correct but with the present essential differences that exo-addition is definitely not the unique grafting mode occurring, is not necessarily dominant quantitatively speaking, and is certainly not the thermodynamically stable addition scheme.

Finally, the existence of the third category of fluorine addends seems in turn not contradicted by a weak absorption tail around the 1050 cm -1 region and below  , but happens to be more difficult to strictly establish since no homologous reference compound is known. However, some authors reported [24] that, at constant final F/C ratio, the comparative fluorination at high temperature of open versus closed-end SWNTs generates a lesser intertubular expansion if tubes are open. This is an indirect experimental evidence that fluorine atoms are able to graft to the inner side of a tube according to mode III. In parallel, the opportunity of formation of endo-"addended" fluorotubes has also been well established by theory [3,25], though less energetic than the former exo-or endo/exo-fluorinations.

 It might be prudently inferred that the tooth at 960 cm -1 might also witness the formation of addends of the third group, but we have also observed [START_REF] Hamwi | [END_REF] such a feature in the IR spectrum of fluorinated multiwall carbon nanotubes endowed with the (CF)n structure, so that this minor peak can arise from addends of the first group too.

Discussion:

Alternatively, it might be argued that bond mixing in F-SWNTs could also be the result of the formation of a mixture of distinct solid phases. Fluorotubes are indeed known to be fragile macromolecular edifices, rather easily turning to multishell carbon phases [13,26,27]. The re-fluorination of such degradation products in the course of the synthesis process could then well produce the (CF)n-like C-F bonds identified, for instance. However, such an eventual mechanism should be limited by the short exposure time used here and above all, we have observed only very scarce multishell particles during our TEM observations, showing that a potential post-conversion to a (CF)n-like phase can not be held responsible for the strong signature at 1205 cm -1 and that the corresponding bonding scheme is therefore intrinsic to a fluorotube.

Interestingly, the present multisite model for the fluorine arrangement on the surface of a SWNT allows to a posteriori justify the varying IR characteristics of fluorotubes depicted throughout different works [12,[14][15][16], by simply considering that the different C-F groups form in different proportions according to the synthesis conditions (see below). The reason why the characterisation of F-SWNTs has often led to varied core level spectroscopic signatures throughout the literature (either multicomponent F1s XPS spectra [13,14] or falsely single F1s profiles as obtained here and elsewhere [START_REF] Claves | Carbon Nanotubes : New Research[END_REF]23,24] or still in many other works) can now be foreseen and also simply depends on the more or less pronounced prevalence of one bonding mode within each sample under study. Indeed, upon implicit but reasonable acknowledgement that changes occurring at the core level also mirror those occurring at the valence level on going from group I to group III C-F bonds, the latter spectroscopy will happen to be highly sensitive to the individual C-F bond energies, i.e. to the individual bond types. Evolution in the binding energies of F1s peaks can then be simply accounted for from charge-independent changes in the C-F bond energy diagram, avoiding recourse to the now obsolete ionocovalent model for fluorocarbons.

The forthcoming paragraphs will deal with the reasons why synthesis conditions should undoubtedly exert a strong influence on the final quantitative constitution of the mixed bonding scheme, giving birth to the above mentioned apparent discrepancies in the characterisation from sample to sample. Some experimental observations [13,16] have already indicated that the synthesis temperature is a key parameter affecting the respective proportions of the so far unidentified various bond types within a fluorotubes sample, but the fluorination method retained (gaseous fluorine vs fluorinating agents, plasma techniques…), the reaction time (see [15]) and some morphological characteristics at the level of the precursor SWNTs batch (average tube diameter, bundled or individual tubes…) may also more or less significantly influence the way the final global fluorine arrangement will look like. It turns then out that the maintenance of the surface motives characterising groups II and III, with respect to the more energetically favourable disposition of group I, must arise from kinetic limitations that are progressively overcome upon increasing the synthesis temperature and/or reaction time, allowing the whole set of addends to evolve toward the most stable of the three subsystems. In this context, the most reliable addition pathway toward the formation of fluorine addends of the first group is a "backside completion" of the groups II and III motives, which additionally implies potential rearrangement if 1-2 addition initially prevails. Indeed, in terms of probability, the direct creation of group I motives seems difficult, because simultaneous attack on opposite sides by two fluorine molecules is required. Therefore, the stepwise creation of addends of the first category should slow down their apparition, so that a high temperature and a significant reaction time will be required to observe their proliferation over the surface.

At last, some complementary mechanistic grounds responsible for a facile development of endo-fluorination can be reasonably anticipated, as well. Indeed, a bundled arrangement of tubes often prevails within SWNTs samples. Owing to the compactness of such nanosized ropes, the diffusion of fluorine molecules into the bulk of the carbon lattice is hindered from the lateral sides. Fluorine admission must then rather occur from the bundles tips, where different apertures pre-exist. Consequently, fluorine species will diffuse into the compact network either via migration in the intertubular channels or directly through the hollow core of a tube. The cross section of a tube being about ten times that of an interstitial channel, the contact of fluorine molecules with the inner side of a tube rather than with the outer side is clearly favoured, facilitating endo-addition. In such conditions, a high temperature route is necessary to initially provide sufficient kinetic energy to the fluorine molecules and carbon lattice, so that to force "de-cohesion" within the ropes and to allow the parallel formation of the other addition modes.

Summary and conclusion:

We have pointed out that the exo-fluorination of SWNTs has so far been established more empirically than rigorously. Furthermore, experimental and theoretical results [17][18][19][START_REF] Claves | Carbon Nanotubes : New Research[END_REF] have shown that the ionocovalent model that has asserted itself over decades to describe chemical bonding in some graphite fluorides was largely erroneous. The extrapolation of such a model to interpret the results of SWNTs fluorination becomes then highly criticisable.

Consequently, all previous experimental observations made on single-walled fluorotubes can rather be accounted for by assuming a mixed bonding scheme that includes graphite fluoridelike, fluorofullerene-like and internally "addended" cylindrical sections.

Figure caption Figure 1 .

 caption1 Figure caption

Figure 2 .

 2 Figure 2. Schematic representation of the overall fluorine addition pattern to SWNTs.

Figure 1 Figure 2

 12 Figure 1