# Trophic conditions influence widespread distribution of Aster-Like nanoparticles within aquatic environments

Maxime Fuster, Hermine H. Billard, Marie Mandart, Johannes Steiger,

Telesphore Sime-Ngando, Jonathan Colombet

► To cite this version:

Maxime Fuster, Hermine H. Billard, Marie Mandart, Johannes Steiger, Telesphore Sime-Ngando, et al. Trophic conditions influence widespread distribution of Aster-Like nanoparticles within aquatic environments. Microbial ecology, 2020, 80 (4), pp.741-745. 10.1007/s00248-020-01541-6. hal-03084202

HAL Id: hal-03084202

https://hal.science/hal-03084202

Submitted on 20 Dec 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 **Title**

2 Trophic conditions influence widespread distribution of Aster Like Nanoparticles

3 within aquatic environments

4

5 **Authors**

6 Maxime Fuster¹, Hermine Billard¹, Marie Mandart¹, Johannes Steiger²,

7 Télesphore Sime-Ngando¹ and Jonathan Colombet¹*

8

9 **Affiliations**

10 ¹Université Clermont Auvergne, CNRS, Laboratoire Microorganismes : Genome,
11 Environnement (LMGE), F-63000 Clermont-Ferrand, France

12 ²Université Clermont Auvergne, CNRS, GEOLAB, F-63000 Clermont-Ferrand, France.

13 * Address correspondence to: jonathan.colombet@uca.fr

14

15 **Abstract**

16 Aster Like Nanoparticles (ALNs) are newly described femto-entities. Their ecology (e.g.
17 geographic distribution, spatial dynamic, preferences, forcing factors) is still unknown. Here
18 we report that these entities, which have largely been ignored until now, can develop or maintain
19 themselves in most aquatic environments in the Loire River catchment, France. We observed a
20 significant influence of the trophic state on ALN ecological distributions. A positive
21 relationship between prokaryotic abundance and ALN (r² =0.72, p<0.01) has been identified,
22 but its exact nature remains to be clarified. Combined with their ubiquitous distribution and
23 high abundances (up to 7.9 x 10⁶ ALNs.mL⁻¹) recorded in our samples, this probably makes
24 ALNs an overlooked functional component in aquatic ecosystems.

25
The discovery of the importance of extracellular vesicles [1], bacteria CPR (Candidate Phyla Radiation), archaea DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea) [2,3] and ‘biomimetic mineralo-organic particles’ [4] permitted to reconsider the diversity and ecological role of the femtoplankton, hitherto confined to the sole viruses, some <0.2µm filterable prokaryotes [5]. The recent discovery of “Aster Like Nanoparticles” (ALNs) in pelagic aquatic environments showed that the femtoplankton still hosts many unknown and undervalued entities [6]. In a previous, unprecedented study on these entities, the authors have shown that ALNs are amorphous star-shaped entities suspected to be organic in nature (composed mainly of carbon, oxygen, calcium and nitrogen with traces of potassium). Pleomorphic, these entities present three main morphotypes, with 4, 11 and 20 arms, and a reduced biovolume, lower than of the smallest known prokaryote. These original characteristics, combined with their sensitivity to biocidal treatments and their ability to grow in the absence of cells, raise questions about their exact nature and origin [6]. Their high seasonal abundances (up to 9.0 ± 0.5 x 10⁷ entities·mL⁻¹), in the range of virus-like particles, may exceed those of prokaryotes by up to about one order of magnitude [6]. Added to observations of phenotypic close contact with prokaryotes [6], such numerical abundance levels probably make ALNs a significant actor in the functioning of aquatic systems. For example, its calcium composition [6] could have a significant impact on calcium homeostasis of aquatic environments. Without yet knowing their full biological nature, we hypothesized that their distribution and dynamics in a wide range of aquatic environments will open a window on the understanding of their ecological significance.

Except for first findings obtained from lake samples [6], we still lack information on the ecological distribution and spatio-temporal dynamics of ALNs, in relation to the potential environmental forcing factors.
Results acquired in this second study demonstrate that ALNs are overlooked femto-entities, widely distributed and that their dynamics are controlled by trophic status, in particular by the biological environment. Identification and quantification of ALNs and of their morphological variants are described in Colombet and collaborators [6]. Twenty-five study sites distributed over the Loire River catchment (France) were selected to represent different aquatic systems (rivers, canal, marshes, lakes, mudflat, and coastal ocean) as well as longitudinal gradient from the source to the estuary (Figure 1a). Sampling sites were classified into three trophic states according to all abiotic and biotic parameters measured in-situ. Further details on the methodology used are provided in the supplementary text and data.

ALNs have the ability to colonize different aquatic environments: lakes, rivers, marshes, coastal oceanic waters, except source areas and altitude lakes (Figure 1). They were found in environments with contrasting physico-chemical (e.g. conductivity, oxygen, nutrients) and biological (e.g. virus, prokaryotes, algae) characteristics (Supplementary Table S1). This defines them as tolerant and ubiquitous entities capable of maintaining themselves in the majority of continental and coastal aquatic environments. The abundances of ALNs recorded (1.1 x 10⁷ ± 0.17 x 10⁷ ALNs.mL⁻¹) indicate preferential development in plain lakes, marshes, Loire estuary and in lesser extent within free-flowing river channels (Figure 1b-c). Detection of ALNs has also been mentioned in the tropical estuarine system of Ha Long Bay (Vietnam) and in the Saloum River in Senegal, thus broadening the habitat preferences and potential distribution of these entities [6].

72

73

74

75
<figure><img src="image_2.png" /><figcaption>**Fig 1.** Sampling site locations (1-25) and distribution of ALNs in different aquatic environments along the watershed of Loire River (France). **a.** Site locations are plotted on the map of the Loire watershed. Each site or river continuum is characterized by ALN concentration range. **b.** Distribution of the ALN concentrations and their different forms along the river continuum. **c.** Comparisons of the ALN concentrations and their different forms between the different aquatic environments sampled. Statistical differences between aquatic environments (test U, *** = p<0.01) are provided in the inserted table (S= Source area, I= Intermediate Loire, D= Downstream Loire, A= Altitude lakes, P=Plain lakes, M=Marshes, NS=Not significant). m= mean.</figcaption></figure>

76

77

78

79

80

81

82

83

84

85

86
However, sampling of these studies was sporadic, and these conclusions must be weighted by
the high seasonal or even daily fluctuations of ALNs recorded previously [6]. The sampling
period, i.e. spring, was based on the results obtained from the unique study on seasonal
monitoring of ALNs in a eutrophic lake [6]. We recognize the need of additional data on the
seasonal variability of ALNs in different aquatic environments in order to optimize sampling
periods.

The presence of ALNs in the coastal ocean samples raises questions about their potential for
development in the marine environment and their possible transfer from continental waters to
the oceans. We suggest that the gradual and significant increase of ALN abundance
(undetectable to 8.8 x 10⁵ ALNs.mL⁻¹) from the source to the estuary (Figure 1b) can be
explained through the ALN downstream transfer along the river continuum, as observed for
other entities or pollutants [7-9]. The occurrence of 11-budding-arm forms in the downstream
area suggests that ALNs could be transported from the upstream catchment to more favorable
conditions for their development downstream. The occurrence of the different forms of ALN
indicates dominance of 20-arm forms (69 % of total ALNs) in marshes, while 11-arm forms
dominate in lakes and rivers (38 % and 47 % of total ALNs, respectively; Figure 1c).
Environmental conditions probably select and/or favor the development of one type in respect
to another, through a process that remains unexplained and which could either reflect the
gradual development from one form to another or the occurrence and development of rather
independent forms. In order to better understand spatial distributions of ALNs, future
investigations will have to demonstrate if a form of resistance distinct from forms of
propagation able to develop over time (i.e. assuming distinct growth stages of the same
organism) [6], exist.

111
112 These observations also point to the hypothesis that ALNs could be present in all aquatic environments but are selected by environmental factors (Baas Becking hypothesis) [10]. Such an assumption is supported by the capacity of ALNs to develop in unrelated biomes, from temperate regions separated by hundreds of kilometers as in our study, to tropical regions [6].

116 According to the parameters recorded, we reported a significant influence of the trophic state on ecological distributions of ALNs(Fig. 2a) with a significant increase from undetectable ALNs.mL⁻¹ in the environments classified in the first trophic state, to 2.2 x 10⁶ ALNs.mL⁻¹ in those classified in the third trophic state [11]. In this study, the trophic state was estimated according to the Trophic State Index, a reference method for the classification of aquatic environments [12].

122

123 Therefore, we suggest that ALNs, in addition to certain microbial components such as phytoplankton communities [13], may serve as a putative indicator of trophic conditions of aquatic environments. Whatever the aquatic system considered, it seems that biological features prevail over physico-chemical ones in the regulation of the distribution and abundances of ALNs (Fig. 2b). The major correlative factor identified is the abundance of prokaryotes (r² =0.72, p< 0.01; Fig. 2b). A significant statistical correlation and the observation of direct physical contact between prokaryotes and ALNs [6], suggest an interaction between these two components. Previous study showed that ALNs can grow without a potential host [6], however, the presence of prokaryotes could promote their development through mechanisms that remain unknown [6]. Therefore, future investigations are needed to elucidate the nature of this mutual relationship which, perhaps, could be related to either bottom-up resource-driven or to top-down host-symbiont interactions.

135

136
<figure><img src="image_3.png" /><figcaption>**Fig 2.** Relationships between ALNs and physical-chemical and biological environment. **a.** Comparison of the average (test U, p<0.01) of ALNs according to the low (state 1 = sites 1, 2, 3, 4), medium (state 2 = sites 5, 6, 8, 11, 14, 15, 16, 18, 19, 20, 24, 25) and high (state 3 = sites 7, 9, 10, 12, 13, 17, 21, 22, 23) trophic levels of the different aquatic environments. **b.** Correlation analysis (Spearman test, p<0.01) between the different physical chemical and biological parameters of the different sampling points. T°C = temperature, O2=dissolved oxygen, P= total organic phosphorus, N= total organic nitrogen, C= total organic carbon.</figcaption></figure>

137

138

139

140

141

142

143

144

145
146 Our findings reinforced the hypothesis of the biological origin of ALNs [6] and the hypothesis
147 that ALNs are ubiquitous and widespread within continental freshwaters and coastal brackish
148 waters. The estimation of the ecological importance of femtoplanktonic entities depends closely
149 on their abundance and functions. This is demonstrated for viruses [14,15], extracellular
150 vesicles, ultramicro-prokaryotes [2,3], and suspected for non-biological entities such as
151 'biomimetic mineralo-organic particles' [4]. The average abundances of 9.9 x 10⁵ and 7.9 x 10⁶
152 we recorded respectively in plain lakes and marshes area (see fig.1), are in the range of those
153 of prokaryotes or viruses. The strong empirical relationship with prokaryotes and the regulation
154 by their environment implies that ALNs are additional and so far, ignored actors in aquatic
155 ecosystems. Furthermore, to fully understand the ecological significance of ALNs in plankton
156 dynamics, it is crucial to validate their biological nature.
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175 Fundings

176 MF was supported by a PhD fellowship from the CPER 2015-2020 SYMBIOSE challenge
177 program (French Ministry of Research, UCA, CNRS, INRA, Auvergne-Rhône-Alpes Region,
178 FEDER). This study is a contribution to the “C NO LIMIT” project funded by the
179 Interdisciplinary Mission of the French National Center of Scientific Research (CNRS)
180 Program X-life, 2018 edition. This research was also financed by the French government IDEX-
181 ISITE initiative 16-IDEX-0001 (CAP 20-25).

182

183 Acknowledgments

184 This study was supported by the CYSTEM platform UCA-PARTNER (University of Clermont
185 Auvergne UCA), Clermont-Ferrand, France) and the Laboratory Microorganisms: Genome and
186 Environment (LMGE, UMR6023 CNRS-UCA, Clermont-Ferrand, France).

187

188 Conflict of Interest

189 The authors declare no conflict of interest.

190

191 Authors contribution

192 MF, HB, MM, JC designed the study and analyzed data. All authors wrote the manuscript,
193 contributed critically and gave final approval for publication.

194

195

196 References

197 [1] Soler N, Krupovic M, Marguet E, Forterre P (2015) Membrane vesicles in natural environments: a major challenge in viral ecology. The ISME Journal. 9(4):793-796. doi:10.1038/ismej.2014.184

199 [2] Castelle C.J, Banfield J.F (2018) Major New Microbial Groups Expand Diversity and Alter our Understanding of the Tree of Life. Cell. 172:1181-1197. https://doi.org/10.1016/j.cell.2018.02.016
200

201 [3] Ghuneim L.-A.J, Jones D.L, Golyshin P.N, Golyshina O.V. (2018) Nano-Sized and Filterable Bacteria and Archaea: Biodiversity and Function. Front Microbiol. 9. doi:10.3389/fmicb.2018.01971

203 [4] Wu C.-Y, Martel J, Wong T-Y, Young D, Liu C-C, Lin C-W, Young J.D (2016). Formation and characteristics of biomimetic mineralo-organic particles in natural surface water. Scientific Reports. 6(1). doi:10.1038/srep28817

205 [5] Sieburth J.M, Smetacek V, Lenz J (1978) Pelagic ecosystem structure: Heterotrophic compartments of the
206 plankton and their relationship to plankton size fractions. Limnology and Oceanography. 23(6):1256-1263.
207 doi:10.4319/lo.1978.23.6.1256

208 [6] Colombet J, Billard H, Viguès B, Balor S, Boulé C, Geay L, Benzerara K, Menguy N, Ilango G, Fuster M, et al
209 (2019) Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization
210 and Dynamics. Frontiers in Microbiology. 10. doi:10.3389/fmicb.2019.02376
211 [7] Harvey E, Gounand I, Little C.J, Fronhofer E.A, Altermatt F (2017) Upstream trophic structure modulates downstream community dynamics via resource subsidies. Ecology and Evolution.7(15):5724-5731.
doi:10.1002/ece3.3144

214 [8] Dann L.M, Smith R.J, Jeffries T.C, McKerral J.C, Fairweather P.G, Oliver R.L, Mitchell J.G (2017) Persistence, loss and appearance of bacteria upstream and downstream of a river system. Marine and Freshwater Research.
215
216 68(5):851. doi:10.1071/mf16010

217 [9] Cho K.H, Pachepsky Y.A, Oliver D.M, Muirhead R.W, Park Y, Quilliam R.S, Shelton, D.R. (2016) Modeling fate and transport of fecally-derived microorganisms at the watershed scale: State of the science and future opportunities. Water Research. 100:38-56. doi:10.1016/j.watres.2016.04.064

220 [10] Baas Becking, L.G.M., and Nicolai, E. (1934). On the ecology of a Sphagnum Bog. Blumea - Biodivers. Evol.
221 Biogeogr. Plants 1, 10-45.

222 [11] Carlson R.E., J. Simpson (1996) A Coordinator's Guide to Volunteer Lake Monitoring Methods. North American
223 Lake Management Society. 96 pp.

224 [12] Carlson, R. E. (1977) A trophic state index for lakes. Limnol. Oceanogr. 22:361-369.

225
226 [13] Martinet J, Guédant P, Descloux S (2016) Phytoplankton community and trophic status assessment of a newly
227
228 impounded sub-tropical reservoir: case study of the Nam Theun 2 Reservoir (Lao PDR, Southeast Asia).
Hydroécologie Appliquée. 19:173-195. https://doi.org/10.1051/hydro/2015006

229 [14] Sime-Ngando T, Colombet J (2009) Virus and prophages in aquatic ecosystems. Can. J. Microbiol. 55:95-109.

230 [15] Brussaard C.P.D (2004) Viral Control of Phytoplankton Populations—a Review. J Eukaryot Microbiol. 51:125-138
231

232