Jérémy Minguez

Tom Maris

Micky Tackx

Charles Gers

Patrick Meire

Luc Legal

Tackx Michèle
email: michele.tackx@univ-tlse3.fr

Genetics of the estuarine copepod Eurytemora affinis with regard to improving water quality

Keywords: Estuaries, Zooplankton, Genetic entities, ISSR, Tributaries

, found little genetic differentiation between the upstream population and downstream one, which is probably at the origin of the upstream population. However, surprisingly, two different genetic entities of the E. affinis complex were present: one in the main stream of the Scheldt estuary and one in its tributaries. This present work aims at understanding how independent tributaries, situated at opposite sides of the estuary, can host a single genetic entity while another one is present only in the main estuary. A dense sampling in the main stream and the two main tributaries was made in May 2014 (12 locations) and four ISSR primers producing 78 polymorphic loci were used to perform a molecular analysis. Bayesian and hierarchical analysis revealed that the results are more mixed and the different genetic entities are less well delimited than reported in our former study based on samples collected in 2012. A genetical mix between tributary and upstream populations is now observed. This suggests that a homogenization process of the E. affinis populations is in progress in parallel to the improvement of water quality. These results are discussed and several explanations are proposed.

Introduction

Estuaries are complex ecosystems at the interface of marine and continental habitats. They are very dynamic as abiotic conditions change strongly both along a longitudinal and a vertical gradient [START_REF] Meire | The Scheldt estuary: a description of a changing ecosystem[END_REF]. Estuaries are also among the most productive ecosystems as they receive large quantities of nutrients and organic matter from their drainage basin.

However, due to their geographic localization between land and sea estuaries have attracted human settlements and hence estuarine ecosystems suffered heavily from anthropogenic pressures such as overexploitation, habitat loss, anthropic management and pollution leading worldwide to the degradation of estuaries [START_REF] Clark | Geochemistry and loading history of phosphate and silicate in the Hudson estuary[END_REF][START_REF] Kennish | Environmental threats and environmental future of estuaries[END_REF].

The Scheldt estuary was considered as one of the most polluted estuaries in the second half of the 20th century [START_REF] Baeyens | General description of the Scheldt estuary[END_REF] due to discharges from industrial activities around the port of Antwerp and the discharge of untreated wastewater of the Brussels agglomeration and many other cities in the catchment. In the seventies and eighties, this led to very low oxygen concentrations in the freshwater and brackish water reach of the estuary [START_REF] Van Damme | Spatial and temporal patterns of water quality along the estuarine salinity gradient of the Scheldt estuary (Belgium and The Netherlands): results of an integrated monitoring approach[END_REF]Cox et al., 2009).

In the Scheldt estuary, a key zooplankton species is the Calanoid copepod Eurytemora affinis (Poppe, 1880), a planktonic suspension feeder, often dominant in temperate estuaries and brackish waters of northern hemisphere. This species is defined as a species complex, formed by sibling species occurring in all Northern Hemisphere estuaries, which cannot be clearly identified with morphological features [START_REF] Lee | Morphological stasis in the Eurytemora affinis species complex (Copepoda: Temoridae)[END_REF][START_REF] Winkler | Genetic heterogeneity among Eurytemora affinis populations in Western Europe[END_REF][START_REF] Sukhikh | Invasion of Eurytemora sibling species (Copepoda: Temoridae) from north America into the Baltic Sea and European Atlantic coast estuaries[END_REF].

Within the E. affinis complex, several genetically distinct lineages are known to exist on the North American continent and within Europe (Bradley, 1978a;[START_REF] Lee | Rapid and repeated invasions of fresh water by the copepod Eurytemora affinis[END_REF][START_REF] Lee | Global phylogeography of a cryptic copepod species complex and reproductive isolation between genetically proximate "populations[END_REF][START_REF] Lee | Morphological stasis in the Eurytemora affinis species complex (Copepoda: Temoridae)[END_REF][START_REF] Lee | Effects of developmental acclimation on adult salinity tolerance in the freshwater-invading copepod Eurytemora affinis[END_REF][START_REF] Alekseef | A new species within the Eurytemora affinis complex (Copepoda: Calanoida) from the Atlantic Coast of USA, with observations on eight morphologically different European populations[END_REF][START_REF] Lee | Pumping ions: rapid parallel evolution of ionic regulation following habitat invasion[END_REF][START_REF] Winkler | Heterogeneity within the native range: population genetic analyses of sympatric invasive and noninvasive clades of the freshwater invading copepod Eurytemora affinis[END_REF][START_REF] Winkler | Genetic heterogeneity among Eurytemora affinis populations in Western Europe[END_REF] Recent awareness of the ecological importance of estuaries and the many ecosystem services they deliver has led to many initiatives to protect and restore estuaries. Since the 1990s, restoration efforts have been implemented in the Scheldt estuary and its drainage basin [START_REF] Van Damme | Spatial and temporal patterns of water quality along the estuarine salinity gradient of the Scheldt estuary (Belgium and The Netherlands): results of an integrated monitoring approach[END_REF]. From 1995 onwards, the OMES project has provided a continuous monitoring of physicochemical and biological factors to evaluate the restoration process and to support long-term management strategies of the Scheldt estuary [START_REF] Maris | Tuning the tide: creating ecological conditions for tidal marsh development in a flood control area[END_REF][START_REF] Cox | A macro-tidal freshwater ecosystem recovering from hypereutrophication: the Schelde case study[END_REF][START_REF] Beauchard | A new technique for tidal habitat restoration: evaluation of its hydrological potentials[END_REF].

Improved wastewater treatment and fertiliser restrictions resulted in a decrease in N concentrations, mainly of NH4, which allowed the development of phytoplankton resulting in an increase in oxygen concentration the fresh-and brackish tidal water stretch from 1996 to 2006, associated with an overall improvement of water quality [START_REF] Van Damme | Spatial and temporal patterns of water quality along the estuarine salinity gradient of the Scheldt estuary (Belgium and The Netherlands): results of an integrated monitoring approach[END_REF]2009;[START_REF] Cox | A macro-tidal freshwater ecosystem recovering from hypereutrophication: the Schelde case study[END_REF].

In the Scheldt estuary, until the 1990s, E. affinis was mainly found in the downstream brackish and marine part of the estuary. This displacement from its optimal salinity zone, which is around the freshwater-brackish water fringe, was explained by the relative bad water quality in the upstream region (oxygen concentration <3mg L -1 ) [START_REF] Soetaert | Spatial and temporal patterns of the zooplankton in the Westerschelde estuary[END_REF][START_REF] Sautour | Comparative spring distribution of zooplankton in three macrotidal European estuaries[END_REF]. [START_REF] Appeltans | Zooplankton in the Schelde estuary (Belgium/The Netherlands). The distribution of Eurytemora affinis: effect of oxygen?[END_REF] have shown that the distribution of E. affinis in the Scheldt is related to the oxygen concentration. [START_REF] Mialet | Spatial spring distribution of the copepod 29 Eurytemora affinis (Copepoda, Calanoida) in a restoring estuary, the Scheldt (Belgium)[END_REF] also demonstrated that E. affinis occurs upstream when oxygen concentration in this region is >4 mg L -1 .

Since 2007, E. affinis has become abundant upstream, reaching even higher abundance (up to 155 000 ind.m -3 ) than previously observed downstream in the estuary [START_REF] Mialet | Spatial spring distribution of the copepod 29 Eurytemora affinis (Copepoda, Calanoida) in a restoring estuary, the Scheldt (Belgium)[END_REF], 2011[START_REF] Chambord | Mesozooplankton affinities in a recovering freshwater estuary[END_REF]. The monthly monitoring of the E. affinis population, ongoing since 1996, revealed no taxonomic differentiation along the estuary (6 stations) and the 4 tributaries, suggesting than the downstream population was the source of the upstream one.

In a former study performed under the direction of some authors of this present work, [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] tried to answer the question: 'Which population is at the origin of the recently occurring upstream population of E. affinis in the Scheldt estuary'? We showed that the downstream population is most probably at the origin of the upstream one, because they are two close genetic entities. The subdivision between downstream and upstream genetic pools could be the result of recent isolation by the 'oxygen barrier effect' during the heavily polluted period. Ecophysiological boundaries may promote isolation resulting in specialized estuarine species (Lee et al., 2003). The study has also revealed that the middle-estuary individuals are a mix of both downstream and upstream populations. [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] have also found that two distinct genetic entities are present in the Scheldt estuary. There is one genetic entity in the Scheldt main stream from the downstream part until the upstream part, and there is another one in the two main tributaries: the Durme and the Rupel. These two tributaries are in the fresh water part of the estuary and on opposite sides. The genetic entity from the two tributaries is morphologically indistinguishable but genetically distinct from that of the Scheldt main stream. Several scenarios were established to give coherent explanations and to understand these former findings. In this present study, we aimed to analyse the population genetic structure of the E. affinis species complex in the Scheldt estuary between sampling points on the main stream and its tributaries at a larger scale to reduce possible bias of our former study. We especially sampled more extensively around, within and between the two tributaries (8 sampling stations versus 2 in our former study).

This study uses ISSR (Inter Simple Sequence Repeats) markers to study the genetic diversity of the E. affinis complex in the Scheldt estuary and its tributaries.

ISSR are advantageous because sequences are known to be abundant, they are also very reproducible and highly informative [START_REF] Bornet | ISSR as new markers for genetic characterization and evaluation of relationships among phytoplankton[END_REF].

Originally conceived for genetic divergence among cultivated plants [START_REF] Wolfe | ISSR techniques for evolutionary Biology[END_REF], they are nowadays used as a new tool for genomic fingerprinting for various terrestrial and marine taxonomic groups: insects [START_REF] Luque | ISSR (inter simple sequence repeats) as genetic markers in noctuids (Lepidoptera)[END_REF], bivalves [START_REF] Casu | Fine-grained spatial genetic structure in the bivalve Gemma gemma from Maine and Virginia (USA), as revealed by Inter-Simple Sequence Repeat markers[END_REF][START_REF] Casu | Inter-simple sequence repeat markers reveal strong genetic differentiation among populations of the endangered mollusc Patella ferruginea (Gastropoda: Patellidae) from two Sardinian marine protected areas[END_REF][START_REF] González-Tizón | Genetic divergence detected by ISSR markers and characterization of microsatellite regions in Mytilus mussels[END_REF], crocodiles [START_REF] Machkour-M'rabet | Between introgression events and fragmentation, islands are the last refuge for the American crocodile in Caribbean Mexico[END_REF].

On the genome, ISSR are localized between microsatellite sequences which are simple regions consisting of repeated di-to tetra-nucleotide sequence motifs flanked by unique sequences [START_REF] Mccouch | Microsatellite marker development, mapping and applications in rice genetics and breeding[END_REF]. The major advantage of ISSR is that no information about the sequences is necessary before amplification (Varela et al., 2007;[START_REF] Machkour-M'rabet | Between introgression events and fragmentation, islands are the last refuge for the American crocodile in Caribbean Mexico[END_REF]. In addition, ISSR allow the analyses of multiple loci in a single reaction [START_REF] Serra | Comparison of ISSR and SSR markers for analysis of genetic diversity in the seagrass Posidonia oceanica[END_REF]. Furthermore, this technique enables to detect genetic variability several years after species introducing [START_REF] Machkour-M'rabet | ISSR (Inter Simple Sequence Repeats) as molecular markers to study genetic diversity in tarantulas (Araneae, Mygalomorphae)[END_REF] and allows working on genetic population structure on a local scale [START_REF] Luque | Apparent influences of host-plant distribution on the structure and the genetic variability of local populations of the Purple Clay (Diarsia brunnea)[END_REF]. The ISSR markers are a useful tool to study the spatiotemporal genetic population structure of organisms. In the restoring Scheldt estuary, it could be used to follow -up the spatio-temporal evolution of the E. affinis species complex in parallel to water quality evolution.

Material and methods

Study site

The Scheldt River originates in Saint-Quentin, North of France. It flows into the North Sea after 355 km in Vlissingen in Netherland (Fig. 1). The Scheldt River becomes the Scheldt estuary under tidal influence at the city of Gent in Belgium at 160 km from Vlissingen. On average 39% of the water coming in from the Scheldt River is deviated towards canals, especially during the dryer summer periods, the main freshwater input at Schelle (91 km from the mouth, where the Rupel enters the Scheldt) comes then from the tributary the Rupel. Being a rain-fed system, the mean river discharge varies seasonally, from an average of 60 m 3 s -1 in the summer to a mean of 180 m 3 s -1 during the winter [START_REF] Meire | The Scheldt estuary: a description of a changing ecosystem[END_REF]. The estuary is vertically well mixed, but there is a pronounced salinity gradient and 3 zones are distinguishable: the marine part (Westerscheldt), a brackish part and a fresh water tidal zone (Fig. 1). [START_REF] Baeyens | General description of the Scheldt estuary[END_REF] and [START_REF] Meire | The Scheldt estuary: a description of a changing ecosystem[END_REF] give a complete description of the Scheldt system and more details on its hydrological characteristics. The two tributaries considered in this study are Durme "Trib 1" and Rupel "Trib 2".

Sampling strategy and DNA extraction

Samples for zooplankton genetics were collected during the OMES monitoring campaign of May 2014. Sampling was carried out at 12 estuarine locations (Fig. 1, Table 1) with 7 samplings in the Scheldt river, 4 in the Rupel and 1 Durme tributaries respectively.

Several hundred litres of water were filtered through a plankton net (150 µm mesh size), the collected zooplankton was conserved in ethanol and frozen at -20°C until analysis. The first step of this study was the identification of the species E. affinis under binocular (magnification 90x), 25 individuals were taken from each sample for genetic analysis.

DNA was extracted individually from the entire body using the QIAGEN DNeasy Tissue kit (QIAGEN) according to the manufacturer's protocol.

A chemical lysis allowed DNA release, pure DNA was then loaded onto the "DNeasy spin columns" which contain a DNA binding silica-gel membrane. The affinity of DNA to membrane silica permits DNA binding, making the removal of residual cell components possible.

The same protocol as in [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] was used, including modifications to the manufacturer's protocol made according to Eberl et al. (2007) to obtain better performance of DNA extraction. DNA concentration was determined by spectrophotometry (NanoDrop ND-1000) at 260 nm. Overall, 300 individuals were analysed.

PCR-ISSR and electrophoresis

The four primers used were the same as in [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF]. PCR amplifications were performed in a 25 ml reaction volume containing 30 ng of template DNA, 1 ml of primer (50 mM; Invitrogen), 1 ml of dNTP (10 mM; Promega), 2.5 ml of 5x PCR Green Buffer (Promega), 3 ml of MgCl2 (25 mM; Promega) and 2.5 units of Taq polymerase (M830A, Promega). All amplifications were carried out using a T3 Thermocycler (Biometra).

The cycling conditions were as follows: initial denaturation step at 94°C for 4 min, 39 cycles of denaturation at 94°C for 45 s, primer annealing at 56°C for 45 s and extension at 72°C for 2 min, followed by a final extension at 72°C for 10 min.

Electrophoresis was performed with 8 ml of amplified products on a 2% agarose gel for 2 h at 120 V. The bands were detected using Ethidium bromide under UV light and digitized (VWR GenoSmart).

Data analysis

For each individual, the presence (1) or absence (0) of bands was scored for each primer and the binary matrix generated was used for analysis. Only bands that could be clearly identified were used and it was assumed that each marker band represented a distinct locus. Band identification and counting was made with Genosoft software (VWR). The obtained binary matrix was then treated with POPGENE version 1.32 [START_REF] Yeh | POPGENE: the Userfriendly Shareware for Population Genetic Analysis[END_REF] under Hardy-Weinberg equilibrium model. Calculated parameters were: percentage of polymorphism (P), Nei's gene diversity (H), the fixation index (FST) and the level of gene flow (Nm). In order to determine the genetic structure and variability within and among populations samples at the different locations, we carried out a non-parametric Analysis of Molecular Variance (AMOVA) and a Mantel test using Arlequin software version 3.0 [START_REF] Excoffier | Arlequin (version 3.0): an integrated software package for population genetics data analysis[END_REF].

Classification method

The distance analysis method was used to treat the binary matrix. A heuristic search for an optimal tree was carried out by TBR (Tree-bisection-reconnection) branch swapping. Distance was measured as the mean character differences, performed with PAUP software version 4.0b10 (Swofford, 2001). The tree was visualized using FigTree version 1.4.2.

Bayesian method

A model-based method developed by [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF] and implemented in the software STRUCTURE 2.3.4 allows the identification of genetically homogenous groups of individuals among populations. This tool attempts to detect the number of clusters by identifying the K populations among samples and to assign each individual to one or more populations with a probability (qi). Several values of K were fixed, from 1 to 12, or the number of sampled populations of E. affinis in this study.

Parameters were set at 50 000 burn-in period steps and 50 000 run-time steps.

We used the admixture model with the correlated allele frequencies model. To estimate the more likely number of populations units K, we graphed the tested K (referred to as Ln p(D) in software output). A plateau was reached at K=5, Individuals were assigned to the same cluster if their proportion of membership (qi) was equal or higher than 0.80. If this was not the case, they were considered as admixture individuals and assigned to different populations.

Self-Organizing Map (SOM) analysis

In order to assess the genetic variability between populations, the same statistical method as [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] was employed: the Self-Organizing Map (SOM) [START_REF] Kohonen | Self-organized formation of topologically correct feature maps[END_REF]. The genetic variability between populations was compared using an artificial neural network method that is a powerful and frequently used tool for exploratory data analysis through a nonlinear projection of multivariate data [START_REF] Pampalk | Using smoothed data histograms for cluster visualization of Self-organizing maps[END_REF][START_REF] Roux | ISSR-PCR: tool for discrimination and genetic structure analysis of Plutella xylostella populations native to different geographical areas[END_REF][START_REF] Bouzid | Genetic diversity of Ligula intestinalis (Cestoda: Diphyllobothriidea) based on analysis of inter-simple sequence repeat markers[END_REF]. Two layers formed by neurons (described by vectorial distances) characterize the model algorithm. Both layers are connected by links that are called weights. The method organizes and visualizes the real vectors according to their similarities. In order to reduce the number of figures, we present here only the hierarchical cluster analysis (Ward Linkage Method) to define the number of groups of outputs neurons [START_REF] Roux | ISSR-PCR: tool for discrimination and genetic structure analysis of Plutella xylostella populations native to different geographical areas[END_REF]).

Results

Genetic polymorphism of populations

For the 12 sampled locations, a very strong polymorphism was estimated at individual level: the percentage of polymorphic loci was evaluated at 100% for the total number of locations. For AMOVA analysis, we gathered individuals from different locations as follows: Downstream group: individuals from "Down1" and "Down2"; "Mid" group: "Mid1" and "Mid2"; Tributaries group: "Trib1", "Trib2a", "Trib2b", "Trib2c" and "Trib2d"; and Upstream group: "Up1", "Up2" and "Up3".

The AMOVA revealed that most of the genetic variance (67.85 %) was evaluated between individuals within locations, 4.65 % of total genetic variance was estimated among groups and 27.5 % among locations within groups. Genetic pairwise differences between populations FST ranged from 0.03 to 0.15 (Table 2). The mean Nei's gene diversity (h) was 0.14 (± 0.09) and Shannon index (H) was 0.22 (± 0.15) (Table 2). The highest value of genetic diversity was found for "Up2" while the lowest for "Down2".

Phenotypic distance between populations

The unrooted tree obtained by distance analysis (Fig. 2) provided 3 main genetic entities in the sampling area. The downstream stations are separated in two different clusters, the first station "Down1" in classified with the middle-estuary stations "Mid1" -"Mid2" in one cluster and the second station "Down2" is classified in a second cluster. Tributaries and upstream locations are also in two related clusters one including "Trib1", "Trib2a", "Trib2d" and "Up3" and a second one composed from "Trib2b", "Up1" and "Up2". Individuals of all the distinct stations were well grouped in a same cluster, except for the individuals from "Trib2c" which are scattered in the two last clusters.

Analysis using neuron network (SOM) of clustering of sampled sites.

The input layer of the SOM was constituted of data (presence/absence) of 74 loci.

The Kohonen map comprised 80 neurons (i.e. a map of 10 * 8 neurons [not shown]).

Five clusters can be deduced from this analysis (Fig. 3), which are equivalent to those found with the Bayesian analysis (see Fig. 4 section 3.4).

Analysis of genotypic differences using Bayesian approach

Individuals from the 12 sampled stations were partitioned into 5 distinct genetic entities (K = 5) on the basis of their membership probabilities (Q) given by STRUCTURE (Table 3, Fig. 4) in accordance to clustering found using SOM (Fig. 3).

Gathering of initial sampled locations was evaluated through the Q values. Most of individuals have impure genome and are allocated to several clusters. Individuals from "Down1" and "Down2" are respectively isolated in cluster 2 and 4 and those of the locations "Mid1" and "Mid2" were found together on the same genetic entity (cluster 5) with an indistinguishable genetic pattern. The individuals found at the rest of the locations (Upstream and Tributaries) are considered as two genetic entities mixed in two clusters (1 and 3). These results given by Structure allow us to evaluate hypothetical hybrid genotypes.

For example, the copepods collected from locations "Trib2c" and "Up1" represented in the cluster 3, show hybridization with different level of introgression: they have respectively 20% and 18% of individuals which show a genotype close to the other genetic entity (cluster 1) of the upstream part of the estuary. Conversely, individuals from "Trib2a" have 14% of hybridization with different level of introgression with the populations from the cluster 3.

Gene flow and dynamic of populations

The higher values of gene flow, reflecting a high level of exchange of individuals, (Table 4) were observed between or among the locations of the two tributaries (4.01 -4.86). The lower values (1.03 -1.06) were observed between middle-estuary locations and upstream or tributaries.

A mantel test was also performed to test the correlation among two distances matrices (geographic and genetic distance). The p-value = 0.32 and the correlation coefficient "r" was 0.08 demonstrating an absence of relation between these two variables.

Discussion

After 40 years of eutrophication and hypoxia, the restoration strategy has resulted in a considerable amelioration of the water quality of the Scheldt estuary [START_REF] Van Damme | Spatial and temporal patterns of water quality along the estuarine salinity gradient of the Scheldt estuary (Belgium and The Netherlands): results of an integrated monitoring approach[END_REF][START_REF] Cox | A macro-tidal freshwater ecosystem recovering from hypereutrophication: the Schelde case study[END_REF]. Since 2007, E. affinis is present in higher abundance in the freshwater than in the brackish water zone [START_REF] Chambord | Mesozooplankton affinities in a recovering freshwater estuary[END_REF].

Genetic diversity

Two-thirds of the genetic variance (67.85 %) occurred between individuals within localities. This proportion is moderate compared to those reported for marine crustaceans, such as, for example 98.7% for the shrimp Aristaeomorpha foliacea, 81.4% for the sand hopper Talitrus saltator [START_REF] Ungherese | Relationship between heavy metals pollution and genetic diversity in Mediterranean populations of the sandhopper Talitrus saltator (Montagu) (Crustacea, Amphipoda)[END_REF][START_REF] Fernández | Analysis of genetic variability in Aristaeomorpha foliacea (Crustacea, Aristeidae) using DNA ISSR (Inter Simple Sequence Repeats) markers[END_REF] and 71.9% and 94.6% for two barnacles species respectively Tesseropora atlantica and Chthamalus stellatus [START_REF] Pannacciulli | Genetic diversity in two barnacle species, Chthamalus stellatus and Tesseropora atlantica (Crustacea, Cirripedia), with different larval dispersal modes in the archipelago of the Azores[END_REF]. AMOVA analysis showed that 4.65 % of total genetic variance in the E. affinis samples was estimated among groups (downstream, middle-estuary, upstream and tributaries) and 27.5 % among localities within these groups. The diversity indices indicate a genetic heterogeneity within localities, and the global level of gene flow Nm (2.08) was low, suggesting a genetic isolation between all/some populations while the pairs values of gene flow indicate the most active populations. The highest gene flow occurred in the tributaries and the upstream estuary, showing that the evolutionary process of the E. affinis species is most dynamic in these locations compared to the rest of the studied locations.

These results may suggest that some of the individuals could not belong to a sole taxon or genetic entity, as it was concluded in our former study, even if they are morphologically undistinguishable [START_REF] Lee | Global phylogeography of a cryptic copepod species complex and reproductive isolation between genetically proximate "populations[END_REF][START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF]. Most genetic characterization works on the E. affinis complex have used mitochondrial gene encoding cytochrome oxidase I (COI) [START_REF] Lee | Rapid and repeated invasions of fresh water by the copepod Eurytemora affinis[END_REF][START_REF] Lee | Morphological stasis in the Eurytemora affinis species complex (Copepoda: Temoridae)[END_REF][START_REF] Winkler | Heterogeneity within the native range: population genetic analyses of sympatric invasive and noninvasive clades of the freshwater invading copepod Eurytemora affinis[END_REF][START_REF] Winkler | Genetic heterogeneity among Eurytemora affinis populations in Western Europe[END_REF]. Using this technique, [START_REF] Winkler | Genetic heterogeneity among Eurytemora affinis populations in Western Europe[END_REF] reported a lack of genetic subdivision among E. affinis from Scheldt, Elbe and Seine estuaries according to the low and non-significant global FST (FST = 0.006).

In this study, the FST pairwise values are lower than in the study of [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF]. It conveys the idea that populations are genetically close. In the same way, comparatively to our former study, there is a genetic heterogeneity decrease going on at the population scale that allows the homogenization of groups. This explains the clear diminution of genetic variability between the several groups in the sampling zone found in this study as compared to the results of [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF].

Genetic structure

In order to better understand the genetic structure of E. affinis in the sampled localities, various methods of classification were adopted. Distance analysis provided 4 main clusters, two related with downstream and middle estuarine stations and another two with a mix of tributaries locations and upstream stations. The observed branch length on the dendrogram shows that distances between individuals are longer than between localities. The branch length between localities is three times closer than in the study of [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF].

This can be explained by the homogenization of the populations in the sampling site during the two-year period between the two studies. The discrimination power of this method was good because few individuals were misclassified.

Mapping individuals according to their ISSR band similarities through the Bayesian analysis revealed 5 clusters, as found in the SOM analysis. The middle-estuary group is located between downstream and upstream and tributaries clusters which is congruent with the results of [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF]. These results show the same main patterns as [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] where tributaries and upstream stations are individualized in two clusters different from the "mid" and downstream stations, but with more mixture between all stations. The recent disappearance of the oxygen barrier allows re-encountering between Scheldt fresh and brackish water populations. This contributes to the decrease of genetic divergences in the populations of the sampling site. Furthermore, [START_REF] Palumbi | Genetic divergence, reproductive isolation, and marine speciation[END_REF] states that, when no barrier to reproduction exists after the bringing back together of allopatric populations, whatever genetic differences had accumulated between isolates will be shared throughout the re-joined population.

Our results show that distances between all E. affinis populations sampled are around three times lower than those found by [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF]. The main clusters are still present in the results but not strictly separated, there has been a homogenization of the populations since the last study.

Population structure

Many factors can explain that the structuration of the different populations is disappearing in the Scheldt estuary:

The sampling, covering 12 stations, is more important than in the previous study ( 5stations in [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF]). This allows to detect more genetic variability in the populations and to have more clues about the recolonization mechanism in the estuary. While the studied estuarine range of [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] was slightly different than the present one, the extreme stations (at 140 km; 128 km and 59 km; 63.5 km from the mouth respectively), are situated in the same hydrological regions of the Scheldt [START_REF] Maris | OMES report 2015[END_REF].

The results of [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] showed that the downstream E. affinis population was most likely at the origin of the upstream one, as they were genetically related.

The genetic distinction between the tributary populations and all main estuarine ones showed that the tributary population were not a reservoir for the upstream population.

In the present study, the populations from tributaries and upstream region are related and grouped, showing that in between the two studies, genetic exchange has occurred between the tributary and the mainstream populations. We may hypothesize that, at first, colonization happened from downstream to upstream in the main Scheldt, while remnant populations of a different genetic identity were present only in tributaries but that recently, these tributary populations were able to interbreed with the upstream populations which had issued from the downstream one. A key point is that compiling the two data sets [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] and the present one), we found that "downstream" and "middle" were genetically comparable while marked differences were observed for "up" and tributaries (Data not shown). [START_REF] Mialet | Spatial spring distribution of the copepod 29 Eurytemora affinis (Copepoda, Calanoida) in a restoring estuary, the Scheldt (Belgium)[END_REF] and [START_REF] Chambord | Mesozooplankton affinities in a recovering freshwater estuary[END_REF] and unpublished monitoring results have shown that E. affinis abundance is strongly influenced by O2 and NH4 concentrations. So, the still ongoing improvement of water quality in the upstream reach may have facilitated interbreeding between tributary and main stream populations.

Estuaries being very dynamic systems, the estuarine flow regime will influence the localisation and the range of a given genotype at each moment of the day. While flows were similar during both sampling (89 m 3 sec -1 in October 2012 and 73 m 3 s -1 in May 2014), the tidal phases of both campaigns could not be synchronised for logistic reasons. This however, does not influence the stronger genetic homogenisation of the populations and the occurrence of mixed tributary and upstream individuals observed now as compared to [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF]. More frequent and fine-gridded sampling would be needed to reveal the exact geographic limits of each genotype.

However, this would be a quasi-impossible task, considering the natural and mandriven dynamics of the estuary and phenomena such as upstream transport.

The fact that sampling in the present study was done after the high-runoff winter months, and the one of [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] before winter, could have favoured mixing of E. affinis overwintering stages during the two winter periods, and thus encounter probability for spring procreating individuals in the present study. It may also explain why the Down and Up mixed genotypes are found more downstream (mainly at Down 1, at 63 km from the mouth) in the present study, whereas they were situated around 78 km from the mouth in [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF].

E. affinis and estuarine restoration

In the Scheldt there are several generations of E. affinis per year with adult abundance peaks between 3 and 10 individuals L -1 [START_REF] Tackx | Selective feeding of Eurytemora affinis (Copepoda, Calanoida) in temperate estuaries: model and field observations[END_REF]2004;[START_REF] Mialet | Spatial spring distribution of the copepod 29 Eurytemora affinis (Copepoda, Calanoida) in a restoring estuary, the Scheldt (Belgium)[END_REF]2011;[START_REF] Chambord | Mesozooplankton affinities in a recovering freshwater estuary[END_REF].The mobility can be high because Eurytemora affinis is able to migrate in several directions with the tide and upstream transport to colonize new sites [START_REF] Morgan | Sink or swim? Copepod population maintenance in the Columbia River estuarine turbidity-maxima region[END_REF][START_REF] Devreker | Trade-offs of the copepod Eurytemora affinis in mega-tidal estuaries. Insights from high frequency sampling in the Seine Estuary[END_REF] These parameters allow the rapid colonizing of new favourable sites after stress periods and explain that a short period between two studies is enough to obtain different results.

There are only two years between the study of [START_REF] Gasmi | Genetic diversity among the Eurytemora affinis species complex in the Scheldt estuary and its tributaries using ISSR-PCR marker assay[END_REF] and this study.

In the former study, we hypothesised that a downstream colonization occurs in direction of upstream while some remnant individuals of another genetically different population managed to survive in the tributaries. A plausible form for such survival could be resting eggs, shown to offer survival capacity to several copepod species over periods of decades (e.g. [START_REF] Ban | Effects of photoperiod, temperature, and population density on induction of diapause Egg production in Eurytemora affinis (copepoda: calanoida) in lake Ohnuma, Hokkaido, Japan[END_REF][START_REF] Dahms | Dormancy in the Copepoda -an overview[END_REF]. Resting eggs of E. affinis have been found in sediments of the Seine estuary, a system submitted to comparable climate and anthropogenic stress than the Scheldt, where E. affinis is also a major component of the plankton community [START_REF] Devreker | Effects of salinity and temperature on the reproduction of Eurytemora affinis (Copepoda; Calanoida) from the Seine estuary: a laboratory study[END_REF]2010;[START_REF] Glippa | Calanoid copepod resting egg abundance and hatching success in the sediment of the Seine estuary[END_REF]. Hatching experiments with these sediments have shown that, when exposed to favourable conditions, the hatching success is high enough for these resting eggs to play a significant role in the dynamics of the E. affinis population in the Seine [START_REF] Glippa | Seasonal fluctuations of the copepod resting egg bank in the middle Seine estuary, France: Impact on the nauplii recruitment[END_REF]. [START_REF] Lee | Rapid and repeated invasions of fresh water by the copepod Eurytemora affinis[END_REF] has shown that the interface between marine and freshwater habitats constitutes a strong barrier that few species have been able to overcome. The copepod E. affinis provides a model system to examine how species overcome boundaries between 'adaptive zones' because this species is able to colonize freshwater habitats within few generations [START_REF] Lee | Rapid and repeated invasions of fresh water by the copepod Eurytemora affinis[END_REF]. Several authors have shown the eco-physiological plasticity of this Calanoid for physiological and genetic regulation under varying conditions of salinity and temperature (Bradley, 1978a, b;[START_REF] Gonzalez | Salinity stress proteins in Eurytemora affinis[END_REF][START_REF] Lee | Pumping ions: rapid parallel evolution of ionic regulation following habitat invasion[END_REF]. The phytoplankton concentration is higher in the Scheldt freshwater reach than in the brackish water reach [START_REF] Cox | Critical transitions in suspended sediment dynamics in a temperate meso-tidal estuary[END_REF] and high phytoplankton concentrations can favour E. affinis development success in freshwater systems [START_REF] Lee | Feasting in fresh water: impacts of food concentration on freshwater tolerance and the evolution of food 3 salinity response during the expansion from saline into fresh water habitats[END_REF]. These studies support that E. affinis has rapid adaptative dynamics and that two years between two studies can be enough to find different results in its population genetic structure on the estuary scale.

As such, genetic analysis of the polymorphism of Copepods allows us to follow the impact of restauration processes, which modified physico-chemical properties of water in estuaries on the (sub) populations of E. affinis. Routine monitoring of zooplankton communities has allowed detecting changes in spatio-temporal variations and abundance of the zooplankton community and its composition at species level (Chambord et al., 2016 and refences cited in this paper). The use of genetic analysis however made it possible to detect management-induced changes in the Scheldt E. affinis population such as mixing of genetically distinct individuals and to suggest hybridisation between historically isolated genotypes. Considering the importance of the E. affinis species complex in temperate estuaries in general, and the present-day efforts in estuarine restoration, the potential of this technique is of general interest to estuarine restoration. Considering the importance of E. affinis as a trophic link between the primary producers and the higher trophic levels [START_REF] Fockedey | Feeding of the hyperbenthic mysid Neomysis integer in the maximum turbidity zone of the Elbe, Westerschelde and Gironde estuaries[END_REF][START_REF] Tackx | Selective feeding of Eurytemora affinis (Copepoda, Calanoida) in temperate estuaries: model and field observations[END_REF][START_REF] Paulsen | Food limited growth of larval Atlantic Herring Clupea harengus recurrently observed in the coastal nursery area[END_REF], these changes in population structure may also involve consequences to the trophic functioning of the estuaries.

In the St Lawrence estuary (Canada), [START_REF] Cabrol | Physiological condition and differential feeding behaviour in the cryptic species complex Eurytemora affinis in the St Lawrence estuary[END_REF] have shown that the two co-existing but spatially separated clades of the E. affinis complex show different feeding behaviour while living in comparable feeding conditions.

Conclusions

The intensive long-term (1996-present) zooplankton community monitoring in the Scheldt provides a rare opportunity to study the spatio-temporal evolution of this 'invasive' reputed species and to reveal dependencies on environmental conditions, which are relevant to estuarine ecology and management in general. The Eurytemora affinis complex of species is indeed a useful tool to study the dynamic mechanisms in a perturbed ecosystem and to better understand the relationship between the different genetic entities.

In this study, two levels of genetic variability were revealed: (1) among upstream freshwater localities, and (2) between freshwater and brackish water localities. These findings complement the work of [START_REF] Winkler | Genetic heterogeneity among Eurytemora affinis populations in Western Europe[END_REF] on E. affinis who, using COI barcoding, reported, a relatively low (1.7-2%) percentage of divergence on the European geographic scale, compared with the important divergence among the four North American clades (up to 15%, [START_REF] Lee | Rapid and repeated invasions of fresh water by the copepod Eurytemora affinis[END_REF][START_REF] Lee | Global phylogeography of a cryptic copepod species complex and reproductive isolation between genetically proximate "populations[END_REF]. As such, the Scheldt case study illustrates the potential of ISSR to detect genetic differentiation at the estuarine drainage-basin scale. Time scale to reach a same level of genetic differentiation between two taxa than the one observed in our study is difficult to estimate, anyway in our experience using spiders 10 to 20 generations are sufficient to observe significant genetic differences using ISSR (Machkour-M'Rabet et al., 2012; distributed between Trib2b and Trib2d). In blue: main estuary (dark blue: "Down" and "Mid"; light blue: "Up"). In pink: tributaries. 3 andTable 4. Cluster 1 corresponds to "down1", green of fig. 3, cluster 2 to "down 2", yellow of fig. 3, cluster 3 to "mid 1" + "mid 2", purple of fig. 4). Black bars delimit sample sites. Cluster 1: Red, Cluster 2: green, Cluster 3: blue, Cluster 4: yellow and Cluster 5: purple (Same colour code as in Fig. 3).

Figure captions

Tables

Fig. 1 .

 1 Fig. 1. Geographic localization and map of the Scheldt estuary with sampling stations

Fig. 2 .

 2 Fig. 2. Unrooted tree obtained by distance analyses of the 12 E. affinis

Fig. 3 .

 3 Fig. 3. Classification of 273 individuals of E. affinis from the 12 sample locations

  3, cluster 4 to trib 2d, Up 3, Trib 1, 80% of Trib 2a and 15% Trib 2c, mainly red with blue individuals (see also Fig. 4) and cluster 5 to Trib 2b, Up 1, Up2, 85% Trib 2c and 20% Trib 2a, mainly blue with red individuals.

Fig. 4 .

 4 Fig. 4. Bayesian analysis of E. affinis performed by STRUCTURE 2.3.4 software.

  Fig. 2

Table 1 .

 1 Circled in blue, the critical zone of genotype mixing.

Table 1 . Geographical coordinates of sampled locations

 1

	Sampling
	location

Table 3

 3 Probability (Q) of membership of individuals of the 12 initial populations of E. affinis to each of the 1 to 5 inferred clusters determined by STRUCTURE. The highest percentage of a sample assigned to one cluster, is indicated in bold + grey. Colouring of the columns: see legend Fig.3.

		Cluster 1	Cluster 2	Cluster 3	Cluster 4	Cluster 5
	Down 1	0.138	0.802	0.012	0.004	0.044
	Down2	0.051	0.014	0.021	0.892	0.021
	Mid1	0.098	0.007	0.048	0.011	0.836
	Mid2	0.025	0.007	0.011	0.004	0.953
	Trib2a	0.736	0.060	0.144	0.009	0.050
	Trib2b	0.043	0.011	0.901	0.006	0.040
	Trib2c	0.203	0.030	0.660	0.011	0.096
	Trib2d	0.951	0.007	0.016	0.007	0.019
	Up1	0.179	0.009	0.720	0.009	0.083
	Up2	0.058	0.010	0.876	0.033	0.024
	Up3	0.933	0.013	0.025	0.003	0.026
	Trib1	0.936	0.008	0.020	0.012	0.023

Table 4

 4 Pairwise comparison between populations of E. affinis of gene flow (Nm, bold, max/min values) above diagonal and geographic distances below. Colours for clusters correspond to those of Figure3 and 4.

	Pop	Down	Down	Mid	Mid	Trib2	Trib2	Trib2	Trib2	Up1 Up2 Up3 Trib
	ID	1	2	1	2	a	b	c	d				1
	Down		1.64	1.71 1.25 2.13	1.32	1.77	2.23	1.5	1.5	1.7	1.81
	1									5	9	9
	Down	21.5		1.47 1.14 1.82	1.57	1.96	1.64	1.9	2.3	1.2	1.69
	2									7	6	5
	Mid1	30.5	9		1.63 1.75	1.82	2.39	1.95	2.2	2.1	1.5	2.03
										9	2	8
	Mid2	35	13.5	4.5		1.67	1.36	1.22	1.35	1.2	1.1	1.0	1.06
										8	2	3
	Trib2a 32.5	11	3	7.5		2.24	2.45	4.01	2.5	2.1	3.9	2.66
										2	4	2
	Trib2b 34.5	13	5	9.5	2		3.32	1.99	2.8	3.7	1.6	1.78
										6	7	5
	Trib2c 38.5	17	9	13.5 6	4		2.50	1.8	3.5	2.6	2.33
										6	1	3
	Trib2d 39.5	18	10	14.5 7	5	1		1.8	2.2	3.9	4.29
										4	6	1
	Up1	40	18.5	9.5	5	12.5	14.5	21.5	22.5		2.8	2.1	1.92
											1	4
	Up2	57.5	36	27	22.5 30	32	39	40	17.		1.8	2.24
										5		1
	Up3	64.5	43	34	29.5 37	39	46	47	24.	7		2.59
										5
	Trib1	50.7	29.2	20.2 15.7 23.2	25.2	29.2	30.2	10.	6.8	13.
										7		8

Acknowledgements

We are indebted to Céline Pélissier (who unfortunately deceased before publication of this work), Frédéric Azémar, Julien Ovaert, Claudine Sossou, Maxime Ferval and for their help during the genetic manipulations and isolation E. affinis individuals. This study was made possible by the OMES monitoring program on the Scheldt estuary, financed by De Vlaamse Waterweg.

Code

Distance from mouth (km)

Latitude Longitude

The


C Caburet

N Farigon

A Mulliez

T Mom

Y Boirie

L Gilain

N Saroul
email: nsaroul@chu-clermontferrand.fr

Impact of nutritional status at the outset of assessment on postoperative complications in head and neck cancer

Keywords: malnutrition, sarcopenia, Clavien-Dindo classification, postoperative complications, head and neck cancer

Introduction

More than half of head and neck cancer patients present with malnutrition at diagnosis [START_REF] Saroul | Which Assessment Method of Malnutrition in Head and Neck Cancer?[END_REF]. In oncology, malnutrition can be defined as a subacute or chronic state of nutritional imbalance associated with the inflammatory tumoral context, altering body composition, with diminished lean mass and muscle mass with or without diminished fat mass, and leading to organ dysfunction, with immune, muscular and cognitive deficits [START_REF] Soeters | A rational approach to nutritional assessment[END_REF]. It is also a source of postoperative infection and delayed healing [START_REF] Pichard | Nutritional assessment: lean body mass depletion at hospital admission is associated with an increased length of stay[END_REF][START_REF] Cosquéric | Sarcopenia is predictive of nosocomial infection in care of the elderly[END_REF].

Despite the growth of chemoradiotherapy, surgery is still a major form of head and neck cancer treatment, about a half of patients undergoing surgery at some point. It entails, however, numerous complications and should not be undertaken without first assessing the full range of possible risk factors, either to reduce their impact or to abstain from surgery.

Risk factors for postoperative complications in oncologic head and neck surgery include: smoking, large tumor size, degree of resection, hypopharyngeal location, long operating time, high ASA (American Society of Anesthesiologists) score, and diabetes. These are all non-independent risk factors for complications or longer hospital stay in head and neck surgery. Prior radiation therapy is also a strong risk factor for complications and for healing disorder in particular [START_REF] Kiliç | Pharyngocutaneous fistulae after total laryngectomy: analysis of the risk factors and treatment approaches[END_REF][START_REF] Timmermans | Predictive factors for pharyngocutaneous fistulization after total laryngectomy[END_REF].

The literature is agreed on the need for early diagnosis and management of malnutrition [START_REF] Dechaphunkul | Malnutrition assessment in patients with cancers of the head and neck: a call to action and consensus[END_REF], but few studies have focused on the impact of nutritional status on postoperative complications in head and neck cancer [START_REF] Leung | Association Between Preoperative Nutritional Status and Postoperative Outcome in Head and Neck Cancer Patients[END_REF]. In particular, there have been no studies on the impact of sarcopenia, although data are plentiful for other forms of cancer, associating sarcopenia with higher rates of postoperative complications, nosocomial infection and lengthened hospital stay in colorectal, esophageal and renal cancer [START_REF] Lieffers | Sarcopenia is associated with postoperative infection and delayed recovery from colorectal cancer resection surgery[END_REF][START_REF] Elliott | Sarcopenia : Prevalence, and Impact on Operative and Oncologic Outcomes in the Multimodal Management of Locally Advanced Esophageal Cancer[END_REF][START_REF] Peyton | Does Sarcopenia Impact Complications and Overall Survival in Patients Undergoing Radical Nephrectomy for Stage III and IV Kidney Cancer?[END_REF].

The present study aimed to assess the correlation between complications and nutritional and muscle status (mass and function) at the outset of treatment (first surgical consultation) ahead of any corrective measures in head and neck oncologic surgery.

Patients and methods

A single-center retrospective observational study included all patients undergoing curative surgery for head and neck squamous cell carcinoma from September 2014 to April 2016. Cases of recurrence, relapse or second cancer and of prior radiation therapy and of salvage surgery were included.

Study data comprised:

• General characteristics: history, smoking status in pack-years, alcohol consumption, anesthesia risk on ASA score (Appendix 1), WHO Performance Status (Appendix 2) and demographics (age and gender).

• Tumor characteristics: UICC (7 th edition) classification and staging, location, classification as primary, recurrent or secondary head and neck cancer, and prior radiation or chemo-therapy.

• Surgical characteristics: type of resection, type of reconstruction, any pharyngeal opening or loss of oral floor tightness. Procedures were categorized by high versus low risk of fistula, with high risk comprising total or partial laryngectomy, total pharyngolaryngectomy, oropharyngectomy with pharyngeal opening, and oral resection with intraoperative loss of oral floor tightness.

Nutritional work-up comprised anthropometric, biological and CT assessment. Anthropometric and biological assessment was performed during the first surgical consultation, and CT examination as part of the extension assessment as soon as possible afterward.

• Anthropometric measurements:

Anthropometric measurements comprised weight 3 months previously, and current weight (kg) and height (cm). Percentage weight-loss was calculated as (weight 3 months previously minus current weight) / current weight x 100; body-mass index (BMI) was calculated as current weight/height². Malnutrition was defined by BMI according to age: ≤ 70 years, <18.5 kg/m 2 ; >70 years, < 21 kg/m 2 [START_REF]Recommandations de la société française de nutrition clinique et métabolisme. Nutrition chez le patient adulte atteint de cancer[END_REF].

• Biological assessment:

Albuminemia and pre-albuminemia thresholds were 35g/l and 0.11 g/l respectively [START_REF]Recommandations de la société française de nutrition clinique et métabolisme. Nutrition chez le patient adulte atteint de cancer[END_REF].

• Composite assessment (NRI) : Biological data were coupled to anthropometric data to calculate a composite Nutrition Risk Index (NRI): 1.519 x albuminemia + (current weight/usual weight) x 100. NRI malnutrition threshold was <97.5, often considered a reference score in head and neck oncology [START_REF] Antoun | Malnutrition, inflammation: which parameter is interesting to detect in planned oncologic surgery?[END_REF].

• CT assessment (quantitative assessment of body composition and especially of muscle mass): CT is a recent means of assessing body composition that has proved highly contributive in oncology [START_REF] Antoun | Malnutrition, inflammation: which parameter is interesting to detect in planned oncologic surgery?[END_REF]. Relative muscle area is determined on a 3 rd lumbar vertebra slice using templates and dedicated software (Slice-O-Matic® V 4.3). It was assessed as part of the extension work-up if an abdominal slice was taken, as in most cases with our institution's nutritional assessment protocol, or else on PET-CT. L3 muscle-mass index (L3MMI) was thus calculated as the ratio between muscle area and height squared, with a threshold of < 52.4cm²/m² for males and < 38.5 cm²/m² for females [START_REF] Baracos | Body composition in patients with non-small cell lung cancer: a contemporary view of cancer cachexia with the use of computed tomography image analysis[END_REF].

• Functional nutritional assessment (qualitative assessment of muscle function)

Functional nutritional assessment used the Short Physical Performance Battery (SPPB), a 3item test scored out of 12 (Appendix 3), with scores <8 indicating severe muscle function disorder, which is a malnutrition severity factor.

Nutritional management was adapted according to malnutrition threshold, following the guidelines of the French-speaking Society of Clinical Nutrition and Metabolism (SFNEP) [START_REF]Recommandations de la société française de nutrition clinique et métabolisme. Nutrition chez le patient adulte atteint de cancer[END_REF].

Complications were assessed over a 30-day postoperative period. Quantitative and qualitative analysis used the 5-grade Clavien-Dindo classification, validated in head and neck surgery [START_REF] Monteiro | Assessment of the Clavien-Dindo classification system for complications in head and neck surgery[END_REF] and quick and easy to implement (Table 1). It requires a definition of normal postoperative course in head and neck oncologic surgery, for which our department uses the Enhanced Recovery After Surgery (ERAS) guidelines to standardize management, and antibiotic prophylaxis in particular [START_REF] Dort | Optimal Perioperative Care in Major Head and Neck Cancer Surgery With Free Flap Reconstruction: A Consensus Review and Recommendations From the Enhanced Recovery After Surgery Society[END_REF]. For analysis, only patients with grade ≥2 complications were counted as having complications. Further data comprised hospital stay (days), compared to the mean for each procedure, and type of discharge (home or to follow-up unit).

Statistical analysis

Qualitative data were reported as number and percentage, and quantitative data as mean ± standard deviation. Intergroup comparison used chi² test or Fisher exact test as appropriate for qualitative variables and Student or Mann-Whitney test for quantitative variables. Complications were analyzed by logistic regression, adjusting on the main clinically and statistically relevant criteria. Analyses used Stata 12 software. All tests were 2-tailed, with the significance threshold set at p <0.05.

RESULTS

92 patients were included: 75 male, 17 female; mean age, 62.3±10 years. 51% were smokers and 43% had chronic alcohol abuse. 6 were ASA 1 (6%), 59 ASA 2 (64%), 26 ASA 3 (28%), and 1 ASA 4 (2%). 11 (12%) had PS (performance status) 0, 67 PS 1 (75%), 10 PS 2 (11%), 1 PS 3 (1%) and none PS 4; in 3 cases, PS was not known. TNM stages were mainly advanced: 9% stage 1, 19% stage 2, 28% stage 3 and 44% stage 4. Table 2 presents procedures, and Table 3 nutritional data. 91 NRI, 68 L3MMI and 92 SPPB data sets could be analyzed. There were no significant differences in tumor location, TNM stage, WHO or ASA score or type of surgery between patients with complete scoring (SPPB, NRI and L3MMI) and those lacking L3MMI. On NRI, 49 patients (54%) were malnourished. On L3MMI, 36 showed sarcopenia (53%). 12 had SPPB <8 (13%). The overall grade ≥2 complications rate was 41.7% (38 patients) and grade ≥3 rate 21.5% (20 patients). The majority of complications were grade 2 (32 patients), the most frequent being surgery-site infection (15 cases: 39%) and delayed healing (18 cases: 47%). There were 11 grade 3 complications: 8 revision procedures for surgery-site infection, 2 cases of surgery-site hemorrhage, and 1 tracheotomy for respiratory distress associated with oral floor hematoma. There were 7 grade 4 complications: 3 following local or general infection (2 pneumopathies, 1 septic shock), 1 following myocardial infarction, and 2 decompensations of smoking-related lung disease. There were 2 deaths: 1 from hemorrhage, and 1 from multiple organ failure following pneumopathy. Figure 1 shows complications types and rates. Risk of surgical complications, taking all procedures together, was significantly higher in case of malnutrition on NRI (OR: 8.5; 63% with vs. 17% without; p<0.001), L3MMI (OR: 4.5; 56% vs. 22%; p< 0.015) and albuminemia (OR: 4.4, 31% vs. 67%; p=0.002), but not on BMI (OR: 2, 40% vs. 57%; p=0.38). 75% (n=9) of the 12 patients with SPPB <8 showed complications (OR: 5.27; p=0.018).

Other risk factors for complications on univariate analysis were non-significant: pharynx or oral floor opening (31% (13 patients out of 41) without versus 49% (25 out of 51) with opening; OR: 2; p=0.09); salvage surgery after radiation or chemo-therapy (55% (10 out of 18) with prior radiation therapy, but for a small sample of 18 patients; p=0.1); tumor stage; performance status; ASA score (16.7% (1 out of 6 patients) with ASA 1 showing complications, versus 40.6% (24 out of 59) with ASA 2, and 50% (13 out of 26) with ASA 3); advanced age >70 years; OR: 1.4; p=0.5); or active smoking or alcohol abuse.

Hospital stay was significantly longer, by 50%, in NRI-malnourished patients (p=0.042). A similar trend could be seen for L3MMI-sarcopenia, although the difference was not significant (p=0.18).

Focusing on severe surgical complications (Clavien-Dindo ≥3, requiring revision surgery or intense care, there were no significantly associated parameters. However, 27% of malnourished patients (13 out of 49) had severe complications, compared to 11% of the others (5 out of 42) (OR: 2.7; p=0.08). On multivariate analysis, NRI malnutrition, L3MMMI sarcopenia and SPPB were the only significant independent risk factors for onset of postoperative complications.

DISCUSSION

Few studies have dealt with surgical complications in head and neck oncology taking all sublocations and procedures together, most focusing rather on some particular type of surgery or, more recently, on free transplants [START_REF] Eskander | Predictors of Complications in Patients Receiving Head and Neck Free Flap Reconstructive Procedures[END_REF][START_REF] Goepfert | Complications, hospital length of stay, and readmission after total laryngectomy[END_REF]. Studies were mainly retrospective, focusing on the most severe complications and thus underestimating the overall rate. The present study used the Clavien-Dindo classification, recently validated in head and neck surgery; its main advantage is to allow comparison between procedures, as it is used in many other specialties. Another advantage is that it does not ignore complications liable to appear to be part of a normal postoperative course; it therefore requires a good definition of "normal" or indeed "ideal" course after a given surgery. Its main drawback is that it is not always well-adapted to head and neck pathology; for example, surgical revision of necrotic free flap is graded 3, the same as for hematoma, despite being much less serious. Nevertheless, in our opinion, lacking a better-adapted classification, surgical complications should be reported on the Clavien-Dindo classification.

The present study found an almost 42% rate of grade ≥2 complications. Surgery site infection and delayed healing were preponderant. The rate reflects the continuous activity of a surgery department over slightly more than 1 year. Comparison with the literature is hampered by the problems mentioned above. Eskander reported a 54% complications rate, with 15% fistula and 11% infection, in a series of 515 patients with free flap reconstruction [START_REF] Eskander | Predictors of Complications in Patients Receiving Head and Neck Free Flap Reconstructive Procedures[END_REF]; independent risk factors comprised mucosal opening and intraoperative filling volume for delayed healing, and age, diabetes and malnutrition for infection. Comparison with the present findings is hindered by Eskander's inclusion of salivary and cutaneous tumor and the systematic use of free flaps. Goepfert focused exclusively on complications of total pharyngolaryngectomy, with a 33% rate of complications overall and 21% for healing complications. The present series showed 26% fistula in procedures with pharyngeal opening, compared to 20-27% for other authors [START_REF] Kiliç | Pharyngocutaneous fistulae after total laryngectomy: analysis of the risk factors and treatment approaches[END_REF][START_REF] Timmermans | Predictive factors for pharyngocutaneous fistulization after total laryngectomy[END_REF]. In recent reports, preoperative nutritional status was an important factor for postoperative complications, although nutritional status assessment modalities were not clearly described and some authors used only preoperative albuminemia, which has proved useful in predicting postoperative complications in both general and in head and neck surgery and also in predicting overall survival in head and neck oncology [START_REF] Danan | Prognostic value of albumin in patients with head and neck cancer[END_REF]. In the present series, it correlated with postoperative complications, but did not emerge as the most important prognostic factor. It is dependent on inflammatory status, varies slowly over time and is a poor indicator of nutritional status in head and neck cancer, as our results confirmed [START_REF] Saroul | Which Assessment Method of Malnutrition in Head and Neck Cancer?[END_REF]. We consider it necessary to associate weight loss by calculating NRI, to increase sensitivity. We showed NRI to correlate well with body composition, and it is an easy assessment to carry out. In the present study, NRI was the parameter correlating most closely with postoperative complications (OR: 8.5).

Interestingly, in the present retrospective study, nutritional status at the outset of treatment was the sole independent risk factor for complications on multivariate analysis. Other classical risk factors (diabetes, prior radiation therapy, smoking) showed no significant correlations. These findings are of course to be taken with caution, given the retrospective design, small study sample and failure to analyze such biological parameters as blood lymphocyte and hemoglobin levels at treatment outset. Complications generally lengthen hospital stay, and in the present study malnutrition at treatment outset was associated with 50% longer hospital stay. The study objective was to assess the impact of sarcopenia on postoperative complications. In is now clear that sarcopenia is a major predictor of postoperative complications and overall survival in digestive, urologic and esophageal oncology [START_REF] Lieffers | Sarcopenia is associated with postoperative infection and delayed recovery from colorectal cancer resection surgery[END_REF][START_REF] Elliott | Sarcopenia : Prevalence, and Impact on Operative and Oncologic Outcomes in the Multimodal Management of Locally Advanced Esophageal Cancer[END_REF][START_REF] Mayr | Sarcopenia predicts 90-day mortality and postoperative complications after radical cystectomy for bladder cancer[END_REF]. The present study showed the same findings, with sarcopenia as an independent risk factor for postoperative complications (OR: 4.5) and a very high (75%) complications rate in case of impaired muscle function (SPPB<8). We find this correlation coefficient abnormally low, and difficult to interpret due to missing data in this retrospective study. Moreover, sarcopenia seems to be a risk factor for serious complications; this could not be shown from the present study, but emerges from the literature [START_REF] Smith | Sarcopenia as a predictor of complications and survival following radical cystectomy[END_REF], and is important for patient management. Diminished muscle mass can be reversible [START_REF] Baeyens | European Working Group on Sarcopenia in Older People. Sarcopenia: European consensus on definition and diagnosis: Report of the European Working Group on Sarcopenia in Older People[END_REF]: appropriate feeding with high-quality amino acids associated to physical exercise seems to be the key to correcting sarcopenia. Two recent Japanese studies showed that elderly sarcopenic patients scheduled for gastric cancer surgery had low protein and calorie intake and incurred more severe postoperative complications [START_REF] Fukuda | Sarcopenia is associated with severe postoperative complications in elderly gastric cancer patients undergoing gastrectomy[END_REF]. The same team demonstrated that adapted nutritional therapy combined with physical activity increased protein and calorie intake in such patients, significantly improving muscle function [START_REF] Yamamoto | Effectiveness of a preoperative exercise and nutritional support program for elderly sarcopenic patients with gastric cancer[END_REF]. Correlation with decreased postoperative complications, however, remains to be proven. In the present series, around 50% of patients showed sarcopenia. This is due to imbalance between muscle protein anabolism and protein catabolism. Pathophysiological hypotheses have been put forward to explain such sarcopenia, and notably muscle protein anabolism resistance in case of neoplasia: inflammation, insulin resistance [START_REF] Guillet | Impaired anabolic response of muscle protein synthesis is associated with S6K1 dysregulation in elderly humans[END_REF][START_REF] Guillet | Changes in basal and insulin and amino acid response of whole body and skeletal muscle proteins in obese men[END_REF], and myofibril fatty infiltration (myosteatosis) [START_REF] Tardif | Muscle ectopic fat deposition contributes to anabolic resistance in obese sarcopenic old rats through eIF2α activation[END_REF]. This resistance is found even without malnutrition (pre-cachexia) [START_REF] Khan | Detection and evaluation of malnutrition in oncology: What tools, what type of cancer and for what purposes?[END_REF]. Studies have confirmed that conventional diet supplements using formulae available on the market are ineffective in stimulating muscle protein synthesis in advanced cancer, and that daily protein dose and the protein and amino acid composition of meals are among the most important factors affecting anabolic response in cancer patients [START_REF] Deutz | Muscle protein synthesis in cancer patients can be stimulated with a specially formulated medical food[END_REF][START_REF] Engelen | Protein anabolic resistance in cancer: Does it really exist?[END_REF]. The present results thus advocate specific preoperative nutritional therapy or nutritional prehabilitation to reduce postoperative complications. Over the last 10 years, most studies of nutritional preparation for surgery have focused on the immune effects of diet. Some studies showed the contribution of pre-and peri-operative immunonutrition (arginine, nucleotides, omega-3 fatty acids) to reducing surgical complications [START_REF] Falewee | Reduced infections with perioperative immunonutrition in head and neck cancer: exploratory results of a multicenter, prospective, randomized, double-blind study[END_REF][START_REF] Van | Effect of perioperative nutrition, with and without arginine supplementation, on nutritional status, immune function, postoperative morbidity, and survival in severely malnourished head and neck cancer patients[END_REF][START_REF] Felekis | Effect of perioperative immunoenhanced enteral nutrition on inflammatory response, nutritional status, and outcomes in head and neck cancer patients undergoing major surgery[END_REF][START_REF] Rowan | Utility of a perioperative nutritional intervention on postoperative outcomes in high-risk head & neck cancer patients[END_REF]. We therefore consider it necessary to develop specific renutrition strategies ahead of head and neck cancer surgery, associating immunonutrition, exercise and formulae stimulating protein anabolism to optimize management of sarcopenia detected at the outset of treatment of patients with head and neck cancer.

CONCLUSION

Malnutrition emerged as an independent risk factor for surgical complications in head and neck cancer surgery. Such complications are frequent, mainly comprising infection and delayed healing. Nutritional assessment at the outset of treatment should be systematic. Particular attention should be paid to muscle tissue, impaired quantity and quality being predictive of complications. In future, muscle function should be restored preoperatively by specific nutritional formulae and physical exercise to reduce the rate of surgical complications. Oral resection with isolated oral approach 6

Disclosure of Interest: The authors declare that they have no conflicts of interest concerning this article

Oral resection with oral and cervical approach 5

Partial glossectomy 7

Table 1 :

 1 Clavien-Dindo ClassificationGRADE 1 Any deviation from the normal postoperative course without pharmacological treatment (other than below) or surgical, endoscopic or radiological interventions.

		Allowed therapeutic regimens are: drugs as antiemetics, antipyretics,
		analgesics, diuretics, electrolytes.
	GRADE 2	Complication requiring pharmacological treatment, including blood/plasma
		transfusion and non-scheduled parenteral nutrition.
	GRADE 3	Complication requiring surgical, endoscopic or radiological interventions
		under non-general anesthesia.
		Complication requiring surgical, endoscopic or radiological interventions
		under general anesthesia.
	GRADE 4	Life-threatening complication requiring intensive care management:
		a) Single-organ dysfunction
		b) Multiorgan dysfunction
	GRADE 5	Death of a patient.

[START_REF] Antoun | Malnutrition, inflammation: which parameter is interesting to detect in planned oncologic surgery?[END_REF]

Table 2 :

 2 Numbers per procedure.

	Procedure	number
	Isolated neck dissection	11
	Extended tonsillectomy (Huet procedure) + neck	12
	dissection
	Intraoral oropharyngectomy + neck dissection	5
	Combined oral and cervical oropharyngectomy	9
	with or without transmandibular approach
	Isolated cervical tongue-base surgery + neck	2
	dissection
	Partial laryngectomy	10
	Total laryngectomy	7
	Total pharyngolaryngectomy	18


Master Turbé Hugo
email: hugo.turbe@ch-le-vinatier.fr

Master Dechelotte Benoit

Overview of prospects for inflammation pathways in autism spectrum disorders

Keywords: Autism, translational psychiatry, inflammation, gut microbiota, neurodevelopment

Aperçu des perspectives des voies de l'inflammation dans les troubles du spectre de l'autisme

Titre court : Voies de l'inflammation et troubles du spectre de l'autisme

Résumé

Les troubles du spectre de l'autisme (TSA) forment un groupe hétérogène de troubles du neurodéveloppement sévères et fréquents. La compréhension des mécanismes physiopathologiques impliqués pourrait permettre l'identification de nouveaux marqueurs et traitements. Un nombre croissant d'études récentes pointent un rôle important du système immuno-inflammatoire dans les TSA. Des revues détaillées s'intéressent déjà à ce sujet. Cependant, ce champ d'investigations est mal connu des praticiens même parmi ceux prenant en charge des patients avec TSA. Notre objectif principal est de fournir une introduction à ces nouvelles perspectives au travers d'une mini-revue de la littérature. Un premier champ d'études anténatal connecte les paramètres du développement foetal aux TSA au travers d'une participation de l'activation immunitaire maternelle associé à la production d'un profil cytokinique pro inflammatoire chez le foetus comprenant notamment IL-1, IL-6 et TNF mais également IL-17. La présence d'auto-anticorps maternels et d'autres troubles du système immunitaire pourraient également altérer le neurodéveloppement foetal. D'autres études postnatales montrent une corrélation entre les TSA et la présence d'autoanticorps chez l'enfant ou encore entre les TSA et l'environnement inflammatoire au travers de niveaux d'interleukines pro inflammatoires altérés (IL-6 étant le plus exploré). Les voies de recherche en pleine expansion liant immunologie et psychiatrie apparaissent prometteuses à plusieurs niveaux dans la compréhension des TSA. Elles devraient permettre à terme de participer au développement de marqueurs diagnostics ainsi qu'à l'émergence de nouvelles thérapeutiques personnalisées adaptées à ces patients.

Mots clés : Autisme ; psychiatrie translationnelle ; inflammation ; microbiote intestinal ; neurodéveloppement

Introduction

Autism spectrum disorders (ASD) represent a heterogeneous group of neurodevelopmental disorders characterized by social interaction impairment as well as repetitive behaviors or limited interests and activities. Exact causes and mechanisms of ASD have not been elucidated even though several genes associated with ASD have been identified. Recent works have pointed out that 40-50% of the variance in ASD is likely due to environmental factors [START_REF] Modabbernia | Environmental risk factors for autism: an evidence-based review of systematic reviews and meta-analyses[END_REF].

Increasing evidence has shown a major role of the immune system and microbiota in the pathophysiology of ASD with dysfunctions occurring during fetal life and/or postnatal periods.

Extensive reviews have already addressed this topic. Yet, this field of investigations is not well known to practitioners even those working with ASD patients. Our main objective is to provide an introduction to these new insights through a mini review of the literature.

Environmental risk factors for autism and inflammation

In their meta-analysis, Modabbernia A, Velthorst E and Reichenberg A recently pointed out that the environmental risk factors with a higher risk of ASD could be advanced parental age and birth complications, mostly hypoxia, trauma and ischemia. A significative but less strong link was found for caesarian section and maternal comorbidities such as obesity and diabetes [START_REF] Modabbernia | Environmental risk factors for autism: an evidence-based review of systematic reviews and meta-analyses[END_REF]. In another study, Parker W, Hornik CD, Bilbo S et al. suggested that these are risk factors for inflammation, and they add early maternal exposure to severe emotional stress as an important risk factor driven by persistent immune dysregulation later in life (increases of basal IL-6 and C-reactive protein and over-secretion of IL-6 after daily stressors) [START_REF] Parker | The role of oxidative stress, inflammation and acetaminophen exposure from birth to early childhood in the induction of autism[END_REF]. Moreover, they emphasized the potential link between ASD and exposure to the widely used acetaminophen during fetal and early development. They suggested that the underlying mechanism of prostaglandin inhibition could impair neurodevelopment and alter resolution of neuroinflammation [START_REF] Parker | The role of oxidative stress, inflammation and acetaminophen exposure from birth to early childhood in the induction of autism[END_REF].

Fetal features and maternal immune conditions

Associations between ASD and congenital infections have been well described with the first trimester being the critical period. There is evidence that it is in reaction to infection, also called maternal immune activation (MIA), rather than infection itself that increases the risk of ASD [START_REF] Meltzer | The role of the immune system in autism spectrum disorder[END_REF]. The mechanisms are not fully understood, yet MIA seems to induce proinflammatory cytokines such as IL-6 which affect placenta and/or dysregulate gene expression in the fetus, leading to de novo cytokine synthesis.

Moreover, presence of maternal autoantibodies is associated with higher incidence of ASD.

They may appear in response to an infection or in a context of autoimmunity in the mother.

A comparative study involving the mothers of 100 ASD childrenand those of 100 healthy children has shown that those of ASD children display a higher rate of antibodies targeting cerebral fetal proteins [START_REF] Jones | Maternal autoantibody related autism: mechanisms and pathways[END_REF]. Another study on 2431 mothers of ASD children has pointed out that they produce four times more specific antibodies against cerebral proteins involved in neurodevelopment than those of healthy children [START_REF] Brimberg | Brain-reactive IgG correlates with autoimmunity in mothers of a child with an autism spectrum disorder[END_REF]. However, no study has yet proven the pathogenic effect of these antibodies in humans nor their clinical interest as biomarkers.

In animal models, mice treated with IgG from mothers of ASD children display significative impaired social interactions compared to mice treated with IgG from mothers of unaffected children [START_REF] Jones | Maternal autoantibody related autism: mechanisms and pathways[END_REF]. Similar results on reduction of social interaction have been replicated in the rhesus monkey [START_REF] Jones | Maternal autoantibody related autism: mechanisms and pathways[END_REF]. The relevance of these autoantibodies is still unknown, and further research on animal models and patients is needed.

Finally, there is growing evidence of a possible specific role for dysregulations of IL-17A pathway participating in the manifestations of some cases of ASD [START_REF] Wong | Maternal IL-17A in autism[END_REF]. In 2016, Choi GB, Yim YS, Wong H et al. observed an IL-17 increase due to administration of polyinosinicpolycytidylic acid (polyI:C) in murine models of MIA. The authors demonstrated that this increase of IL-17 could be linked to a higher synthesis of IL-6 and may produce an autistic phenotype [START_REF] Choi | The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring[END_REF].

The different ways maternal condition may impact the fetal neurodevelopment are shown in Figure 1.

Postnatal immunity Autoimmunity

Direct correlation of serum autoantibodies with autistic symptoms has not yet been clearly

established. However, the presence of neuronal targeted autoantibodies could be correlated with a more severe form. Recently, an association between ASD and an autoantibody related to autoimmune hepatitis was reported, suggesting a possible link between autism and other autoimmune pathologies [START_REF] Zhang | Mitochondrial DNA and anti-mitochondrial antibodies in serum of autistic children[END_REF].

Inflammatory environment also plays a role. Mice with overexpression of IL-6 showed behavioral impairment [START_REF] Wei | Brain IL-6 and autism[END_REF]. In a comparative study, autistic children displayed increased levels of different cytokines, especially IL-6, which were linked to severity of symptoms. A possible diagnostic test with a combination of serotonin and IL-6 has been shown to have good sensitivity (84,9%) and specificity (96,8%) in autism diagnosis [START_REF] Yang | The combined role of serotonin and interleukin-6 as biomarker for autism[END_REF].

It has also been demonstrated that autistic patients could display higher rates of neurotensin, a vasodilating peptide found both in neurons and gut, which can induce mast cell degranulation. This may explain the higher prevalence of allergic symptoms, especially digestive ones, among these patients [START_REF] Lyall | Asthma and Allergies in Children With Autism Spectrum Disorders: Results From the CHARGE Study: Asthma and allergies in autism[END_REF].

Microbiota

Microbiota represents a promising lead in autism pathophysiology. Gut bacteria can modulate the gut-brain axis through various secretions such as GABA or cytokines. ASD children often display a modified microbiota with an increase of strains such as Suterella spp or Ruminococcus torques [START_REF] Li | The microbiota-gut-brain axis and its potential therapeutic role in autism spectrum disorder[END_REF]. Moreover, 3-Hydroxy-3-(3-hydroxyphenyl) propanoic acid secreted by a Clostridium strain is associated with ASD symptoms.

Microbiota could also be a therapeutic target. In their review, Li Q and Zhou J-M noted a significant reduction of anxiety in ASD children receiving Bacteroides fragilis strains [START_REF] Li | The microbiota-gut-brain axis and its potential therapeutic role in autism spectrum disorder[END_REF]. In another double-blind randomized placebo controlled study with Lactobacillum plantarum administration, improvements of ASD children behavioral scores were observed [START_REF] Li | The microbiota-gut-brain axis and its potential therapeutic role in autism spectrum disorder[END_REF].

More recently, in an open label study, Kang D-W, Adams JB, Gregory AC et al. have shown the potential effectiveness of a microbiota transfer therapy (MTT) on 18 ASD patients who had significantly lower scores of gastrointestinal symptoms and ASD symptoms after treatment. These effects persisted after 8 weeks of follow-up and were associated with an increased overall bacterial diversity and a higher abundance of Bifidobacterium, Prevotella, and Desulfovibrio taxa [START_REF] Kang | Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study[END_REF].

The potential disruptions in the brain-gut axis involved in ASD are summed up in Figure 2.

Conclusion

ASD display wide clinical presentations. Its pathophysiology seems multifactorial and unclear, but progress has been made to explore the immunological hypothesis. The fetal period appears to be critical since higher rates of ASD children are found in pregnant women displaying inflammation or immune-related conditions. Immunity dysregulation is also involved during postnatal periods, with identification of more specific cytokines and inflammatory profiles, and seems to be more associated with severe forms. Gut microbiota is also a promising lead to better understand pathways of neuronal dysregulation in ASD and opens the way for future therapeutic approaches. Maternal immune activation, which can be caused by congenital infection, may lead to a fever response, production of interleukins (IL-1, IL-6, TNF, IL-17), immunoglobulins or anti neuronal autoantibodies that may alter or cross the placenta to affect the fetal neurodevelopment directly or through a de novo fetal pro-inflammatory state. In healthy individuals gut microbiota can influence the brain homeostasis through secretion of neurotransmitters (such as GABA), pro-inflammatory cytokine production and vagal nerve regulation. In individuals with autism spectrum disorders, gastrointestinal symptoms are frequent. The microbiota taxonomic composition is impaired and intestinal permeability is not provided (leaky gut) allowing anaerobic bacteria to proliferate and leading to an alteration of short-chain fatty acids metabolism.

Figure 1 .

 1 Figure1. Maternal immune activation and its effect on fetal neurodevelopment. Maternal immune activation, which can be caused by congenital infection, may lead to a fever response, production of interleukins (IL-1, IL-6, TNF, IL-17), immunoglobulins or anti neuronal autoantibodies that may alter or cross the placenta to affect the fetal neurodevelopment directly or through a de novo fetal pro-inflammatory state.

Figure 2 .

 2 Figure 2. Mechanisms of brain-gut axis in autism spectrum disorders.

Disclosure of interest

The authors declare that they have no competing interest.