Ophélie De Rycke

Anne-Laure Védie

Giovanni Guarneri

Frédéric Nin

Céline De Flori

Olivia Hentic

Salim Idri

Alain Sauvanet

Vinciane Rebours

Jérôme Cros

Anne Couvelard

Philippe Ruszniewski

Dr Louis De Mestier
email: louis.demestier@aphp.fr

O-positive blood type is associated with prolonged recurrence-free survival following curative resection of pancreatic neuroendocrine tumors

Keywords: pancreas, neuroendocrine tumors, prognosis, blood type ABO antigen, Carcinoid, Prognosis, Surgery

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pancreatic neuroendocrine tumors (PanNET) are relatively rare neoplasms, although their incidence has increased in the past decades, probably due to better detection with improved imaging techniques and increased awareness by professionals [START_REF] Hallet | Exploring the rising incidence of neuroendocrine tumors: A population-based analysis of epidemiology, metastatic presentation, and outcomes: Neuroendocrine Tumor Epidemiology[END_REF][START_REF] Dasari | Trends in the Incidence, Prevalence, and Survival Outcomes in Patients With Neuroendocrine Tumors in the United States[END_REF]. Surgical resection is the reference treatment for localized PanNET because it provides a good chance of cure [START_REF] Falconi | ENETS Consensus Guidelines Update for the Management of Patients with Functional Pancreatic Neuroendocrine Tumors and Non-Functional Pancreatic Neuroendocrine Tumors[END_REF]. Nevertheless, recurrence occurs in 15% to 20% of patients following curative-intent surgery [START_REF] Ausania | Can we predict recurrence in WHO G1-G2 pancreatic neuroendocrine neoplasms? Results from a multiinstitutional Spanish study[END_REF][START_REF] Kim | Time-trend and recurrence analysis of pancreatic neuroendocrine tumors[END_REF]. The main features influencing postoperative PanNET recurrence are tumor size, Tstage, lymph-node metastases, vascular invasion, Ki67 proliferation index and tumor grade [START_REF] Falconi | ENETS Consensus Guidelines Update for the Management of Patients with Functional Pancreatic Neuroendocrine Tumors and Non-Functional Pancreatic Neuroendocrine Tumors[END_REF][START_REF] Ausania | Can we predict recurrence in WHO G1-G2 pancreatic neuroendocrine neoplasms? Results from a multiinstitutional Spanish study[END_REF][START_REF] Kim | Time-trend and recurrence analysis of pancreatic neuroendocrine tumors[END_REF][START_REF] Rindi | Competitive Testing of the WHO 2010 versus the WHO 2017 Grading of Pancreatic Neuroendocrine Neoplasms: Data from a Large International Cohort Study[END_REF].

The ABO blood type system is defined by the expression of human blood group antigens A, B, AB or O on the surface of cells, and the presence of anti-B and/or anti-A antibodies in serum. Although the precise underlying mechanisms are not fully understood, the ABO blood type could play a role in the development and progression of cancer. Indeed, numerous studies have reported an association between non-O blood type and negative outcomes in various types of malignancies [START_REF] Zhang | ABO blood groups and risk of cancer: a systematic review and meta-analysis[END_REF][START_REF] Franchini | The prognostic value of ABO blood group in cancer patients[END_REF][START_REF] Iodice | ABO blood group and cancer[END_REF] and patients with O blood-type were found to have a significantly decreased risk of death in a large cohort of patients operated on for pancreatic ductal adenocarcinoma(HR 0.78, p=0.037) [START_REF] Rahbari | AB0 blood group and prognosis in patients with pancreatic cancer[END_REF].

On the other hand, the impact of ABO type has not been extensively explored in PanNET. Surprisingly, it has been suggested that O blood type is associated with a higher rate of PanNET in patients with type 1 multiple endocrine neoplasia (MEN1) [START_REF] Weisbrod | Association of Type-O Blood with Neuroendocrine Tumors in Multiple Endocrine Neoplasia Type 1[END_REF] and von Hippel-Lindau disease (VHL) [START_REF] Weisbrod | Association of Type O Blood with Pancreatic Neuroendocrine Tumors in Von Hippel-Lindau Syndrome[END_REF]. Nevertheless, these results were contested in other studies evaluating either patients with MEN1 [START_REF] Nell | No Association of Blood Type O With Neuroendocrine Tumors in Multiple Endocrine Neoplasia Type 1[END_REF][START_REF] Bartsch | Higher Risk of Aggressive Pancreatic Neuroendocrine Tumors in MEN1 Patients With MEN1 Mutations Affecting the CHES1 Interacting MENIN Domain[END_REF] or sporadic PanNET [START_REF] Iodice | ABO blood group and cancer[END_REF][START_REF] Ben | Association between ABO blood types and sporadic pancreatic neuroendocrine tumors in the Chinese Han population[END_REF]. Moreover, the prognostic role of the ABO type has not been explored in PanNET.

Thus, we performed a retrospective study to evaluate the impact of the ABO and Rhesus blood types on the risk of recurrence in patients who underwent curative surgical resection of PanNET.

Patients and Methods

Patients

All consecutive patients operated on for non-metastatic PanNET in Beaujon University Hospital from January 1 st , 2003 to January 1 st , 2018 were included in the study. Pathological proof was available in all cases. Patients with poorly-differentiated carcinoma, mixed neuroendocrine-non-neuroendocrine neoplasms or with metastatic disease identified before or during surgery were excluded.

All patients were operated on in the same surgery unit with standardized techniques and monitoring. The preoperative work-up included contrast-enhanced cross-sectional imaging (CT-scan or MRI) and somatostatinreceptor imaging. The postoperative follow-up was standardized and included clinical, biological and morphological examinations every 6 months for 2 years then yearly thereafter.

Data collection

We collected retrospective epidemiological, surgical, pathological, and follow-up data in all patients. Tumor grade was evaluated retrospectively according to the 2019 WHO classification (G1 with a Ki-67 index < 3%, G2 with a Ki-67 index of 3-20%, and G3 with a Ki-67 index > 20%) [START_REF]WHO Classification of Tumours[END_REF]. All PanNET stages were determined retrospectively according to the 2017 UICC Tumor-Nodes-Metastasis classification [START_REF] Bierley | International Union against Cancer[END_REF].

Blood type (A, B, AB or O), rhesus (D, C, E, c, e) and Kell systems were determined preoperatively in all patients and centrally collected in the Etablissement Français du Sang database of Beaujon University Hospital.

Patients without complete blood type data were excluded from the study.

Data collection was performed after receiving patient agreement according to the Declaration of Helsinki and the study was approved by the Institutional Review Board (IRB 00006477) of Paris University, France.

Statistical analyses

Quantitative variables were expressed as medians (25-75 interquartile range) and compared using the Mann-Whitney test. Qualitative variables were expressed as frequencies (percentages) and compared using the Chisquare test. The distribution of ABO groups in our cohort was compared to that in the French population [START_REF][END_REF].

The primary endpoint was recurrence-free survival (RFS), which was defined as the time between the date of PanNET surgical resection and of recurrence (local, regional or distant, identified during regular clinical and morphological follow-up) or that date of the last news. Patients without recurrence at the final follow-up visit were censored. The end date of the study was July 1, 2019. Survival rates were estimated using the Kaplan-Meier method and compared using the log-rank test. Factors associated with RFS were evaluated using the Cox proportional hazard models. All relevant baseline variables were entered into a univariate analysis, and then all noncollinear variables with p values < 0.15 were entered into a multivariate analysis. All analyses were twosided. P values < 0.05 were considered to be significant. Analyses were performed with SPSS® (version 20; IBM) software.

Results

Patients

Ninety-eight out of 398 patients operated on for PanNET between 2003 and 2018 were excluded (72 with metastases, 11 with non-metastatic poorly-differentiated carcinoma or mixed neuroendocrine-nonneuroendocrine neoplasms and 15 patients without blood type characterization). Thus, 300 patients were included in the study.

The F/M ratio was 1.34 and the median age was 53.6 years old (Table 1). Forty patients had an oncogenetic syndrome (24 with MEN1, 13 with VHL, 2 with tuberous sclerosis and 2 with type 1 neurofibromatosis). A functional syndrome existed in 53 patients (17.7%), including hypersecretion of insulin, gastrin, glucagon, thyrocalcitonin and parathormone-related protein in 40, 9, 2, 1 and 1 patients, respectively. Nearly half of the patients underwent parenchyma-sparing surgical procedures (enucleation or median pancreatectomy).

Pancreaticoduodenectomy and distal pancreatectomy were performed in 22.7% and 30.3%, respectively.

The median tumor size at pathological examination of the resected specimens was 2 cm, and a proportion of patients presented with factors usually associated with recurrence. Notably, 37.7% of PanNET were G2 or G3, 19.4% were pT3-pT4 and 25.7% had regional lymph-node metastases.

Blood type characterization and association with tumor features

The most prevalent blood types were O (45%) and A (42%) (Table 2). As expected, the B and AB types were much rarer (10% and 3%, respectively). Eighty-three percent of patients were positive for Rhesus D (RhD+).

There was no difference in the distribution of ABO blood types between our cohort and the French population, for all blood types (p=0.95) or O RhD+ vs. other types (p=0.74) (Table 3).

There was no significant correlation between blood type and tumor features: functioning syndrome, oncogenetic syndrome, ki67, tumor size and stage, lymph node status and pathological characteristics. Patients with O RhD+ type tended to have a lower median Ki67 (1% [START_REF] Hallet | Exploring the rising incidence of neuroendocrine tumors: A population-based analysis of epidemiology, metastatic presentation, and outcomes: Neuroendocrine Tumor Epidemiology[END_REF][START_REF] Dasari | Trends in the Incidence, Prevalence, and Survival Outcomes in Patients With Neuroendocrine Tumors in the United States[END_REF][START_REF] Falconi | ENETS Consensus Guidelines Update for the Management of Patients with Functional Pancreatic Neuroendocrine Tumors and Non-Functional Pancreatic Neuroendocrine Tumors[END_REF][START_REF] Ausania | Can we predict recurrence in WHO G1-G2 pancreatic neuroendocrine neoplasms? Results from a multiinstitutional Spanish study[END_REF] vs. 2% [START_REF] Hallet | Exploring the rising incidence of neuroendocrine tumors: A population-based analysis of epidemiology, metastatic presentation, and outcomes: Neuroendocrine Tumor Epidemiology[END_REF][START_REF] Dasari | Trends in the Incidence, Prevalence, and Survival Outcomes in Patients With Neuroendocrine Tumors in the United States[END_REF][START_REF] Falconi | ENETS Consensus Guidelines Update for the Management of Patients with Functional Pancreatic Neuroendocrine Tumors and Non-Functional Pancreatic Neuroendocrine Tumors[END_REF][START_REF] Ausania | Can we predict recurrence in WHO G1-G2 pancreatic neuroendocrine neoplasms? Results from a multiinstitutional Spanish study[END_REF], p=0.063) and Rhesus c positive patients tended to have fewer lymph-node metastases (23.2% vs. 37.2%, p=0.057).

Recurrence-free survival analyses

The median total postoperative follow-up was 43.9 months (17.0-77.8). Recurrence occurred in 50 patients (16.7%). The 5-and 10-year RFS rates were 77.6 ± 3.1% and 67.1 ± 5.6%, respectively.

O RhD+ type was associated with a decreased risk of recurrence (HR= 0.51, 95% CI [0.27-0.97], p=0.035) on univariate analysis (Figure 1 and Table 4). The 5-and 10-year RFS rates were 85.4 ± 3.9% and 71.2 ± 13.4% in O RhD+ patients, versus 72.4 ± 4.4% and 62.6 ± 5.8% otherwise, respectively. Conversely, no significant survival impact was identified with A type (HR 1,32, 95%CI [0.76-2.31], p=0,32), B type (HR 1,67, 95% CI [0.75-2.73], p=0,20) or AB type (HR 0,046, 95% CI [0-27.12], p=0,34), alone or combined with RhD (Table S1).

The O RhD+ blood group was independently associated with a significantly decreased risk of recurrence on multivariable analysis adjusted for the main prognostic factors and the existence of an oncogenetic syndrome (HR 0.34, 95% CI [0.15-0.75], p=0.007) (Table 4). Older age (HR 1.04, 95% CI [1.01-1.06], p=0.010), higher Ki67 (HR 1.11, 95% CI [1.06-1.16], p<0.001), larger PanNET size (HR 1.23, 95% CI [1.11-1.37], p<0.001) and lymph-node metastases (HR 3.50, 95% CI [1.75-7.01], p<0.001) were also independent factors of a poor prognosis, while there was a statistical tendency for a poorer prognosis with the presence of a functional syndrome and R1 margin status (Table 4).

Discussion

To the best of our knowledge, this is the first study to show that ABO blood type has a significant prognostic impact in patients with PanNET. Our results show that O RhD+ patients may have a decreased risk of PanNET recurrence following curative-intent resection, independent from classic prognostic factors, which are mainly related to tumor characteristics.

The classification of human blood based on the inherited properties of red blood cells (erythrocytes) is

determined by the presence or absence of the antigens A and B, which are carried on the surface of the red cells.

Persons may thus have type A, type B, type O, or type AB blood. The ABO genes have three main allele forms: A, B, and O. Alleles A and B encode for a glycosyltransferase that enzymatically modifies the H antigen to create the A and B antigens. ABO blood groups are determined by the presence of the A and B antigens on the surface membrane of erythrocytes and anti-B and/or anti-A antibodies in serum. Conversely, the O allele encodes for a nonfunctional glycosyltransferase and is characterized by both anti-A and anti-B antibodies in serum. The expression of glycoproteins forming these blood groups is not only found in erythrocytes but can also be found in normal tissue throughout the body, including the pancreas. In addition, blood group antigens are expressed on the surface of malignant digestive tumor cells, although their expression is frequently altered [START_REF] Itzkowitz | Cancer-associated alterations of blood group antigen expression in the human pancreas[END_REF][START_REF] Pour | Expression of blood group-related antigens ABH, Lewis A, Lewis B, Lewis X, Lewis Y, and CA 19-9 in pancreatic cancer cells in comparison with the patient's blood group type[END_REF].

Numerous studies and meta-analyses have reported an association between non-O blood type and an increased risk of various malignancies [START_REF] Zhang | ABO blood groups and risk of cancer: a systematic review and meta-analysis[END_REF][START_REF] Franchini | The prognostic value of ABO blood group in cancer patients[END_REF], including pancreatic [START_REF] Iodice | ABO blood group and cancer[END_REF][START_REF] Wolpin | ABO blood group and the risk of pancreatic cancer[END_REF], renal [START_REF] Joh | ABO blood group and risk of renal cell cancer[END_REF], gastric [START_REF] Wang | ABO blood group system and gastric cancer: a case-control study and meta-analysis[END_REF], biliary [START_REF] Zhou | Evaluation of risk factors for extrahepatic cholangiocarcinoma: ABO blood group, hepatitis B virus and their synergism[END_REF], breast [START_REF] Miao | Influence of ABO blood group and Rhesus factor on breast cancer risk: a meta-analysis of 9665 breast cancer patients and 244,768 controls[END_REF] and nasopharyngeal carcinomas [START_REF] Sheng | ABO blood group and nasopharyngeal carcinoma risk in a population of Southeast China[END_REF]. Additional studies have reported a positive prognostic impact of the O blood type. Rahbari et al. [START_REF] Rahbari | AB0 blood group and prognosis in patients with pancreatic cancer[END_REF] reported its association with a decreased risk of death (HR 0.78, p=0.037) in 627 patients operated on for an exocrine pancreatic carcinoma. Similarly, non-O blood type was associated with increased risks of death (HR 1.68, p=0.004) and recurrence (HR 1.53, p=0.065) in 900 patients undergoing renal-cell carcinoma resection [START_REF] Kaffenberger | ABO blood group is a predictor of survival in patients undergoing surgery for renal cell carcinoma[END_REF], as well as increased risks of death and recurrence in 333 patients operated on for non-small cell lung cancer [START_REF] Fukumoto | The ABO blood group is an independent prognostic factor in patients with resected non-small cell lung cancer[END_REF]. Thus our results are consistent with the results in the literature.

Although ABO gene products may play a role in carcinogenesis, the precise underlying mechanisms have not been fully investigated. Altered ABO antigen expression in tumor cells has been associated with the development and prognosis of cancer [START_REF] Franchini | The prognostic value of ABO blood group in cancer patients[END_REF]. The ABO-glycosyltransferase or the glycosylation pattern itself may also play a role. Indeed, changes in glycosylation favor the acquisition of biological features essential for tumor progression [START_REF] Pinho | Glycosylation in cancer: mechanisms and clinical implications[END_REF]. Pro-oncogenic glycosyltransferase modification may occur only in A and B blood types, since O blood type lacks functional glycosyltransferase. Indeed, Wolpin et al. [START_REF] Wolpin | Variant ABO blood group alleles, secretor status, and risk of pancreatic cancer: results from the pancreatic cancer cohort consortium[END_REF] showed that glycosyltransferase activity had a greater prognostic value than blood type itself. The risk of pancreatic cancer in patients with the A² phenotype characterized by reduced glycosyltransferase activity and fewer complex A antigens on the cell surface, was similar to that in O-type patients, and both of these groups had a decreased risk compared to those with an A 1 phenotype, whose enzymatic activity is normal (p=0.004). Moreover, preclinical data have suggested that A and B antigens mediate resistance to apoptosis and escape from immune surveillance [START_REF] Marionneau | Expression of histo-blood group A antigen increases resistance to apoptosis and facilitates escape from immune control of rat colon carcinoma cells[END_REF], suggesting that A and B antibodies have a protective function in patients with O blood type [START_REF] Fukumoto | The ABO blood group is an independent prognostic factor in patients with resected non-small cell lung cancer[END_REF]. Another hypothesis is that ABO gene products are associated with alterations in tissue inflammatory state (TNFα) and intercellular adhesion (ICAM1), both mechanisms that play a major role in tumor progression [START_REF] Paré | Novel association of ABO histoblood group antigen with soluble ICAM-1: results of a genome-wide association study of 6,578 women[END_REF]. Although the ABO system has been associated with the occurrence and prognosis of pancreatic exocrine cancer (and other malignancies), there are very few studies in PanNET. In a study including 181 patients with von Hippel-Lindau disease (VHL), O blood type was associated with the occurrence of PanNET and larger tumor size [START_REF] Weisbrod | Association of Type O Blood with Pancreatic Neuroendocrine Tumors in Von Hippel-Lindau Syndrome[END_REF]. However this study may not be fully relevant because PNET were not pathologically confirmed but defined as any solid or complex pancreatic lesions. This may explain in part, the unusually high prevalence of "PanNET" in this VHL cohort (72%, vs. 15-20% in the literature), which may have included a proportion of misdiagnosed patients. This same team published a similar study in 105 patients with type 1 multiple endocrine neoplasia (MEN1) [START_REF] Weisbrod | Association of Type-O Blood with Neuroendocrine Tumors in Multiple Endocrine Neoplasia Type 1[END_REF]. O blood type was associated with the occurrence of neuroendocrine tumors of all types (duodenum, stomach, pancreas, lung, thymus). The prevalence of NET was 53% in O-type and 28% in non-Otype MEN1 patients (p=0.01). Once again, the pathological confirmation of these NET was not systematic.

However, these results were disputed in other studies. In one study the ABO blood type of 200 MEN1 patients from the Dutch National MEN1 database was not correlated to the occurrence of NET [START_REF] Nell | No Association of Blood Type O With Neuroendocrine Tumors in Multiple Endocrine Neoplasia Type 1[END_REF]. The prevalence of NET was 66% and 69% in patients with O or non-O blood groups, respectively (p=0.67). Another study of 62 patients also did not confirm the association between blood type and neuroendocrine tumors in MEN1 patients [START_REF] Bartsch | Higher Risk of Aggressive Pancreatic Neuroendocrine Tumors in MEN1 Patients With MEN1 Mutations Affecting the CHES1 Interacting MENIN Domain[END_REF]. No association was reported between blood group and the occurrence of PanNET in one study that included more than 15,000 subjects, although the number of patients with PanNET was limited (n=14) [START_REF] Iodice | ABO blood group and cancer[END_REF]. The same results were reported in a Chinese case-control study including 387 patients with PanNET [START_REF] Ben | Association between ABO blood types and sporadic pancreatic neuroendocrine tumors in the Chinese Han population[END_REF].

In conclusion, patients with O RhD+ blood type have a significantly decreased risk of recurrence following PanNET curative-intent resection, independent from classical prognostic features. Thus, we confirm that tumorrelated features are highly important to evaluate the risk of recurrence in patients with PanNET, and show for the first time that patient-related characteristics such as blood type should also be taken into account and could be used to adapt the postoperative follow-up in these patients.

Figure 1 .

 1 Figure 1. Comparison of recurrence-free survival following PanNET resection, between patients with or without O-RhD+ blood type.

Table 1 .

 1 Characteristics of 300 patients operated on for PanNET 14

	6 (45-63.7)

Table 2 .

 2 Blood type characterization of 300 patients operated on for PanNET

		Univariate		Multivariate
		HR	95% CI	p-value	HR	95% CI	p-value
	Age (each additional
		1.02	1.00-1.05	0.048	1.04	1.01-1.06	0.010
	year)
	Oncogenetic syndrome 0.49	0.19-1.24	0.133	0.55	0.17-1.81	0.326
	Functioning syndrome 1.67	0.83-3.35	0.148	2.33	0.91-5.98	0.078
	Ki67 (each additional
		1.15	1.10-1.20	<0.001	1.11	1.06-1.16	<0.001
	%)
	Size (each additional
		1.28	1.20-1.38	<0.001	1.23	1.11-1.37	<0.001
	cm)
	N1 status	4.55	2.57-8.05	<0.001	3.50	1.75-7.01	<0.001
	R1 margins	7.03	3.21-15.38	<0.001	2.16	0.87-5.32	0.097
	O-RhD+ group	0.51	0.27-0.97	0.035	0.34	0.15-0.75	0.007

Table 4 .

 4 Univariate and multivariate Cox proportional hazard analyses of factors influencing the risk of recurrence following pancreatic neuroendocrine tumors curative-intent surgical resection. The following variables had p<0.15 at univariate analysis but were collinear to other variables and were thus not entered into the multivariate model: multiple tumors, incidentalomas, tumor grade, T stage, vascular emboli and perineural invasion.

Acknowledgements

The authors would like to thank Mrs Dale Roche-Lebrec for technical assistance in the preparation of the manuscript.

Funding sources

None

Disclosure statement

The authors have no potential conflicts of interest to declare.

Statement of Ethics

Data collection was performed following patient agreement according to the declaration of Helsinki and Institutional Review Board approval (CEERB Paris Nord, IRB n° 00006477-15-073).

Blood type

Frequency in the present study (%)

Frequency in French population (%) [START_REF][END_REF] O-RhD-


Laila Somoue

Herve Demarcq

Ahmed Makaoui

Karim Hilmi

Abdelouahed Omar Ettahiri

Ben Mhamed

Aziz Agouzouk

Tarik Baibai

Said Jamila Larissi

Said Charib

Abdelkrim Kalmouni

Mohamed Laabir
email: mohamed.laabir@umontpellier.fr

Influence of Ocean-Lagoon exchanges on spatio-temporal variations of phytoplankton assemblage in an Atlantic Lagoon ecosystem (Oualidia, Morocco)

Keywords: Oualidia Lagoon, Phytoplankton, Environmental factors, African Atlantic coast, Ocean -Lagoon exchange Dinophysis, Ostreopsis, Karenia, Coolia

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Coastal Lagoons are among the most productive marine ecosystems, however they remain fragile and are often exposed to multiple natural and anthropogenic constraints [START_REF] Kjerfve | Coastal lagoon processes[END_REF]. Lagoons are highly productive areas that are located in the transitional areas at the land-ocean boundary (Perez-Ruzafa et al., 2012). These areas have become important because they provide the key to understanding the general dynamics of the seas they are connected with. Their existence and their influence on the coastal zones have become a fundamental study topic in many disciplines [START_REF] Basset | A unifying approach to understanding transitional waters: Fundamental properties emerging from ecotone ecosystems[END_REF]. A better knowledge of the functioning of these ecosystems is required to ensure their sustainable management [START_REF] Rharbi | Caractéristiques hydrologiques et écologiques de la lagune de Oualidia, milieu paralique de la côte atlantique marocaine[END_REF][START_REF] Rosa | Temporal variability of the mass exchanges between the main inlet of Ria Formosa lagoon (southwestern Iberia) and the Atlantic Ocean[END_REF]. The Oualidia Lagoon, located on the Atlantic coast of Morocco (Africa), was registered as a RAMSAR site (International convention of wetlands conservation) since 2005 [START_REF] Maanan | A long-term record of land use change impacts on sediment in Oualidia lagoon, Morocco[END_REF] because of its great ecological and socioeconomic importance. It holds an increasing touristic activity and it is one of the most important Moroccan zones for oyster farming since 1950 [START_REF] Rharbi | Caractéristiques hydrologiques et écologiques de la lagune de Oualidia, milieu paralique de la côte atlantique marocaine[END_REF]. Other socioeconomic activities in this area includes intensive agriculture, livestock, fishing, and salt mining. Local residents exploit mussels (Perna perna and Mytilus galloprovincialis) fixed on the rocks and reef flats and collect clams (Ruditapes decussatus) [START_REF] Maanan | A long-term record of land use change impacts on sediment in Oualidia lagoon, Morocco[END_REF][START_REF] Jayed | Accumulation of Organochlorines in the European Clam (Ruditapes decussatus) and Sediment of the Oualidia Lagoon (Morocco)[END_REF]. Phytoplankton community in coastal Lagoons are a major component of the food web structure and functioning and supply the major source of organic carbon [START_REF] Gaikwad | Diversity of Phytoplankton and zooplankton with respect to pollution status of river Tapi in North Maharastra region[END_REF]. Phytoplankton sensitivity to environmental changes and the fluctuation of its specific composition are precious indicators of alterations of the whole ecosystem [START_REF] Devassy | Phytoplankton community structure and succession in tropical estuarine complex (Central West Cost of India)[END_REF]. Phytoplankton species diversity is sensitive to environmental parameters, a slight modification in the state of the environment could modify this diversity (Ghsoh et al., 2012). As an example, nutrients supply, driven either from land or from the ocean through tidal influence have been shown to influence the phytoplankton activity, and consequently the functioning of communities in Lagoons [START_REF] Sylaios | Hydrology and nutrient enrichment at two coastal lagoon systems in northern Greece[END_REF].

To our knowledge, studies on phytoplankton in African Atlantic coastal ecosystems are rare.

The only study on qualitative and quantitative distribution of phytoplankton in Oualidia Lagoon was carried out from January to December 1997 by [START_REF] Bennouna | Étude des populations phytoplanctoniques de la lagune de Oualidia (Maroc), dinoflagellés potentiellement nuisibles[END_REF]. They showed that diatoms were the dominant organisms at most times (70 to 98% of the phytoplankton population). However, the performed studies in Oualidia focused mainly on Harmful Algal Blooms (HABs) species [START_REF] Bennouna | Etude du phytoplancton du complexe lagunaire Oualidia-Sidi Moussa[END_REF][START_REF] Rharbi | Importance des paramètres hydrologiques et phytoplanctoniques sur la croissance de l'huître Crassostrea gigas en élevage dans la lagune de Oualidia[END_REF][START_REF] Zourarah | Les processus côtiers actuels et leur impact sur l'environnement littoral des Doukkala (côte atlantique marocaine) : Approche hydrodynamique, Morphologique, Sédimentologique et Géochimique[END_REF] and were carried out in a limited number of stations. Taleb et al. (2002) showed that maximum Paralytic Shellfish Poisoning (PSP) toxin level recorded in mussel from Oualidia Lagoon during the November 1994 was up to 2500 µg Eq STX.100 g -1 of shellfish meat which is much higher than the regulatory international threshold of 80 µg Eq STX 100 g -1 of shellfish meat. Both the dinoflagellates Alexandrium minutum and Gymnodinium catenatum were suspected to be the causative species but without formal identification. [START_REF] Bennouna | Eau colorée à Lingulodinium polyedrum (Stein) Dodge dans une zone aquacole du littoral du Doukkala (Atlantique marocain)[END_REF] reported the occurrence of the dinoflagellate Lingulodinium polyedrum causing red tides along the Moroccan Atlantic coast including Oualidia Lagoon in July 1999. More recently, [START_REF] Daghor | The first occurrence of Red Tide caused by Karenia sp. in the Atlantic Moroccan Coast (Oualidia Lagoon)[END_REF] reported an intense bloom of the dinoflagellate Karenia sp. the Oualidia Lagoon with concentrations up to 1.04 × 10 7 cells L -1 . Here we conducted a field study covering for the first time the entire Lagoon from downstream to upstream during four seasons in 2011 with three main objectives : 1) to highlight the diversity of microphytoplankton species of the Oualidia Lagoon on a seasonal basis, 2) to investigate the effect of environmental factors on the spatio-temporal variation of phytoplankton communities and 3) to highlight the influence of ocean -Lagoon exchange on spatio-temporal variations of phytoplankton assemblage in this African Atlantic Lagoon ecosystem.

Material and Methods

Study area

The Oualidia Lagoon located 76 km south of El Jadida and 67 km north of Safi (Fig. 1) is one of the most important coastal ecosystems on the Moroccan Atlantic coast. This Lagoon is 7 km long and 0.5 km wide, with a total area of 3.5 km 2 [START_REF] Hilmi | Etude courantologique de la lagune de Oualidia (Maroc) en automne[END_REF]2009 ;[START_REF] Maanan | A long-term record of land use change impacts on sediment in Oualidia lagoon, Morocco[END_REF] and widely connected with the Ocean through a major inlet (150 m wide and 2 m deep) and a secondary pass active in open sea during the highest tides [START_REF] Mejjad | Sediment geochronology and geochemical behavior of major and rare earth elements in the Oualidia Lagoon in the western Morocco[END_REF][START_REF] Maanan | A long-term record of land use change impacts on sediment in Oualidia lagoon, Morocco[END_REF]. The Lagoon is composed of a network of very narrow dendritic channels, connected to a main channel of 6.5 km long and 2 m depth in average with a maximum of 5 m during high tides [START_REF] Bidet | Etude sédimentologique de la lagune de Oualidia (Maroc)[END_REF]. The intertidal zone (75% of the Lagoon surface) is predominantly sandy with rare slicks. The upper part of the Lagoon (0.6 km 2 ) is composed of salt marshes. The Oualidia climate is arid to semi-arid, maximum temperatures of up to 40°C in summer were recorded when an Eastern warm wind (Chergui) blows. However, generally, the mean daily atmospheric temperature varies between 21°C and 22°C in summer and between 14°C and 15°C in winter [START_REF] Bennouna | Eau colorée à Lingulodinium polyedrum (Stein) Dodge dans une zone aquacole du littoral du Doukkala (Atlantique marocain)[END_REF]. The low and seasonal rainfalls account for 1% of the fresh water entering the Lagoon and the rest is coming from groundwater. The annual cumulative rainfall in 2011 are 442.3 mm (maximum of 331.9 mm during January-June 2011 and 110.4mm during July-December; data from National Meteorological Services). The annual hygrometric deficit was 650 mm. The predominant wind directions are WSW to NW during the wet season and NNE to NE during the dry season [START_REF] Zourarah | Les processus côtiers actuels et leur impact sur l'environnement littoral des Doukkala (côte atlantique marocaine) : Approche hydrodynamique, Morphologique, Sédimentologique et Géochimique[END_REF][START_REF] Zourarah | Fifty-year sedimentary record of heavy metal pollution in the lagoon of Oualidia (Moroccan Atlantic coast)[END_REF][START_REF] Mejjad | Sediment geochronology and geochemical behavior of major and rare earth elements in the Oualidia Lagoon in the western Morocco[END_REF]. The hydrological regime of the Lagoon is tightly associated with the tidal rhythm [START_REF] Orbi | Lagoon ecosystem study trough two cases: Oualidia (Atlantic) and Nador (Mediterranean) -Morocco[END_REF]Hilmi et al., 2005[START_REF] Hilmi | Hydrodynamisme de la lagune de Oualidia (Maroc) durant l'été et l'automne 2005[END_REF]. A high nutrient input is favored by rising tides in the Lagoon, which increases organic production and improves aquaculture yields [START_REF] Maanan | A long-term record of land use change impacts on sediment in Oualidia lagoon, Morocco[END_REF]. [START_REF] Makaoui | L'upwelling de la côte atlantique du Maroc entre 1994 et[END_REF] reported that he Lagoon is more influenced by the oceanic input of nutrients particularly the case of PO4 in reason of upwelling events. [START_REF] Mejjad | Sediment geochronology and geochemical behavior of major and rare earth elements in the Oualidia Lagoon in the western Morocco[END_REF] suggested that seasonal and diurnal nutrient variability in the Oualidia Lagoon results from the influence of the water continental inputs, precipitation and evaporation regimes as well as oceanic-Lagoon exchanges. There are no river discharging into the Lagoon, but several authors have mentioned the existence of underground freshwater seepage probably in the first part of the Lagoon and upstream [START_REF] Carruesco | Genèse et évolution à l'holocène de trois lagunes de la façade atlantique : Moulay Bousselham, Oualidia (Maroc) et Arcachon (France)[END_REF][START_REF] Hilmi | Etude courantologique de la lagune de Oualidia (Maroc) en automne[END_REF][START_REF] Hilmi | Hydrodynamisme de la lagune de Oualidia (Maroc) durant l'été et l'automne 2005[END_REF][START_REF] Rharbi | Caractéristiques hydrologiques et écologiques de la lagune de Oualidia, milieu paralique de la côte atlantique marocaine[END_REF].

Several authors [START_REF] Hilmi | Etude courantologique de la lagune de Oualidia (Maroc) en automne[END_REF]2009[START_REF] Koutitonsky | L'étude du comportement hydrosédimentaire du système lagunaire Oualidia par la modélisation mathématique, Volet 1: synthèse des données et simulation de la réfraction des houles[END_REF]2012) have studied the tidal regime and the water circulation in the Oualidia Lagoon. They concluded that this marine system is governed by the semi-diurnal tide (M2 tide) which dominates in the Atlantic Ocean. The tide's amplitude reaches around 3 m at the entrance of the Lagoon during the spring tides, and around 0.8 m during the neap tides. Due to the complex topography and the small depths observed upstream of the Lagoon, tides are asymmetric in nature and the amplitude of M2 tide is decreasing due to the friction on the bottom. On average, the maximum and minimum depths in the Lagoon are 5 m and 1.5 m, respectively [START_REF] Bennouna | Eau colorée à Lingulodinium polyedrum (Stein) Dodge dans une zone aquacole du littoral du Doukkala (Atlantique marocain)[END_REF]. A maximum of 77% or 52% of the channel volume is flushed during one spring or neap tide, respectively [START_REF] Hilmi | Etude courantologique de la lagune de Oualidia (Maroc) en automne[END_REF]. [START_REF] Carruesco | Genèse et évolution à l'holocène de trois lagunes de la façade atlantique : Moulay Bousselham, Oualidia (Maroc) et Arcachon (France)[END_REF] estimated a renewal of 89% or 72% of the Lagoon waters during one spring or neap tidal cycle, respectively. Using 2D hydrodynamic model, [START_REF] Hilmi | Etude courantologique de la lagune de Oualidia (Maroc) en automne[END_REF] found that tidally averaged renewal time for the whole Lagoon was 7 days, while the local renewal time at the upstream end of the Lagoon is 25 days. Oyster farming is the most widespread aquaculture activity in the Oualidia Lagoon.

The average annual production of oysters is estimated to be 250 tons [START_REF] Rharbi | Importance des paramètres hydrologiques et phytoplanctoniques sur la croissance de l'huître Crassostrea gigas en élevage dans la lagune de Oualidia[END_REF].

Sampling and measurements

Six stations along Oualidia Lagoon were sampled monthly from downstream to upstream during representative months of the four seasons of 2011: winter (February), spring (May), summer (August) and fall (October) (Fig. 1). Water sampling was performed using an hydrobiological bottle at subsurface (-0.5 m depth). The maximum depths of the stations ranged between 0.5 to 3.5m at low tide and 2 to 6.5m at high tide.

Abiotic factors

Temperature, salinity and nutrients (nitrate, ammonium and phosphates) were measured in all stations during low and high tides. Temperature and salinity were determined using a probe WTW LF195. 500 ml of seawater was filtered (0.45 µm) and conserved at -20 °C until the analyses of nutrients performed spectrophotometrically according to the method of [START_REF] Aminot | Hydrologie des écosystèmes marins. Paramètres et analyses[END_REF].

Biotic factors

Chlorophyll a (Chl-a) measurements were performed from 500 ml seawater samples filtered throughout 47 µm Whatman GF/F filter during low and high tides. Chl-a was extracted from filters immerged in 10 ml 90 % acetone for 24 h in the dark at -4 °C [START_REF] Strickland | A practical handbook of seawater analysis[END_REF]Parsons, 1972, Linder,1974), and analyzed using a fluorometer 10-AU (Turner Design).

Determinations of phytoplankton species and abundances were made from 100 ml of sea water fixed using Lugol's iodine. Phytoplankton counts were done for samples of only high tides. Phytoplankton counts were carried out according to the Utermöhl (1958) method and the determination of the different taxa was made by inverted light microscopy (Nikon) with appropriate identification keys (Trégouboff and Rose, 1957;[START_REF] Nezan | Guide pratique à l'usage des analystes du phytoplancton[END_REF][START_REF] Tomas | Identifying Marine Phytoplankton[END_REF][START_REF] Botes | Phytoplankton Identification Catalogue Saldanha Bay, South Africa[END_REF]. Phytoplankton abundance was expressed in cells L -1 . The frequency of taxa, expressed in%, was calculated using formula : F = (μi / μT) *100 (μi = number of samples in which species is present and μT = total number of samples).

Data analyses

Each station was characterized by a specific assemblage of microphytoplankton described by its species richness (RS) index (number of species recorded), total density (D), Shannon diversity H index [START_REF] Shannon | The mathematical theory of communication[END_REF].

Species diversity (H) was calculated using Shannon's formula:

H= ∑ ni/N S i=1 *log 2 ni/N
Where, S = specific richness (number of species); ni = abundance of species i and N = total abundance of all species.

PCA and Co-inertia analysis were performed with the ADE4 package in the R software [START_REF] Dray | The Ade4 Package: Implementing the Duality Diagram for Ecologists[END_REF] to evaluate the associations between species composition and environmental variables. A redundancy analysis (RDA) as developed by [START_REF] Van Den Wollenberg | Redundancy Analysis an Alternative for Canonical Correlation Analysis[END_REF] was carried out in place of the co-intertia analysis and have given very similar results. The considered taxa were diatoms and dinoflagellates with percentage of occurrence ≥ 40%. The abbreviated names of species are given in table 2. Only data related to high tide sampling were considered for the environmental parameters, since phytoplankton was only taken at high tide period. The abundances were transformed into log (X + 1) to minimize differences in numbers.

Results

Abiotic factors and chlorophyll a

Temperature and salinity

In May (spring) and October (autumn), the temperature did not undergo diurnal variations both upstream and downstream and temperature ranged between 20 and 22.5 °C at low tides (LT) and high tides (HT). In August (summer season) at HT, upwelling marine waters cool the Lagoon waters with the lowest registered temperature (15.5 °C), while at LT the temperature ranged between 20 °C and 24 °C, at downstream and upstream, respectively. In February (winter), marine inputs tend to warm the Lagoon waters and temperature increased from 15 °C to 18 °C (Fig. 2a,b and Appendix 1). The Lagoon is highly influenced by marine waters (salinity of 35) at HT, with salinity exceeding 35 at all stations (a maximum of 36.5) except at station 6 (located upstream) where an average salinity of 30 was recorded. In contrast, at LT, the Lagoon waters were characterized by a salinity increasing from 23 at upstream to 36 at downstream of the Lagoon (Fig. 2).

Nutrients

The Oualidia Lagoon was characterized by relatively high nutrient concentrations, generally at LT, with values increasing upstream (Fig. 3). Nitrate (NO3) showed the highest concentration in August and October (up to 30 µM and 20 µM, respectively) at HT (Fig. 3b).

At LT, February and May were characterized by the highest concentrations with values of up to 30 and 40 μM respectively at station 6 upstream (Fig. 3a). Phosphates (PO4) ranged between 1.3 and 4 µM at HT (in February, May and August) and between 0.8 and 2.5 µM at LT (in August and May, Fig. 3c andd). October was globally the least rich month in PO4, especially at HT (< 1µM) and February at LT (Fig. 3d). Temporal variation in ammonia (NH4) concentration was observed with high levels (up to 30 μM) in August and October at HT (Fig. 3f). NH4 concentrations remain low in February and May (< 3 µM) during HT (Fig. 3f). At LT, the highest levels of NH4 (17-24 μM, maximum in May) were recorded (Fig. 3e), whereas all other concentrations were lower than 6 μM during all other seasons.

Chlorophyll a

The highest chlorophyll a (Chl-a) concentrations during the survey were observed in August with maximal values of 6 μg L -1 at LT and 3.89 μg L -1 at HT. During this period, Ch-a at all stations, was > 4 μg L -1 at LT and < 4 μg L -1 at HT. For the other seasons, Chl-a concentrations were < 2.1 μg L -1 (Fig. 4). The maximum Ch-a for each period was observed in LT when compared to HT.

Microphytoplankton

Taxonomic composition

The phytoplankton of the Oualidia Lagoon covers six groups and 114 taxa. Diatoms and Dinoflagellates were the most represented in term of species, with 68 and 40 taxa, respectively. In contrast, Silicoflagellates, Euglenophytes and Raphidophytes were poorly represented (Table 1). Diatom species dominated the microphytoplankton in all stations and seasons (Fig. 5), with a relative abundance exceeding 80 %. However, Dinoflagellates accounted for 50 % of microphytoplankton in St2 in May and St6 in August and were represented mainly by Scrippsiella sp. and Peridinium quadridentatum.

Specific richness and specific diversity

The number of taxa recorded per station varied between 13 and 42. October and particularly August showed the highest numbers of taxa (generally ≥ 32) in contrast with February and May (13-33 taxa) situations (Fig. 6a). The highest specific richness was observed upstream, at station 5 (27-40 taxa). The Shannon (H) index values of phytoplankton were generally > 3 during all periods. In summer, microphytoplankton was more diversified (H > 4), mainly downstream (maximum of 4.7) compared to upstream (3.3). The lowest diversity (2.5) was observed at Station 5 in May, due to the important proliferation of the diatom Nitzschia spp (Fig. 6b).

Distribution of microphytoplankton densities

The distribution of phytoplankton abundance was very heterogeneous along the Lagoon. The highest densities (Fig. 7) were observed in October (2.20×10 4 cells L -1 and 4.46×10 4 cells L -1 ) and August (1.42×10 4 to 3.09×10 4 cells L -1 ), with a peak in St6 (6.92×10 4 cells L -1 ) due to the proliferation of several diatom species (Thalassiosira spp., Surirella sp., Chaetoceros spp…) and the dinoflagellate Peridinium quadridentatum. Low densities were recorded in February and May (0.4×10 4 cells L -1 and 1.95×10 4 cells L -1 ).

Effects of the environmental factors

The links between species composition and environmental variables was established using a co-inertia analysis. The necessary preliminary step was to perform a centered PCA (Principal Component Analysis) in order to evaluate the spatiotemporal distribution of taxa independently of the environmental variables (Fig. 8). The analyzed matrix includes observations from all stations as summarized in Table 2. The abundances were transformed into log (X + 1) to account for the data distribution skewness and make them closer to a normal distribution. The first two axes of the factorial plane F1 X F2 represented 41% of the total inertia for the PCA. The PCA revealed important differences in species associations (Fig. 8a) between seasons and few differences between stations (Fig. 8b). The species are well scattered in the F1 x F2 factorial plane. Two main groups of taxa have emerged: Group I mostly associated to August and October periods and was represented mainly by marine species frequently encountered in Atlantic coastal waters. Some of them are considered to be upwelling indicators (Chaetoceros, Pseudo-nitzschia, Thalassiosira, Leptocylindrus danicus and Gymnodinium : [START_REF] Elghrib | Phytoplankton distribution in the upwelling areas of the Moroccan Atlantic coast localized between 32°30'N and 24[END_REF]. Group II was mainly associated with February and May periods (Fig. 8a), and was mainly represented by brackish or freshwater species belonging to Surirella, Paralia and Navicula genera, frequently observed in this Lagoon. The equivalent PCA was performed on the environmental variables only (plot not shown) and indicated that the environmental parameters (72% of the variability accounted for the first two axis) were contrasted between seasons, driven by an axis of variable salinity (46%) and Temperature axis (26%) with nutrients evenly balanced between both.

The co-inertia analysis revealed the seasonal effect of environmental factors of the species associations (Fig. 9). The first axis F1 was described by NO3 and mainly NH4. There was a clear separation between the salinity and nutrients particularly NO3 and NH4. Temperature contributed significantly to the formation of the F2 axis. It was opposite to the nutrient especially to the PO4 (Fig. 9a). A separation between the different periods was also clearly visible. The stations of each period, with few exceptions, formed a single group (Fig. 9c).

August and October periods are highly diversified and correspond to an important development of many phytoplankton taxa resulting from a NO3 and NH4 supply from the sea.

In these two periods, the close relationship between environmental factors and taxa is generally well marked (Fig. 9c). August was characterized by low temperatures ranging between 15 °C and 17 °C and high levels of nutrients mainly NO3 (from 9 to 11 μM with a maximum of 33.3 μM at station 6). This upstream station was characterized by highly contrasted environmental and biological parameters including low salinities (29.5), high temperature (22.8 °C) and high levels of nitrogen nutrients (32-33.3 µM). In August (Fig. 9b) several taxa (Group II) such as Navicula, Diploneis, Pleurosigma and Surirella were dominant whereas their abundance in the other periods were generally low; which suggest their preference for cold waters and the availability of nitrogen nutrients mainly NO3.

October was characterized by high temperatures (20 °C to 21.2°C), very low levels of PO4 (<1 μM) and high levels of nitrogen mainly in NH4 (31 μM). This month was marked by the proliferation of dinoflagellates taxa (Fig. 9b) such as Scrippsiella (700 cell L -1 ), Protoperidinium (800 cells L -1 ), and harmful or potentially toxic taxa such as Pseudonitzschia (9700 cells L -1 ), Prorocentrum (900 cells L -1 ), and Dinophysis species (400 cells L - 1 ) including Dinophysis caudata; Dinophysis acuminata and Dinophysis fortii. February and May were characterized by low levels of NH4 (0.4-8 µM) but an important level in phosphates (1.4-3.7 μM), compared to August and October (PO4 : 0.4-2 μM). At February and May, phytoplankton richness was low (Fig. 9b) where a few taxa (Group III) such as Diplopsalis, Thalassionema nitzschoides and Alexandrium showed relative high abundance.

Discussion

Data showed that Oualidia Lagoon is characterized by important tidal variations of the environmental parameters in all sampled stations and across seasons, with consequences on the dynamic of phytoplankton assemblages. Tidal differences in temperature were highly marked in February and August. In the summer months, the seasonal upwelling of the Atlantic coast cools the Lagoon waters and water fill the entire Lagoon at high tides. The salinity at HT was similar to that prevailing in the open Atlantic Ocean, with decreasing values from downstream to upstream (St1 to St6). At LT, the decreasing gradient of the salinity from St1 to St6 was more pronounced. The permanent occurrence of freshwater resurgences [START_REF] Rharbi | Elaboration d'une stratégie d'élevage de l'huître Crassostrea gigas dans la lagune de Oualidia (Maroc) sur la base de l'étude des relations trophiques[END_REF][START_REF] Hilmi | Hydrodynamisme de la lagune de Oualidia (Maroc) durant l'été et l'automne 2005[END_REF] in the Lagoon influences the distribution of salinity, mainly upstream where desalination reached its maximum (22.9). Nutrient concentrations, particularly nitrates, increased from downstream to upstream. At LT, the present study confirmed the results of several authors [START_REF] Mejjad | Sediment geochronology and geochemical behavior of major and rare earth elements in the Oualidia Lagoon in the western Morocco[END_REF][START_REF] Rharbi | Elaboration d'une stratégie d'élevage de l'huître Crassostrea gigas dans la lagune de Oualidia (Maroc) sur la base de l'étude des relations trophiques[END_REF] who indicated the presence of an increasing gradient downstream-upstream in nutrients and a decreasing gradient for salinity. This is due to the hydrodynamic characteristics of the Lagoon [START_REF] Mejjad | Sediment geochronology and geochemical behavior of major and rare earth elements in the Oualidia Lagoon in the western Morocco[END_REF][START_REF] Hilmi | Etude courantologique de la lagune de Oualidia (Maroc) en automne[END_REF][START_REF] Hilmi | Hydrodynamisme de la lagune de Oualidia (Maroc) durant l'été et l'automne 2005[END_REF] as the marine influence is marked downstream because of the change to Lagoon-oceanic connection (Fig. 1). The stations located upstream were more influenced by the continental enrichment together with freshwater resurgences likely rich in nutrients in this part of the Lagoon. This enhances the development and the richness of phytoplankton upstream. The upstream zone is enriched in nitrogen due to agricultural activities and even downstream area is enriched through tidal currents [START_REF] Rharbi | Elaboration d'une stratégie d'élevage de l'huître Crassostrea gigas dans la lagune de Oualidia (Maroc) sur la base de l'étude des relations trophiques[END_REF][START_REF] Bennouna | Etude du phytoplancton du complexe lagunaire Oualidia-Sidi Moussa[END_REF]. These authors suggested that Chl-a concentration increased upstream and this is confirmed by our observation mainly at LT for chlorophyll recorded values. Tidal currents were shown to be higher downstream of the Oualidia Lagoon [START_REF] Hilmi | Etude courantologique de la lagune de Oualidia (Maroc) en automne[END_REF][START_REF] Hilmi | Hydrodynamisme de la lagune de Oualidia (Maroc) durant l'été et l'automne 2005[END_REF][START_REF] Koutitonsky | L'étude du comportement hydrosédimentaire du système lagunaire Oualidia par la modélisation mathématique, Volet 1: synthèse des données et simulation de la réfraction des houles[END_REF]. Thus, the considerable reduction in the hydrodynamic intensity in the upstream area could favor not only the phytoplankton development as shown in our study but also the benthic fauna as suggested by other authors [START_REF] Bidet | Etude sédimentologique de la lagune de Oualidia (Maroc)[END_REF]Elasri et al., 2015Elasri et al., , 2017)). [START_REF] Kamara | Recherches préliminaires au développement de l'élevage de la palourde européenne (Ruditapes decussatus) sur les côtes marocaines et au repeuplement des sites surexploités[END_REF] pointed out that the upstream part of the Lagoon was a stable area and was therefore suitable for Clams growth.

In terms of seasonal variability, the waters of the Lagoon were rich in nitrates and ammonium during all seasons of 2011. The higher concentrations occurred generally at LT, especially in spring (May), where NO3 and NH4 were at LT > 35µM and 20µM, respectively. They did not exceed 9 µM at HT. The registered high concentrations of NO3 at LT are in favor of anthropogenic origin due mainly to agriculture, freshwater resurgence and urban discharges.

High levels of PO4 are observed at HT, particularly in February, with a maximum of 3µM, reflecting the significant oceanic input of PO4 during this season, and probably NH4 during August and October. These conclusions are corroborated with the study of [START_REF] Makaoui | L'upwelling de la côte atlantique du Maroc entre 1994 et[END_REF] who reported that the Lagoon is more influenced by the oceanic input of nutrients particularly PO4 in reason of upwelling events. [START_REF] Mejjad | Sediment geochronology and geochemical behavior of major and rare earth elements in the Oualidia Lagoon in the western Morocco[END_REF] suggested that seasonal and diurnal nutrient variability in the Oualidia Lagoon results from the influence of the water continental inputs, precipitation and evaporation regimes as well as oceanic-lagoon exchanges.

The observed variability in nutrients concentrations have direct effect on the development of phytoplankton with high Chl-a concentrations observed in August (values of 3.89 µg.l -1 at HT and 6.52 µg.l -1 at LT). Interestingly the values of Chl-a are high despite moderate microphytoplancton concentrations in Oualidia. This could be explained by the potential contribution of other groups as pico and nano-phytoplankton. Further studies have to focus on the distribution and abundance of these groups, their contribution to the total chlorophyll biomass and to quantify potential relationships linking their temporal changes to environmental factors. Our results corroborated those of Garcia Olivia et al. ( 2018) who suggested that the functioning of the coastal lagoons and their biological assemblages are strongly determined by the environmental conditions of each Lagoon and by the connectivity that these environments maintain with the adjacent sea. At the same time, the hydrodynamic behavior of coastal lagoons plays a crucial role in their functioning, not only in terms of water quality conditions, but also in terms of environmental range for species inhabiting the Lagoons, species connectivity, and fishing capacities [START_REF] Pérez-Ruzafa | Fisheries in coastal lagoons: An assumed but poorly researched aspect of the ecology and functioning of coastal lagoons[END_REF]2018[START_REF] Gamito | Effects of Changing Environmental Conditions on Lagoon Ecology[END_REF]. Our results show that most of the environmental variables including nutrients are influenced by hydrodynamic and tidal rhythm in the Oualidia Lagoon.

Studies on phytoplankton diversity and dynamic in Oualidia are rare; the obtained data characterizing the spatio-temporal variations of abundance and diversity of microphytoplankton would help us to better understand the functioning of this human impacted ecosystem but also may contribute to sustainable management of the aquacultural resources as the reared mollusk Crassostrea gigas. Our results suggest that in terms of phytoplankton, the Lagoon of Oualidia is a highly diversified ecosystem, well structured and balanced in phytoplankton populations during all the periods and particularly in August. The Shannon index values ranged between 3 and 4.69 bits suggesting the influence of oceanic waters on the phytoplankton populations of the Lagoon. [START_REF] Ghosh | Diversity and seasonal variation of phytoplankton community in the Santragachi lake, West Bengal, India[END_REF] suggested that high diversity indices reflect a healthy ecosystem when the opposite is a sign of degraded environment. Our data corroborated those of [START_REF] Bennouna | Etude du phytoplancton du complexe lagunaire Oualidia-Sidi Moussa[END_REF][START_REF] Rharbi | Importance des paramètres hydrologiques et phytoplanctoniques sur la croissance de l'huître Crassostrea gigas en élevage dans la lagune de Oualidia[END_REF] who showed that the diversity indices of phytoplankton in Oualidia were high (3 to 4.5 bits) and approached those observed in oceanic environment. The phytoplankton of Oualidia Lagoon was represented by five groups, with diatoms and dinoflagellates being the most dominant taxa when considering both species number and density. During our survey, diatoms dominated upstream and downstream during the different seasons, with the exception of St2 in May and St6 in August which showed an important development of two dinoflagellate species Scrippsiella sp. and P.

quadridentatum. These results corroborated those of [START_REF] Elghrib | Phytoplankton distribution in the upwelling areas of the Moroccan Atlantic coast localized between 32°30'N and 24[END_REF] and [START_REF] Demarcq | Oceanographic and biological features in the Canary Current Large Marine Ecosystem[END_REF] who showed that diatoms are dominating in Moroccan Atlantic coastal waters. [START_REF] Bennouna | Etude du phytoplancton du complexe lagunaire Oualidia-Sidi Moussa[END_REF][START_REF] Rharbi | Importance des paramètres hydrologiques et phytoplanctoniques sur la croissance de l'huître Crassostrea gigas en élevage dans la lagune de Oualidia[END_REF] reported that the Oualidia Lagoon was characterized by the dominance of diatoms almost 10 years ago. Other studies showed that diatoms and dinoflagellates dominate the phytoplankton in Moroccan Atlantic coastal ecosystems such as Dakhla Bay [START_REF] Saad | Impact of economic development on the dynamics of phytoplankton and physic-chemical quality of Dakhla Bay (South of Morocco). South. Asian[END_REF], Moulay Bousselham Lagoon [START_REF] Loumrhari | Succession de la population phytoplanctonique en fonction des paramètres physicochimiques (sites Mehdia et Moulay Bousselham)[END_REF] and Cintra Bay (unpublished data) but also in Moroccan Mediterranean marine ecosystems (the coastal waters M'diq Bay or Oued Laou : Rijal leblad et al., 2013 and the Nador Lagoon : El Madani et al., 2011) but also in the Tunisian Mediterranean lagoons of Bizerte [START_REF] Armi | Seasonal phytoplankton responses to environmental factors in a shallow Mediterranean lagoon[END_REF] and the Cullera Estany spanish Lagoon (Pachès et al., 2014). [START_REF] Badylakande | Spatial and temporal patterns of phytoplankton composition in a subtropical coastal lagoon, the Indian River Lagoon, Florida, USA[END_REF] reported that the relatively high level of diatoms dominance in lagoons may in part be attributable to tidal mixing energy and tidal water in flux. Diatoms are often more dependent on and tolerant of environments characterized by strong vertical mixing energy, while the turbulence of the water column at these sites may have a negative impact on the relative success of dinoflagellates [START_REF] Margalef | Functional morpho-logy of organisms involved in red tides, as adapted todecaying turbulence[END_REF][START_REF] Smayda | Community assembly inmarine phytoplankton: application of recent models to harmfuldinoflagellate blooms[END_REF]. At the species level, another feature of tidally mixed regions of the Lagoon is the presence of phytoplankton taxa considered oceanic or neritic such us Thalassionema nitzschioides and Skeletonema costatum. Overall, there was a general tendency for dinoflagellates to bloom during the warm season, while the dominant diatoms bloomed over a broader temperature range [START_REF] Badylakande | Spatial and temporal patterns of phytoplankton composition in a subtropical coastal lagoon, the Indian River Lagoon, Florida, USA[END_REF].

Phytoplankton in the Oualidia Lagoon was represented by 114 taxa, mainly dominated by marine species, such as Leptocylindrus danicus, Leptocylindrus minimus, Pseuonitzschia delicatissima, Pseudo-nitzschia seriata, Thalassiosira, Chaetoceros, Dinophysis, Protoperidinium. Brackish or freshwater taxa were faintly encountered such as Bacillaria paxillifera, Epithemia, Euglena. We also noted the presence of benthic species such as Amphora, Cocconeis, Licmophora, Nitzschia indicated a mixing of the water column with a sediment resuspension from the bottom favored by the hydrodynamic regime and the shallow depth of the lagoon [START_REF] Bennouna | Étude des populations phytoplanctoniques de la lagune de Oualidia (Maroc), dinoflagellés potentiellement nuisibles[END_REF][START_REF] Rharbi | Importance des paramètres hydrologiques et phytoplanctoniques sur la croissance de l'huître Crassostrea gigas en élevage dans la lagune de Oualidia[END_REF]. Our results suggest that the oceanic waters substantially influence the Oualidia lagoon. The present study highlights the influence of the tidal currents in the Oualidia Lagoon on phytoplankton composition with marine species entering at HT periods from the Atlantic Ocean.

In general, our results corroborated those obtained in macrotidal Atlantic Lagoons and differed from those of Mediterranean ecosystems. In terms of seasonality, [START_REF] Rosa | Temporal variability of the mass exchanges between the main inlet of Ria Formosa lagoon (southwestern Iberia) and the Atlantic Ocean[END_REF] showed in their study on Ria Formosa lagoon (southwestern Iberia) that this Lagoon acted as a source of material during Spring and Summer seasons, which contributed to increase the biological productivity of the coastal ocean. Upwelling events that occurred more evidently during the Autumn survey drove an import amount of nutrients into the Lagoon, enhancing its biological productivity. [START_REF] Glé | Variability of nutrients and phytoplankton primary production in a shallow macrotidal coastal ecosystem (Arcachon Bay, France)[END_REF] showed that nutrient levels in Arcachon Bay (a mesotidal coastal lagoon of 174 km2 on the southwest Atlantic coast of France) seem to play an important role in the control of phytoplankton primary production rates during the productive period and explain their spatial, seasonal and inter-annual variability. [START_REF] Bennouna | Étude des populations phytoplanctoniques de la lagune de Oualidia (Maroc), dinoflagellés potentiellement nuisibles[END_REF] revealed that phytoplankton development in the Oualidia Lagoon, begins in May and is marked by two peaks: in June (maximum 11.9×10 4 cells L -1 ) and July (7.6×10 4 cells L - 1 ). In August, phytoplankton concentrations are again low (0.25×10 4 to 0.71×10 4 cells L -1 ), then increase and fluctuate to give an autumnal peak in October and November. In Moulay Bousselham Lagoon (located in Northern Moroccan Atlantic Ocean), [START_REF] Loumrhari | Succession de la population phytoplanctonique en fonction des paramètres physicochimiques (sites Mehdia et Moulay Bousselham)[END_REF] emphasized that a maximum phytoplankton abundance was recorded from March to September with a maximum of 3.6×10 4 cells L -1 . The minimum phytoplankton abundance was recorded in February (9×10 3 cells L -1 ). In the Nador Lagoon (Moroccan Mediterranean), El Madani et al, ( 2011) have listed 311 phytoplankton species belonging to seven groups with 133 diatoms and 169 dinoflagellates species. The maximum phytoplankton abundance was found in August due to the bloom of Nitzschia longissima (1.7×10 7 cells L -1 at station located in the N-W Beninsar area). The minimum abundance was recorded in November. In the Tunisian North Lagoon of Bizerte, [START_REF] Armi | Seasonal phytoplankton responses to environmental factors in a shallow Mediterranean lagoon[END_REF] reported the importance of environmental factors and nutrient inputs in structuring the biomass of phytoplankton communities. According to [START_REF] Kjerve | Comparative oceanography of coastal lagoons, in Estuarine Variability[END_REF][START_REF] Kjerfve | Coastal lagoon processes[END_REF] and [START_REF] Umgiesser | Comparative hydrodynamics of 10 Mediterranean lagoons by means of numerical modeling[END_REF], coastal lagoons can be subdivided into choked, restricted, and leaky systems based on the degree of water exchange between lagoon and ocean. This exchange greatly influences the variability of abiotic factors, thus controlling the abundance and composition of phytoplankton populations and consequently the upper trophic levels in the lagoons. Oualidia Lagoon is considered to be a leaky system [START_REF] Hilmi | Hydrodynamisme de la lagune de Oualidia (Maroc) durant l'été et l'automne 2005[END_REF], and is subject to a very significant oceanic influence.

In our study, the highest phytoplankton species diversity (> 4 bits) and density (> 400×10 2 cells L -1 ) were found in summer and autumn in the entire lagoon, particularly at St5 and St6. This was due to the higher nutrient concentrations (>30 µM) measured in the stations located upstream and confirmed by the regularly high values of Chl-a recorded at all stations in summer at LT. This zone was also exceptionally exposed to the sediment suspension rich in organic matter, caused by the dredging of the sediment trap set up upstream in February 2011. This event could be responsible of the high levels of ammonium and nitrate measured during May 2011, which could stimulate the phytoplankton development observed in August and October 2011. Also, the nutrients input originating from continental shelf together with freshwater resurgences and from Atlantic waters related to upwelling characterizing this region mainly in summer and persisting in autumn [START_REF] Makaoui | L'upwelling de la côte atlantique du Maroc entre 1994 et[END_REF] are probably responsible of the observed enrichment of the Oualidia Lagoon waters. In contrast, Winter (February) and Spring (May) periods showed the lowest values of species diversity and phytoplankton cell abundances. Our results corroborated those of [START_REF] Rharbi | Importance des paramètres hydrologiques et phytoplanctoniques sur la croissance de l'huître Crassostrea gigas en élevage dans la lagune de Oualidia[END_REF][START_REF] Botes | Phytoplankton Identification Catalogue Saldanha Bay, South Africa[END_REF] who reported that the Oualidia Lagoon is under the influence of the upwelling, causing a drop in temperature together with high nutrient concentrations enhancing phytoplankton development during spring and summer. Our results showed that nutrients seem to be the main environmental abiotic factors determining the development of several phytoplankton populations. In Oualidia, the phytoplankton diversity seems to be favored by a wide range of temperature and salinity related to intense water exchanges with the Atlantic Ocean.

Phytoplankton showed a rapid response to modified nutrient levels through changes in biomass and composition [START_REF] Reynolds | The Ecology of Phytoplankton[END_REF]. Our field results show that nitrogenous compounds (NO3 and NH4) could be responsible for the growth of many taxa such as Thalassiosira, Scrippsiella, Chaetoceros, Prorocentrum, Protoperidinium and Surirella mainly in August and October, although they are less represented in space and during all periods. Potential toxic or harmful species [START_REF] Lassus | Toxic and harmful microalgae of the World Ocean. Micro-algues toxiques et nuisibles de l'Océan Mondial[END_REF][START_REF] Moestrup | IOC-UNESCO Taxonomic Reference List of Harmful Micro Algae[END_REF], which appear in the 'harmful algal bloom' list of the Intergovernmental Oceanographic Commission of UNESCO, belonging to different genera such as Pseudo-nitzschia, Alexandrium,

Figures legend

Fig. 1 .

 1 Fig. 1. Sampled stations from downstream to upstream of the Oualidia Lagoon (Atlantic coast, Morocco). Parks (1, 3, 5, 7 and 8) and Past indicate the oyster farming zones and the Ocean/lagoon connection, respectively.

Fig. 2 .

 2 Fig. 2. Spatio-temporal variations of temperature (°C) and salinity at low tide (a and c) and high tide (b and d) periods in the sampled stations of Oualidia Lagoon, upstream and downstream for station 1 and 6.

Fig. 3 .

 3 Fig. 3. Spatio-temporal variations of phosphate, nitrate and ammonium concentrations (µM) at low tide (a,c and e) and high tide (b, d and f) in the sampled stations of Oualidia Lagoon.

Fig. 4 .

 4 Fig. 4. Spatio-temporal variation of chlorophyll a concentrations (μg L -1 ) measured at low (a) and high tide (b) in the sampled stations of Oualidia Lagoon.

Fig. 5 .

 5 Fig. 5. Spatio-temporal variation of percentages (%) in term of abundance of different phytoplankton groups in Oualidia lagoon.

  Fig. 1

  Fig.5

Fig. 7

 7 Fig.7

Fig. 8

 8 Fig.8

Fig. 9

 9 Fig.9

Table 1 .

 1 Inventory and percentage frequency of taxa encountered at the Oualidia lagoon

Table 2 .

 2 The codes assigned to the hydrological and phytoplankton communities for the Co inertie and PCA analyses.

Appendix 1: Table

a

. Spatio-temporal variation of the temperature (a1) and salinity (a2) at high (HT) and low tides (LT). Table

b

. Spatio-temporal variation of the concentrations in µM of nitrate (b1), Phosphate (b2) and ammonium (b3) at high (HT) and low tides (LT)

Table 1 Diatoms (% Frequency taxa) February May August October

 1

	Asteromphalus Ehrenberg. 1844	0.0	0.0	33.3	0.0
	Adoneis Andrews & Rivera. 1987	16.7	0.0	0.0	0.0
	Actinocyclus Ehrenberg. 1837	16.7	0.0	33.3	0.0
	Amphora Ehrenberg ex Kützing. 1844	16.7	0.0	83.3	50.0
	Bacillaria paxillifera (Müller) Marsson 1901	0.0	0.0	16.7	0.0
	Bellerochea Van Heurck. 1885	0.0	16.7	33.3	16.7
	Chaetoceros Ehrenberg. 1844	33.3	50.0	83.3	100.0
	Cocconeis Ehrenberg. 1836	16.7	0.0	0.0	16.7
	Coscinodiscus Ehrenberg. 1839	83.3	0.0	100.0	50.0
	Cyclotella (Kützing) Brébisson. 1838	0.0	0.0	66.7	50.0
	Cerataulina pelagica (Cleve) Hendey 1937	0.0	0.0	0.0	16.7
	Cylindrotheca closterium (Ehrenberg) Reimann &	66.7	0.0	50.0	100.0
	Lewin 1964
	Dactyliosolen Castracane. 1886	0.0	16.7	33.3	33.3
	Detonula Schütt ex De Toni. 1894	0.0	0.0	16.7	16.7
	Diploneis bombus (Ehrenberg) Ehrenberg 1853	83.3	33.3	100.0	50.0
	Diploneis crabro (Ehrenberg) Ehrenberg 1854	0.0	0.0	33.3	33.3
	Diploneis spp	100.0	16.7	83.3	100.0
	Diatomée sp	16.7	16.7	16.7	0.0
	Ditylum brightwellii (West) Grunow in Van Heurck 1885 16.7	0.0	0.0	0.0
	Entomoneis Ehrenberg. 1845	0.0	33.3	33.3	33.3
	Fragilaria Lyngbye. 1819	66.7	16.7	66.7	16.7
	Eucampia Ehrenberg. 1839	0.0	0.0	0.0	33.3
	Epithemia Kützing. 1844	0.0	0.0	50.0	33.3
	Grammatophora Ehrenberg. 1840	0.0	50.0	50.0	83.3
	Guinardia flaccida (Castracane) Peragallo 1892	0.0	0.0	33.3	0.0
	Guinardia striata (Stolterfoth) Hasle. 1996	16.7	0.0	33.3	50.0
	Guinardia sp1	16.7	0.0	33.3	16.7
	Guinardia sp2	0.0	33.3	16.7	16.7
	Gyrosigma Hassall. 1845	16.7	0.0	50.0	83.3
	Hemiaulus proteus Heiberg. 1863	33.3	0.0	33.3	33.3
	Helicotheca tamesis (Shrubsole) Ricard. 1987	16.7	0.0	50.0	16.7
	Lauderia annulata Cleve. 1873	16.7	0.0	50.0	50.0
	Leptocylindrus danicus Cleve. 1889	66.7	66.7	66.7	83.3
	Leptocylindrus minimus Gran 1915	83.3	33.3	100.0	100.0
	Leptocylindrus mediterraneus (Peragallo) Hasle 1975	16.7	0.0	0.0	16.7

Acknowledgements

We thank the staff of the marine environment monitoring station INRH in the Oualidia. This work is dedicated to the memory of Doctor Jaouad Lakhdar Idrissi who died while carrying out the first missions of a comprehensive integrated program of rehabilitation of the Oualidia Lagoon and whose present work is part. Thanks to IRD (Institut de Recherche pour le Développement France) for funding Dr Mohamed Laabir stay in INRH (Institut National de Recherche Halieutique, Morocco) laboratory in Casablanca during 2017.

Prorocentrum, Dinophysis, Ostreopsis, Karenia, Coolia, Gonyaulax, Gymnodinium, Dictyocha and Chattonella were present in Oualidia, particularly in October. Even if their concentrations were relatively low (unpublished data), they are subject to regular monitoring program as Oualidia Lagoon holds important oyster farming and recreational activities.

Consequently, the ecology, the biology and the toxicity of these HABs species have to be investigated.