Isabelle Rogowski
email: isabelle.rogowski@univ-lyon1.fr

Matthieu Degot

Dimitri Juré

Jean-Philippe Hager

Lionel Neyton

Yoann Blache

Shoulder functional status in rugby union players with and without history of shoulder problems

Objectives: To assess the effects of (1) rugby union practice, (2) history of injury managed nonoperatively, and (3) history of injury managed operatively on shoulder functional status in male rugby union players.

Design: Cross sectional study Setting: Clinical Participants: 86 male athletes were assigned into four groups: multisport athletes, rugby union players without shoulder problems, with history of shoulder injury managed nonoperatively and with history of shoulder injury managed operatively.

Main outcome measures: SI-RSI questionnaire, maximal isometric glenohumeral internal and external rotator strength, unilateral seated shot put test, upper quarter Y balance test.

Results: Healthy players presented higher internal (p=0.03) and external (p=0.04) rotator strength than multisport athletes. History of shoulder injury managed nonoperatively did not impair physical abilities but limited player's psychological readiness (p<0.001). After 4.5-months, shoulder stabilization surgery impaired maximal muscle strength and upper quarter body stability and mobility (p<0.001 for all).

Conclusions: The shoulder functional status in rugby union player presented increased glenohumeral rotator strength when compared to non-collision sport athletes. In rugby union players, psychological concerns remained in the long-term after a shoulder injury managed nonoperatively, and psychological and physical readiness seemed not be reached at 4.5 months postoperatively to respond to rugby union practice demand.

INTRODUCTION

Rugby union playing demands shoulder-region specific adaptations [START_REF] Edouard | Influence of Rugby Practice on Shoulder Internal and External Rotators Strength[END_REF] in order to resist to constraints applied during the rugby union specific tasks, such as tackle, ruck, or scrum. Such tasks become injurious when both the upper arm is placed in hazardous position and the shoulder is subject to a compressive or posteroinferior force [START_REF] Montgomery | Video analysis of shoulder dislocations in rugby: Insights into the dislocating mechanisms[END_REF]. Such mechanisms may conduct to shoulder traumatic injuries [START_REF] Montgomery | Video analysis of shoulder dislocations in rugby: Insights into the dislocating mechanisms[END_REF]. Both the non-operative and operative managements are used to restore rugby union player's shoulder integrity [START_REF] Bohu | The epidemiology of 1345 shoulder dislocations and subluxations in French rugby union players: a five-season prospective study from 2008 to 2013[END_REF].

The effects of such managements however remain to be explored in order to bring new knowledge to healthcare and conditioning specialists in relation with shoulder injury rehabilitation.

Regardless of the level of rugby union practice, the most common shoulder injuries are acromioclavicular and glenohumeral subluxations/dislocations [START_REF] Bohu | The epidemiology of 1345 shoulder dislocations and subluxations in French rugby union players: a five-season prospective study from 2008 to 2013[END_REF]Singh, Trewartha, Roberts, England & Stokes, 2016), which account for the most play-time loss [START_REF] Headey | The epidemiology of shoulder injuries in English professional rugby union[END_REF]. Because the acromioclavicular joint injuries occurring during contact sports are mainly of type I and II [START_REF] Funk | Sports injuries of the shoulder[END_REF], nonoperative treatment is first prescribed.

The return-to-sport decision-making is mainly based on the player's perception on shoulder comfort and ability to perform sport-specific tasks, and the return-to-sport occurs between two and eight weeks according to injury severity [START_REF] Dragoo | Acromioclavicular joint injuries in National Collegiate Athletic Association Football: Data from the 2004-2005 through 2008-2009 National Collegiate Athletic Association injury surveillance system[END_REF]. The relevance of operative treatment is considered when the athlete is not coping or progressing with rehabilitation [START_REF] Funk | Sports injuries of the shoulder[END_REF], or for acromioclavicular joint injuries from type III [START_REF] Funk | Sports injuries of the shoulder[END_REF]. Little is however known on the effects of such history of injuries on shoulder functional status. For glenohumeral anterior instability, rugby union player can choose the nature of the treatment, which depends on the time of season and specific sporting requirements [START_REF] Funk | Sports injuries of the shoulder[END_REF]. The nonoperative treatment results in many recurrences [START_REF] Funk | Sports injuries of the shoulder[END_REF], and shoulder stabilization surgery is considered in primary care, especially when players are young [START_REF] Boone | First-time anterior shoulder dislocations: has the standard changed[END_REF]. In contact sports, the shoulder stabilization surgery is increasingly based on the Latarjet procedure [START_REF] Funk | Sports injuries of the shoulder[END_REF], which consists of the coracoid process transfer to the antero-inferior part of the glenoid. The coracoid graft healing is commonly achieved at 3 months postoperatively (Young et al., 2010), allowing theoretically return to sport. However, in practice, some professional rugby union players can return to sport at this time [START_REF] Neyton | Surgical treatment of anterior instability in rugby union players: clinical and radiographic results of the Latarjet-Patte procedure with minimum 5-year follow-up[END_REF]Colegate-Ston, van der Watt & de Beer, 2015) but delay up to 12 months from surgery are also reported in the literature [START_REF] Ciccotti | Return to play criteria following surgical stabilization for traumatic anterior shoulder instability: A systematic review[END_REF]. Consequently, additional knowledge on functional status of shoulder with and without history of problems in rugby union players needs to help the decision-making for returning to the same sport at preinjury level in the shortest time while reducing risk of reinjury.

Before returning to sport, the restoration in shoulder physical abilities appears to be obvious after an injury episode. The progression in physical abilities from injury to return to sport at preinjury level follows a continuum to achieve return to participation then return to sport and finally return to performance [START_REF] Ardern | Consensus statement on return to sport from the First World Congress in Sports Physical Therapy, Bern[END_REF]. However, the psychological dimension may also play a role like the patient's emotional health affects the outcomes of nonoperative and operative treatments [START_REF] Ayers | The role of emotional health in functional outcomes after orthopaedic surgery: extending the biopsychosocial model to orthopaedics: AOA critical issues[END_REF]; and the injury episode could result in fear of reinjury and limited self-efficacy and motivation [START_REF] Ardern | Consensus statement on return to sport from the First World Congress in Sports Physical Therapy, Bern[END_REF]. Characterising the psychological readiness of athlete to return to sport following shoulder injury in association with the physical abilities may also highlight the decision making to return to full performance.

The aim of this study was three folds: to assess the effects of (1) rugby union practice, (2) history of injury managed nonoperatively, and (3) history of injury managed operatively on shoulder functional status in male rugby union players. It was hypothesized that rugby union practice would result in specific adaptations in shoulder functional status. It was also hypothesized that full restoration in physical and psychological abilities would be observed after a history of shoulder injury managed nonoperatively, while history of shoulder stabilization surgery would impair both the psychological and physical abilities in rugby union players.

MATERIALS & METHODS

Population

Eighty-six male athletes (age: 22.3 ± 3.9 years [15;38], height: 1.79 ± 0.07 m [1.65;2.00], mass : 82.4 ± 14.3 kg [55.5;121.0], weekly sport practice: 5.7 ± 2.8 hours [1.5;12.0]) volunteered to participate in this study, which was approved by the Ethical Committee (#2018-A03013-52). The distance from the C7 spinous process to the most distal tip of the third finger was measured both the sides, using a tape, when the upper limb abducts at 90° in the frontal plane.

The athletes' shoulders were assigned into four groups. Both the shoulders of 18 multisport athletes (soccer = 8; running = 5; basketball = 4; yoga = 1) were included in the reference group (MS,n=36) and those of 21 rugby union players were included in the healthy rugby group (HR, n=42). Shoulders of 17 rugby union players with history of shoulder injury managed nonoperatively constituted the third group (RNO, n=19; 14 dominant shoulders; acromioclavicular sprain stage I or II = 12, glenohumeral subluxation = 6, sternoclavicular sprain = 1). Finally, shoulders of 30 rugby union players with history of shoulder injury managed operatively with stabilization surgery by Latarjet procedure were assigned into the fourth group (RSS, n=30; 18 dominant shoulders). Inclusion and exclusion criteria for the four groups of shoulders are detailed in Table 1. **** Insert Table 1 near Isometric maximal voluntary contractions when arm abducted at 90° and elbow flexed at 90° in supine position were performed according to the procedure described by [START_REF] Gillet | Adaptive alterations in shoulder's range of motion and strength in young tennis players[END_REF]. The maximal strengths were measured for glenohumeral external (ER) and internal (IR) rotator muscles in each side, then normalized by body mass (expressed in N/kg) and used to compute the strength ratio ER/IR per shoulder.

Each participant then performed the upper-quarter Y-Balance Test (YBT) according to the procedure found in Degot, Blache, Neyton & Rogowski (2019a). The maximal distances reached in mediolateral, inferolateral and superolateral directions were measured for each upper limb then normalized by the respective distance between C7 and third finger (expressed in % of upper limb length), and averaged.

Finally, each participant performed the unilateral seated shot-put test (USSPT) according to the procedure described in [START_REF] Chmielewski | Normalization considerations for using the unilateral seated shot put test in rehabilitation[END_REF]. For each upper extremity, the distances of three pushing shots were averaged and normalized allometrically when dividing the mean distance per limb by body mass 0.35 [START_REF] Chmielewski | Normalization considerations for using the unilateral seated shot put test in rehabilitation[END_REF].

Statistical analysis

All data are presented by mean ± standard deviation. Effect sizes (ES) were computed, with ES=0.2 for small effect, ES=0.5 for medium effect and ES≥0.8 for large effect; and independent t-tests were applied to compare demographic characteristics and shoulder functional status between MS and HR.

Analyses of variance (ANOVA) with one independent factor were applied to highlight the group effect (HR vs. RNO vs. RSS) on demographic characteristics and shoulder functional status. In case of significant group effect, post hoc C-Dunnett tests were applied to compare, on the one hand, HR and RNO, and on the other hand, HR and RSS. All the statistical tests were made using SPSS 11.0 software and the level of significance was set at p≤0.05.

RESULTS

MS presented significantly higher age and weekly training hours than HR (Table 2; ES= 0.71; medium-to-large effect; p=0.004 and ES= 0.50; medium effect; p=0.03, respectively), while HR was heavier than MS (ES=1.04 ; large effect; p=0.01). When compared rugby union player groups, no significant differences were observed in demographic and sport characteristics, except for weekly training hours for which RSS practiced significantly more hours per week at the time of injury than HR (ES= 2.1; large effect; p<0.001). **** Insert Table 2 near here **** Regarding the shoulder functional status, MS and HR presented similar SI-RSI, ER/IR, YBT and USSPT scores, while normalized maximal ER and IR strengths were significantly higher in HR than in MS (Table 3; ES= 0.42; low-to-medium effect; p=0.04 and ES= 0.53; medium effect; p= 0.03, respectively). When comparing the three groups of rugby union players, ANOVA revealed a significant group effect for SI-RSI (p<0.001), normalized maximal ER (p<0.001) and IR strengths (p<0.001), mean YBT score (p=0.001) and YBT score in inferolateral direction (p<0.001). SI-RSI score were significantly lower in RNO than in HR (ES=11.9; large effect; p<0.001), while no other significant differences were reported between HR and RNO. SI-RSI score (ES=22.3; large effect; p=0.001), normalized maximal ER (ES=1.23; large effect; p<0.001) and IR strengths (ES=0.64; medium-to-large effect; p<0.001), mean YBT score (ES=0.90; large effect; p<0.001) and YBT score in inferolateral direction (ES=1.84 ; large effect; p<0.001) were significantly lower in RSS than in HR. **** Insert Table 3 near here ****

DISCUSSION

The aim of this study was to investigate the effects of practice and history of shoulder injury managed nonoperatively or operatively on the shoulder functional status in male rugby union players. The main findings were that rugby union practice demands enhanced glenohumeral muscle maximal strength.

The history of shoulder injury managed nonoperatively did not impair physical abilities but limited the rugby union player's psychological readiness. When assessed 4.5-months postoperatively, the shoulder stabilization surgery by Latarjet procedure impaired maximal glenohumeral muscle strength and upper quarter body stability and mobility.

Rugby union specific tasks place high demand onto the shoulder region in order to resist to compressive and posteroinferior forces (Usman, McIntosh & Fréchède, 2011). Although our healthy rugby players were heavier than our non-collision-sport athletes, score values were comparable as they were expressed related to body dimensions [START_REF] Crewther | The Ratio and Allometric Scaling of Speed, Power, and Strength in Elite Male Rugby Union Players[END_REF], i.e. body mass for maximal isometric strengths and medicine ball push-throw test [START_REF] Chmielewski | Normalization considerations for using the unilateral seated shot put test in rehabilitation[END_REF], and upper limb length for upper quarter Y balance test [START_REF] Gorman | Upper Quarter Y Balance Test: Reliability and Performance Comparison Between Genders in Active Adults[END_REF]. Healthy rugby union players presented with higher maximal isometric strength at glenohumeral joint than athletes practicing non-collision sports, with similar anteroposterior muscular balance [START_REF] Edouard | Influence of Rugby Practice on Shoulder Internal and External Rotators Strength[END_REF], and similar abilities in upper quarter body stability, mobility and power. Such increased glenohumeral muscle strength may need to resist to the repeated substantial forces applied to the shoulder in the tackle (Usman et al., 2011). Mean maximal internal and external rotator muscle strengths reported for our healthy rugby union players were however lower than the thresholds predictive of in-season shoulder injury occurrence proposed by [START_REF] Ogaki | Preseason muscle strength tests in the assessment of shoulder injury risk in collegiate rugby union players[END_REF]. This does not mean that our healthy players should be at high risk for shoulder injury because the specificity of the thresholds remains low [START_REF] Ogaki | Preseason muscle strength tests in the assessment of shoulder injury risk in collegiate rugby union players[END_REF] and the force applied onto the shoulder region during game depends on the skill level [START_REF] Mcintosh | Tackle characteristics and injury in a cross section of rugby union football[END_REF], which is difficult to compare between a squad from one Japanese university club [START_REF] Ogaki | Preseason muscle strength tests in the assessment of shoulder injury risk in collegiate rugby union players[END_REF] and our convenient sample of rugby union players. Our findings then confirmed that rugby union practice demands increased maximal muscular strength at the glenohumeral joint and showed no specific adaptations in upper quarter body stability, mobility and power in comparison with non-collision sport athletes when performance was normalized by body dimensions. Like the shoulder functional status presented with rugby-unionspecific adaptations, studying the effects of history of shoulder problems may demand to compare injured players in reference to healthy rugby union players.

Impact on shoulder during rugby union tackle or sudden arm horizontal extension are the main mechanisms for shoulder subluxation and dislocation [START_REF] De Beer | Shoulder injuries in rugby players[END_REF][START_REF] Montgomery | Video analysis of shoulder dislocations in rugby: Insights into the dislocating mechanisms[END_REF]. Such injuries result in joint instability due to a distension or rupture of the ligaments stabilizing the joint [START_REF] De Beer | Shoulder injuries in rugby players[END_REF], and non-operative management consists of recovering the joint stability within surrounding muscular tissues, especially the glenohumeral rotator muscles. The physical abilities of our shoulders with history of injury managed nonoperatively appeared to be restored when compared to our healthy shoulders. Restoration in maximal strength and upper quarter body stability, mobility and power is crucial to return to competition at preinjury level or above [START_REF] Ardern | Consensus statement on return to sport from the First World Congress in Sports Physical Therapy, Bern[END_REF]. However, the psychological readiness to practice rugby was still impaired in players having experienced a shoulder injury episode. Limited positive emotions and confidence, and reinjury anxiety [START_REF] Gerometta | The Shoulder Instability-Return to Sport after Injury (SIRSI): a valid and reproducible scale to quantify psychological readiness to return to sport after traumatic shoulder instability[END_REF] may have beneficial effects on the player to raise awareness of how preventing strategies are important during training and competition (Wadey et al., 2014). They may also act as negative competitive stressors, which are known to have deleterious effects on athlete's performance [START_REF] Sarkar | Psychological resilience in sport performers: a review of stressors and protective factors[END_REF]. It then appeared that the shoulder injury managed nonoperatively in rugby union players may be fully compensated physically, and the effects of partially restored psychological abilities on prevention and performance remained to be investigated.

When glenohumeral dislocation with glenoid bony lesion occurs, shoulder stabilization surgery by Latarjet procedure is advocated to limit the shoulder instability recurrence [START_REF] Boone | First-time anterior shoulder dislocations: has the standard changed[END_REF].

The tip of the coracoid process is transferred through the subscapularis muscle onto the antero-inferior glenoid, and satisfactory graft healing is achieved at 3 months postoperatively (Young et al., 2010).

The healing of the coracoid graft is a primary criterion to authorize a progressive return to sport, including contact sports (Young et al., 2010), occurring between 3 and 5 months after surgery in professional rugby union players (Colegate-Ston et al., 2015). After serious injury, return to the same sport at preinjury level demands psychological and physical readiness [START_REF] Gerometta | The Shoulder Instability-Return to Sport after Injury (SIRSI): a valid and reproducible scale to quantify psychological readiness to return to sport after traumatic shoulder instability[END_REF], which was not met in our amateur rugby players assessed at 4 months postoperatively. Indeed, 71% for SI-RSI score remained lower than scores above 76% commonly admitted for return to sport [START_REF] Müller | Predictive parameters for return to pre-injury level of sport 6 months following anterior cruciate ligament reconstruction surgery[END_REF], and scores reported for our healthy rugby union players. Physically, our operated shoulders presented with deficits in strength and upper quarter stability and mobility, as attested by significant lower values for maximal glenohumeral internal and external rotator muscle strength and YBT composite score when compared with our healthy shoulders.

Shoulder stabilization surgery seemed to unalter the muscular balance at glenohumeral joint, but the decrease reported in maximal strength may jeopardize the stability when shoulder must counteract the force generated by impact during specific rugby union tasks (Usman et al., 2011). Given that the upper quarter YBT is proposed to assess upper quarter stability, mobility and strength [START_REF] Gorman | Upper Quarter Y Balance Test: Reliability and Performance Comparison Between Genders in Active Adults[END_REF], it can be thought that the lower YBT composite score in surgically stabilized shoulder may be related to deficit in stability. Such a hypothesis may however not be confirmed when focusing on each YBT component, as the deficit occurred in inferolateral direction only. Despite stability deficits may occur in certain directions, another hypothesis concerned the limitation in glenohumeral mobility. During YBT, it may be supposed that trunk rotates to help the moving hand to reach the maximal distance in inferolateral direction, hence placing the supporting arm in horizontal adduction. At 4 months after Latarjet procedure, the extension in osseous glenoid cavity by coracoid bone transfer, extending the osseous glenoid cavity, may results in a non-anatomical barrier limiting shoulder mobility in horizontal adduction. It could be then interesting to monitor YBT performance beyond 4 months postoperatively to assess progress in shoulder mobility, and to investigate the mechanisms underlying the reduced YBT performance in inferolateral direction. These findings however showed that rugby union player's shoulder at 4 months postoperatively presented with reduced psychological and physical abilities, which still demanded to be restored before returning to full rugby union playing.

The main limitations of this study were related to its retrospective nature, which resulted in (1) the lack of information on the rehabilitation program following the shoulder injury managed nonoperatively, on the return to sport in rugby union with history of shoulder stabilization surgery, and on the playing position and experience in rugby union players, and (2) the interindividual variability in delay from injury in rugby union players with history of shoulder injury managed nonoperatively.

However, this study was the first to address the shoulder functional status through psychological and physical issues in rugby union players with and without history of injury or surgery. Further studies need to assess the effects of return-to-sport programs involving both the psychological and physical dimensions on shoulder functional status restoration and whether the shoulder functional status assessed at 4 months postoperatively can predict the return to sport in rugby union players.

To conclude, the findings of this study were that the functional status of rugby union player's shoulder presented increased glenohumeral rotator muscle strength when compared to non-collision sport athletes. In rugby union players, psychological concerns remained in the long-term after a shoulder injury managed nonoperatively, and psychological and physical readiness were not reached at 4.5 months after shoulder stabilization surgery to respond to rugby union practice demand. These findings bring new knowledge to clinicians and sport coaches on the restoration in psychological and physical * for significant differences between MS and HR at p≤0.05 † † † for significant differences between HR and RNO at p≤0.001 ‡ ‡ ‡ for significant differences between HR and RSS at p≤0.001

  here **** Procedure Each athlete began the testing session by completing the French version of SI-RSI questionnaire (Gerometta, Klouche S, Herman, Lefevre & Bohu, 2018) (for each shoulder in MS and HR), then performed the warm-up described in Degot et al. (2019b), and finally performed three performance physical tests.

Table 2 :

 2 Mean ± standard deviation [minimum ;maximal] demographic values in the four groups, namely, MS for multisport athletes, HR for healthy rugby union players, RNO for rugby union players with history of shoulder injury managed nonoperatively, and RSS for rugby union players with history of shoulder stabilization surgery.

		MS	HR	RNO	RSS	p
	n	18	21	17	30
	Age (years)	23.6 ± 3.3

Table 3 :

 3 Mean ± standard deviation shoulder functional status outcome values in the four groups, namely, MS for multisport athletes, HR for healthy rugby union players, RNO for rugby union players with history of shoulder injury managed nonoperatively, and RSS for rugby union players with history of shoulder stabilization surgery.

		MS	HR	RNO	RSS	p
	n	36	42	19	30
	SIRSI (%)	98,50 ± 3,15	99,70 ± 1,26	84,69 ± 20,19	71,73 ± 15,14	† † †, ‡ ‡ ‡
	ER (N.kg -1 )	2,21 ± 0,39	2,37 ± 0,43	2,40 ± 0,37	1,85 ± 0,48	*, ‡ ‡ ‡
	IR (N.kg -1 )	2,11 ± 0,39	2,32 ± 0,53	2,33 ± 0,54	1,80 ± 0,52	*, ‡ ‡ ‡
	ER/IR	1,07 ± 0,24	1,06 ± 0,23	1,08 ± 0,27	1,05 ± 0,16
	YBTC	0.90 ± 0.09	0.87 ± 0.11	0.84 ± 0.08	0.77 ± 0.08	‡ ‡ ‡
	YBTML	1,02 ± 0,09	1,00 ± 0,10	0,98 ± 0,10	0,98 ± 0,09
	YBTIL	0,90 ± 0,12	0,88 ± 0,14	0,83 ± 0,12	0,62 ± 0,12	‡ ‡ ‡
	YBTSL	0,77 ± 0,11	0,74 ± 0,13	0,71 ± 0,08	0,72 ± 0,12
	USSPT	67,75 ± 9,78	70,49 ± 14,12	76,02 ± 11,54	71,29 ± 13,24

SIRSI: Shoulder Instability Return to Sport after Injury; ER: maximal glenohumeral external rotator strength; IR: maximal glenohumeral internal rotator strength; mediolateral (YBTML), inferolateral (YBTIL), superolateral (YBTSL) and average (YBTC) normalized distances for upper quarter Y balance test; USSPT: unilateral seared shot put test)

Fundings

This study was supported by the 'Groupement de Coopération Sanitaire Ramsay Générale de Santé pour l'Enseignement et la Recherche' (COS-RGDS-2018-06-037).

community rugby union. International Journal of Sports Medicine, 37, 659 -664. Usman, J., McIntosh, A. S., & Fréchède, B. (2011). An investigation of shoulder forces in active shoulder tackles in rugby union football. Journal of Science and Medicine in Sport, 14, 547-552. Wadey, R., Podlog, L., Hall, M., Hamson-Utley, J., Hicks-Little, C., & Hammer, C. (2014)


Laury Açaf
email: laury.acaf@hotmail.com

Marie Abboud-Abi

Saab

Mirella Khoury-Hanna

Rodolphe Lemée

Bloom dynamics of the newly described toxic benthic dinoflagellate Ostreopsis fattorussoi along the Lebanese coast (Eastern Mediterranean)

Keywords: Ostreopsis fattorussoi, Benthic HABs, Dinoflagellates, microphytobenthos, Lebanese coast, Eastern Mediterranean

This study involves monitoring both benthic and planktonic abundances of the toxic dinoflagellate Ostreopsis fattorussoi in 5 stations along the Lebanese coast (Eastern Mediterranean); it extended from June 2014 to July 2016 and aims to study the dynamics of its development. Results revealed its presence year round along the coast, presenting seasonal blooms, both epiphytic and planktonic. A major bloom (maximum of 880 694 cells/g FW and 18 000 cells/L) occurred early in the summer (June-July), followed by a minor one in the fall (October-November) (≤ 50 000 cell/g FW). Its development seemed influenced by a combination of multiple environmental parameters. Water temperature seemed to play a major role in its development, as it appeared to be a thermophilic species, with maximum abundances per site reached between 27°C and 30.5°C. Its development could be facilitated by some agitation resulting from winds. Results concerning the role of nutrients and other environmental parameters remain inconclusive.

INTRODUCTION:

The geographical distribution of the benthic dinoflagellate Ostreopsis spp. covers tropical and subtropical areas (Ballantine et al., 1988;Grzebyk et al., 1994;Parsons and Preskitt, 2007;Tindall and Morton, 1998) as well as temperate areas in New Zealand (Chang et al., 2000;Rhodes et al., 2000), Australia (Pearce et al., 2001), Japan (Taniyama et al., 2003) and the Mediterranean Sea (Aligizaki and Nikolaidis, 2006;Mangialajo et al., 2011;Vila et al., 2001). Some studies suggest that it was possibly introduced from tropical to temperate areas via ship ballast water (Butrón et al. 2011), or as a result of the anthropogenic global climate change and warming of sea water (Maso and Garcés, 2006;Miraglia et al., 2009), whereas others suggest it could be indigenous to the Mediterranean Sea (Tognetto et al., 1995).

Two species, Ostreopsis cf. siamensis J. Schmidt and Ostreopsis cf. ovata Fukuyo, have been known for years in the North Western Mediterranean Sea; the latter in particular has formed blooms in the area (Mangialajo et al., 2011;Tognetto et al., 1995) and is the subject in most available literature. Its presence was first reported in the Mediterranean Sea in the bay of Villefranche-sur-mer (France) in 1972(Taylor, 1979). Both species are known to produce palytoxin-like metabolites. Palytoxin (PTX) is among the most potent known naturally produced toxins of marine origin even at extremely low concentrations (Amzil et al., 2012;Moore and Scheuer, 1971) and has been linked to several human intoxications in tropical areas (Alcala et al., 1988).

Since the early 2000s, Ostreopsis spp. has emerged as a threat to public health in the Mediterranean Sea. Cases of human intoxication have been linked to this genus in the Western Mediterranean Sea (Durando et al., 2007;Illoul et al., 2012;Kermarec et al., 2008;;Pfannkuchen et al., 2012;Vila et al., 2008 and2016). In some cases, more than 200 individuals were affected in one bloom episode, e.g. Genoa in 2005(Durando et al., 2007) ) and Algeria in 2009(Illoul et al., 2012). The absence of reports of intoxication in the Eastern Mediterranean Sea could be due to undiagnosed cases or is simply a reflection of the lack of related studies in the area. The intoxication occurs either through direct contact with cells or through inhalation of aerosols (Ciminiello et al., 2006 and2014;Sansoni et al., 2003;Tartaglione et al., 2015;Tichadou et al., 2010). The affected population is therefore not limited to swimmers and people involved in water activities, but also includes residents and people in the vicinity of the bloom area who are not in direct contact with the water. Reported symptoms in the Mediterranean Sea are mild and include irritation to the eyes, nose, skin and respiratory system, fever, while respiratory distress was reported on a few rare cases (Ciminiello et al., 2006;Simoni et al., 2003;Tichadou et al., 2010;Vila et al., 2016;Durando et al., 2007). In addition, PTX and PTX-like metabolites have been found in edible marine organisms from different trophic levels (Biré et al., 2013(Biré et al., & 2015)). For these reasons, despite the fact that cases of intoxications through ingestion of seafood have not yet been reported in the Mediterranean Sea, they remain a potential risk. These toxins also represent a threat to the ecosystem itself, as they have been linked to a mass mortality (Illoul et al., 2012), decrease in population (Blanfune et al., 2012;Shears and Ross, 2009) and a negative impact on reproduction of some marine fauna (Guidi-Guilvard et al., 2012). As the occurrence of Ostreopsis blooms is accompanied by closing of beaches (Tichadou et al., 2010) and negative press, the consequences extend into the economy.

An estimated "worst case scenario" could translate into losses of up to millions of euros in tourist areas such as the South East of France ( Lemée et al., 2012) and this could be expected in other areas where the economy is based on fishing, aquaculture, ports and/or seasonal tourism attracted by coastal activities. As a result, the genus Ostreopsis has been studied extensively in the Western Mediterranean Sea in the last two decades. The biotic and abiotic factors driving its development are still not fully understood. In the Eastern Mediterranean Sea, related studies remain scarce. In Lebanon, the genus Ostreopsis has been detected since 1979 (Abboud-Abi Saab, 1989). In a previous study, it was identified as Ostreopsis cf. siamensis, based on morphological characteristics (Abboud -Abi Saab et al., 2013); but in 2016, Accoroni et al., using molecular analysis, found mainly the new species named Ostreopsis fattorussoi Accoroni, Romagnoli & Totti and very few Ostreopsis cf. ovata. The presence of this last species was almost negligible (Casabianca S. and Penna A. pers. comm., in Accoroni et al., 2016). It was then likely, but not certain, that Ostreopsis cf. siamensis cited by Abboud-Abi Saab et al. (1989) was a misidentification. The geographic distribution of O. fattorussoi also includes Cyprus, and the same genotype has also been reported prior to its description in Greece, and outside the Eastern Mediterranean in Spain and in Puerto Rico (Penna et al., 2010(Penna et al., ,2014;;David et al., 2013;Accoroni et al., 2016, Tartaglione et al., 2016). As for toxicity, three of the five strains of O. fattorussoi from Lebanon were found to be toxic, producing ovatoxins (OVTX): OVTX-a and structural isomers OVTX-d and -e, so far found only in O. cf. ovata (Accoroni et al., 2016). The same was found for other strains from Cyprus, whereas others produce three exclusive palytoxin-like compounds (OVTX-i, OVTX-j1, OVTX-j2, and OVTX-k;Tartaglione et al., 2016). It is therefore crucial to study the ecology of this newly described species. The present work is the first to study the ecology of O. fattorussoi in Lebanese coastal waters (Eastern Mediterranean) based on both epiphytic and planktonic abundances, through investigation of their temporal and spatial distribution along the coast and their relation to environmental factors. It extends from June 2014 to July 2016, thus covering 3 summer bloom periods in 5 sites along 172 km of the coastline.

MATERIALS AND METHODS

Study area and sampling frequency

Lebanon is situated in the East central part of the Levantine Basin (Eastern Mediterranean), with 220 km of coast, from Aarida in the north to Naqoura in the South. The continental shelf is very narrow 3 to 4 km (Pfannenstiel, 1960), with a mean depth of 20 m to 40 m (Goedicke, 1972). The bottoms are rough with wide rocky patches (Majdalani, 2004), potentially providing support for proliferation of benthic biota. The general circulation along the coast of Lebanon is dominant northwards during most of the year along with the general counterclockwise current gyre of the Eastern Mediterranean (Goedicke, 1972). This current progressively loses force as it approaches eastwards (Nakhlé, 2003). The tide is semidiurnal and is among the lowest in the Mediterranean with a maximum range of 15 to 20 cm (Abboud-Abi Saab, 1985). The current is locally modified by the configuration of the coastline and the topography of the narrow continental shelf. Water movement along the coast is also strongly related to surface currents and seasonal meteorological factors (Goedicke, 1972). As a result, the small scale hydrodynamism is dominated by local winds.

All sampling sites were in exposed rocky shores where west winds can easily be associated to waves and then be a representative of local agitation. Preliminary sampling was conducted in May 2014 at eleven sites in order to survey most rocky beaches of the Lebanese Coast. It revealed the presence of O. fattorussoi in all eleven sites. The monitoring campaign that followed starting June 2014 was limited to five of the sites due to logistic constraints (such as site accessibility year round). The five sites were distributed from North to South and are thus representative of the entire coast. They are labeled as follows (North to South): TRI in Anfeh (close to Tripoli), BAT in Batroun, BYB in Byblos-Jbeil, BEY in Beirut and NAQ in Naquoura (Figure1, Table 1). Sampling was conducted on weekly basis at BAT, and on monthly basis at the remaining sites until December 2015. Additional sampling was conducted at TRI and BAT in the spring and summer of 2016 (Table 1). Streams nearby BYB and TRI are seasonal as most of the water comes from rain and snow melting in the nearby mountains. They potentially bring in municipal wastewater and agricultural effluents rich with nutrients. In the presence of Northern winds, BAT can be affected by the discharges from a chemical plant located 3.9 Km its North (Abboud-Abi Saab, 1985). BYB and TRI are nearby beach resorts. NAQ and BEY are in proximity of small ports.

Environmental parameters:

Air temperature (Tair) was measured in situ using a mercury thermometer. Wind speed (WS) in m/s, and total precipitation in the week preceding sampling date (Rain) were obtained from the website windguru.cz. Wind speed was used as a proxy of water agitation at the sampling date.

Maximum wind speeds reached in the previous week (WSmax) of sampling were also used in each sampling site in order to analyze influence of previous conditions on Ostreopsis development.

Water temperature (Twater) was measured at each site using a mercury thermometer at 10 cm depth. Salinity was measured using the Beckman © RS7-C salinometer. Samples were also collected for the measurements of three nutrients: Nitrites (NO2 -), Nitrates (NO3 -) and Orthophosphates (P-PO4).

They were kept at -20˚C until they were analyzed within a month. Nitrites (NO2 -) concentrations were measured following the methodology described by (Bendschneider and Robinson, 1952) and Nitrates (NO3 -) were obtained according to the methodology described by (Strickland and Parsons, 1968) with a slight modification by Grasshoff (1964). Orthophosphates (P-PO4) concentrations were measured following the methodology described by (Murphy and Riley, 1962).

Biological parameters

Sampling

Ostreopsis fattorussoi being a tychoplanktonic species, both benthic (macroalgae) and planktonic (sea water) samples were collected, in triplicates at three sampling stations assigned 10 m apart on each site. Planktonic samples were collected first, at 20 cm depth (30 cm above the macroalgae) using plastic bottles of 250 ml, with minimal disturbance to the substrate in order to avoid the resuspension of epiphytic O. fattorussoi cells into the water column. For the benthic samples, 9.5 ± 6.0 g of macroalgae were collected along with some surrounding water at the same spot at 50 cm depth, using plastic bottles of 250 ml. Typically, the red macroalgae Ellisolandia elongata (J.Ellis & Solander) K.R.Hind & G.W.Saunders was collected. The choice of macroalgae (Table 1) was based on dominance per site and availability on all sites, and was maintained for the most part throughout the sampling period to limit the influence of different substrata that has been reported by several authors (Totti et al., 2010;Vila et al. 2001). Lugol solution at 1% vol/vol was added to the samples for preservation and they were stored at 4°C.

Quantification of O. fattorussoi

For planktonic samples, after homogenization, subsamples of 50 ml were settled in a counting chamber for 24 hours, according to Utermöhl (1958). Counting was performed using an inverted microscope Wild M40. Cell abundances were expressed in number of cells per liter (cells/L). For benthic samples, the sample was shaken vigorously for 10 seconds in order to dislodge the cells from the macroalgae. The macroalgae was then separated from the water using a 200 µm mesh sieve, and rinsed twice with 100 ml of filtrated sea water, for the removal of any remaining epiphytic O. fattorussoi cells. The total water (sample + rinsing water) was homogenized using a Motoda box. This box is a splitting apparatus (often used for zooplankton samples) allowing the separation of homogeneous subsamples (Motoda, 1959). One final 50 ml subsample was kept at 4°C. Counting was done using a 1ml Sedgwick-Rafter © counting chamber and an optic microscope (Motic BA 400). Cell abundance was expressed in number of cells of O. fattorussoi per gram of fresh weighed macroalgae cells/g FW. Identification of Ostreopsis cells at the species level was carried out in some samples through observation of thecal plates' arrangements using calcofluor staining and epifluorescence microscope (Figure 2), since O. fattorussoi does show some morphological features that lead unequivocally to its identification (Accoroni et al., 2016).

At least two samples per site, selected from different bloom events during different seasons were observed for this purpose.

Statistical analysis:

All statistical analysis was applied using R programming language version 3.3.2 and its user interface R studio. Weekly data obtained in BAT were included in the graphs but excluded from statistical analysis as certain corresponding environmental data were not available; monthly data from this site were included in the analysis. As O. fattorussoi was sampled in triplicates, the site average was used in the dataset. The Shapiro test of normality was applied to each variable (benthic and planktonic O. fattorussoi abundances as well as all available environmental variables).

Accordingly, the non-parametric spearman rank correlation test was applied to a matrix including all the above mentioned variables in order to determine their relationships. The same test was repeated per site. Kruskal-Wallis non-parametric test of the analysis of variance was applied to both planktonic and and benthic abundances of O. fattorussoi in order to evaluate possible spatial variations between the sites; a Friedman non-parametric repeated measurement test was also applied at each site to determine temporal variability. A PCA (Principle Component Analysis) was applied using environmental variables as active variables, benthic and planktonic abundances of O. fattorussoi as quantitative illustrative variables, and sites as qualitative illustrative variable.

Analyses were based on Euclidean distance-transformed data.

RESULTS

Environmental parameters

Climatological parameters

Overall, measured air temperature (Tair) varied between a minimum of 14°C (BAT and BYB, Jan 2015) and a maximum of 36.3°C (NAQ, May 2015) (Table 2). It varied following a typical seasonal pattern in all 5 stations as the highest temperatures were recorded in the summer and lowest in winter (Figure 3 followed by a max of 79.9 mm/week in BAT (Jan 2015) (Figure 3(d)). Summer was the dry season and maximum precipitation for each site was in January.

Hydrological parameters

Water temperature (Twater) ranged between a minimum of 17 °C (BAT, Mar 2015) and a maximum of 32°C (BAT, Aug 2014). In all 5 sites, Twater variation followed a typical seasonal pattern with the highest temperatures in the summer and lowest in winter (Figure 4

Nutrients

Nitrites concentration ranged between a minimum of 0.008 µmol/L (TRI, Jun 2015) and a maximum of 0.37 µmol/L (NAQ, Aug 2014) (Table 2). They were generally highest in BEY, and lowest in TRI and NAQ (Figure 6 (a)). Nitrates concentration ranged between a minimum of 0.06 µmol/L (TRI, Mar 2016) and a maximum of 25.12 µmol/L (NAQ, Jan 2015) (Table 2) 2). They remained below 0.5 µmol/L in all sites except for occasional peaks in the summer (Figure 6(c)).

Temporal and spatial variation in O. fattorussoi abundances

Observation of thecal plates' arrangements confirmed the exclusive presence of a single species:

Ostreopsis fattorussoi. The species demonstrated a perennial presence in all the years of the study along the Lebanese coast, both in planktonic and benthic samples, although it was occasionally undetected in some of the sites during the winter (Figures 7 &8). Lowest abundances were recorded typically in winter months, and increases in abundance were noted starting early spring (Figure 8). The most important benthic blooms in terms of maximum abundances reaching more than 200 000 cells/g FW (i.e. major blooms) occurred almost exclusively during the summer. Less intense blooms with maximum abundance not exceeding 50 000 cells/g FW (i.e. minor blooms) occurred in autumn, with the exception of one autumn bloom at NAQ in 2014 which reached 228 257 cells/g FW. In 2014, important benthic summer blooms occurred at all of the sites (often more than 200 000 cells/g FW), whereas in 2015, such abundances were only recorded at TRI, BYB and NAQ. In 2016, data was only available for TRI and BAT; in both cases, summer blooms of 2016 (TRI and BAT) were minor in terms of abundance (< 50 000 cells/g FW). In addition, minor spring blooms were detected in BAT (April and May). Planktonic abundances generally followed the same pattern as the benthic ones. In addition, planktonic blooms were observed in BAT and BEY in the summer of 2015, where benthic blooms were not detected (Figure 7). Yearly maximum abundances were variable; in 2014, they reached 880 694 cells/g FW (BEY, June) and 9 000 cells/L (BAT, June & July), as opposed to 397 242 cells/g FW (BYB, July) and 18 065 cells/ L (NAQ, June) in 2015, then 53 228 cells/g FW and 4 346 cells/L (BAT) in 2016. The ANOVA Friedman test showed no significant difference in cell abundances in either of the sites, neither in terms of seasons nor in terms of years (p > 0.05). Spearman rank correlation test showed a very strong significant correlation between benthic and planktonic O. fattorussoi (r = 0.88, p < 0.01) (Table 3).

In 2015, weekly sampling in BAT allowed the detection of two consecutive major blooms, during the second half of June then during the second half of July. In 2016, minor summer blooms occurred in both sampled sites, reaching their maximum abundances of 32 262 cells/g FW (BAT) and 45 398 cells/g FW (TRI) mid-June. Planktonic abundance followed the same pattern with a relatively high maximum of 4 346 cells/L (BAT). Spring blooms were detected in BAT starting in April and reached a maximum benthic abundance of around 50 000 cells / g FW both in April and in May.

The Kruskal-Wallis test showed that no significant difference was noted between the 5 sites for both benthic (Kruskal-Wallis chi-squared = 104.04, p-value > 0.05) and planktonic abundances (Kruskal-Wallis chi-squared = 70.061, p > 0.05).

Relation between O. fattorussoi abundances and environmental parameters

Water temperature was strongly correlated with benthic abundances of O. fattorussoi (r = 0.61, p < 0.01) and moderately correlated with planktonic abundances (r = 0.49, p < 0.01) when considering all 5 sites; however, it varied between the sites from very strong in TRI (r =0.8, p < 0.05) to non-significant in NAQ (p > 0.05) (Table 3). A weak positive correlation was noted between wind speed and planktonic abundances (r = 0.26, p < 0.01), but none with WSmax. Strong negative correlation with precipitation was noted only in BEY (Table 3). No significant correlation was noted with the measured nutrients, with the exception of a moderate negative correlation between nitrates and benthic abundances in BYB (r = -0.53, p < 0.05) and BEY (r = -0.59, p < 0.05) (Table 3). The first two principal components (PC) of the PCA analysis (Figure 9) amount to a cumulative percentage of the variance of 61.5%. The first PC (29.37%) is primarily a measure of Twater and Salinity, and is strongly but negatively correlated to Nitrates. The second PC (18.95%) mainly reflects wind speed, and is strongly correlated to WSmax. The PCA did not reveal a significant influence of a specific environmental parameter on O. fattorussoi abundances.

DISCUSSION

Temporal and spatial variation in O. fattorussoi abundances

During this study, bloom occurrences of O. fattorussoi were site and year specific, but a clear seasonal pattern was established. Typically, cells of Ostreopsis fattorussoi were barely detectable in winter. An increase in abundance was visible starting early spring, and blooms occurred in the summer with maximum abundances exceeding 200 000 cells / g FW occurred, followed by less intense autumn blooms in terms of maximum abundances as they remained at 50 000 cells / g FW or below. This succession of a summer bloom followed by a less intense autumn bloom has been reported in 2001 in the same area in a study that surveyed planktonic abundances only Abboud -Abi Saab et al., 2013) (Table 4); it has also been reported for O. cf. ovata in the North Western Mediterranean Sea (Mangialajo et al., 2011). In terms of timing of the blooms, summer blooms of O. fattorussoi detected in this study occur early compared to all other Mediterranean sites (Table 4). In addition, a couple of blooms were recorded as early as April and May, making them -along with one occurrence in the Aegean Sea (Aligizaki and Nikolaidis, 2006), the only spring blooms of Ostreopsis spp. reported in the Mediterranean Sea (Table 4). In terms of bloom intensity, maximum abundances reached during blooms of a species belonging to genus Ostreopsis in the Mediterranean Sea are variable. Both benthic and planktonic abundances recorded during this study remained considerably lower than those of Northern Mediterranean Sea, where the species O. cf. ovata is predominant, as they reach millions of cells/g FW in benthic samples, and exceed 10 5 cells/L in planktonic samples (Table 4). In this study however, maximum abundances remained at 18 000 Cells/L and below 400 000 cells / g FW, with only one exception (880 694 x10 5 cells / g FW at BEY). It was worth indicating that as maximum abundances are year and site specific in NW Mediterranean sea, some sites presented lower maximum abundances than neighboring sites (Mangialajo et al., 2011), that were similar to those of O. fattorussoi in this study. In contrast, maximum O. fattorussoi abundances reached in this study exceed those reported in the Eastern and Southern Mediterranean. Outside the Mediterranean Sea, maximum abundances are variable.

Compared to this study, they were much higher in New Zealand (Shears and Ross, 2009), and much lower in Mexico (Okolodkov et al., 2007) and in the Sea of Japan (Selina et al., 2014).

Nevertheless, comparing maximum benthic abundances of Ostreopsis species is always difficult, since biotic substrates preferences can heavily affect this parameter, even at low spatial scale (Cohu et al., 2011). Data from the more frequently sampled site (BAT) suggest that bloom duration is around one month, and bloom decline occurs within a week from reaching maximum abundances. Longer blooms such as the ones occurring in NAQ are not uncommon in the North western Mediterranean Sea (Mangialajo et al., 2011).

Influence of environmental factors on O. fattorussoi abundances

Temperature:

Our data suggests that water temperature plays a major role in the development of O. fattorussoi.

We found a moderate and strong positive correlation between water temperature and planktonic and benthic cell abundances, respectively. Maximum abundances per site were reached at relatively high water temperature (27°C < TCmax < 30.5°C). In the Mediterranean Sea, several studies have linked optimum growth of Ostreopsis spp. to relatively high temperatures (Cohu et al., 2013;Mangialajo et al., 2008;Sansoni et al., 2003;Simoni et al., 2003;), whereas others did not find a correlation between temperatures and its abundances (Accoroni et al., 2011(Accoroni et al., & 2012;;Cohu et al., 2011;Mangialajo et al., 2011;Totti et al., 2010;Vila et al., 2001). In the present study, O. fattorussoi was found year round, thus in all the measured range of temperature during the study (17°C to 32°). We might consider 18°C (threshold temperature associated with cell abundances of ≥1 000 cell/g FW) triggers a swifter proliferation of available cells in their vegetative state. In the present study, maximum abundance was recorded as water temperature was still increasing, whereas in Adriatic Sea, maximum abundances of O. cf. ovata were recorded as water temperature is decreasing (Accoroni et al., 2012(Accoroni et al., & 2015;;Totti et al., 2010). In both cases, water temperature corresponding to the maximum of abundance of O. fattorussoi TCmax is lower than maximum temperature recorded Tmax (in the present study in each of the 5 sites). The different TCmax per site could be due to the presence of different strains of O. fattorussoi on different sites; in fact, this is not unusual for different species, as several studies have showed species, clades or subclades to respond differently in terms of temperature (Guerrini et al., 2010;Pezzolesi et al., 2012;Tawong et al., 2014).

Nitrites, Nitrates and orthophosphate

In this study, taking into account the entire dataset, we found no correlation between O. fattorussoi abundances and any of the measured nutrients. Orthophosphate concentrations in BAT did not seem to be affected by the nearby chemical factory. We did however find a moderate negative correlation when the correlation test was performed on site basis, between nitrates and benthic abundances (in BEY and BYB). These results are inconsistent with a previous study in the same area (3 sites in common) that found a positive correlation of O. cf. siamensis abundances with Nitrites and Nitrates, and a negative correlation with orthophosphate (Abboud -Abi Saab et al., 2013). Results were also contradictory in the Mediterranean, and the link between nutrients and Ostreopsis spp. has yet to be established. Several studies found no evidence of a role of nutrients in the bloom dynamics of Ostreopsis spp. (Accoroni et al., 2011(Accoroni et al., & 2012;;Vila et al., 2001). (Carnicer et al., 2015) found a negative correlation between O. cf. ovata and nitrates, whereas, despite the absence of correlation, (Cohu et al., 2013) has reported a positive association between Phosphate concentration, (rather than nitrogen or silicate), and O. cf. ovata. Accoroni et al. (2015) found that N/P ratio around the Redfield value was necessary for cell proliferation of O. cf. ovata. The lack of a statistically significant correlation in these studies as well as the present one was not unexpected, as during the bloom cells were presumably consuming the nutrients (Accoroni et al., 2015). Ammonium was not analyzed in the present study, while this nutrient was found to be important Ostreopsis species growth in a recent study (Jauzein et al., 2017).

This could have influenced the present interpretation. More generally, analysis of every supplementary parameter, including organic sources, could be useful for interpretation when studying the ecology of Ostreopsis species, since at least one species has been proved to be mixotrophic (Ostreopsis cf. ovata;Jauzein et al., 2017).

Cell Resuspension and Wind

In this study we found a strong positive correlation between benthic and planktonic abundances of O. fattorussoi, as confirmed by Vila et al. (2001) for Ostreopsis sp. also on samples of the macroalgae Ellisolandia elongata. A relatively good correlation between the two is also reported in the Mediterranean Sea for O. cf. ovata and O. cf. siamensis (Aligizaki and Nikolaidis, 2006;Mangialajo et al., 2008Mangialajo et al., & 2011)). This could be due to re-suspension of epiphytic cells into the water column caused by waves (Mangialajo et al., 2011;Totti et al., 2010;Vila et al., 2001) as reflected by the positive correlation between planktonic abundances and wind speed, which in turn influences water agitation on a local level. Re-suspension is also brought on due to human activity causing movement of the substrata (Mangialajo et al., 2011), or due to daily cycle vertical migrations (Vila et al., 2008). Cell resuspension might partially explain the absence of a major benthic bloom at BAT and BEY in the summer of 2015, despite the occurrence of a planktonic one. Another explanation could be due to the heterogeneity of Ostreopsis sp. distribution on a small scale (within a few meters) (Jauzein et al., 2018), revealed in this study by high standard deviation of O. fattorussoi abundance between triplicate samples (Figures 7 and8). In addition, there was a positive correlation between wind speed and benthic abundances of O. fattorussoi at TRI; as water agitation at each site could be influenced by local winds, these results suggest that the species might prefer some agitation, as also observed for Ostreopsis spp. in other studies (Selina et al., 2014;Vila et al., 2001). The effects of water agitation on blooms of Ostreopsis spp. in the Mediterranean Sea might be variable, as it has been suggested that has a negative effect as higher abundances are found in sheltered or calm areas (Accoroni et al., 2012;Barone, 2007;Mangialajo et al., 2008;Totti et al., 2010), as wind and waves could disperse the bloom (Cohu et al., 2011;Accoroni and Totti, 2016). A single environmental parameter did not solely influence the development of O. fattorussoi, but it is a combination of several factors; the PCA did not highlight the influence of a specific environmental factor on its abundance. This has also been reported for O. cf. ovata (Accoroni et al., 2015;Mangialajo et al., 2008).

Conclusion

The present study found that O. fattorussoi is present year round in Lebanese coastal waters, with seasonal blooms along the coast. A major bloom occurred early in the summer (June-July), followed by a second minor one in the fall (October-November). Results showed that a combination of several environmental factors affect its development. It was revealed to be a thermophilic species, as the highest concentrations were reached between 27°C and 30.5°C depending on the site. A minimum of 18°C is suggested as a threshold temperature promoting proliferation of O. fattorussoi. Moreover, its development seemed slightly facilitated by some water agitation due to wind. Results concerning the role of nutrients and other environmental parameters still inconclusive. Major blooms coincide with the summer where beaches are most frequented and minor blooms coincide with autumn, a busy period for recreational fishing (Sharif Jemaa, personal communication, June 6 th , 2016). It is therefore recommended to maintain a monitoring program throughout the year, focusing on benthic samples as they are more conservative (Lemée et al., 2012). It should be intensified mid spring until the end summer in order to allow more thorough ecological studies of all phases of the bloom. It is also important to assess its toxins in the food chain, as it is customary for local inhabitants of the coastal zone to consume certain species of macroalgae and seafood including filtrating sea urchins and shellfish (Patella sp.). Cases of intoxication, if any, would have gone undiagnosed due to the lack of awareness on the issue in public health officials and medical practitioners as well as the general public in Lebanon.

Declarations of interest: none

Acknowledgments:

The present work was done with the financial assistance of the European Union under the ENPI CBC Mediterranean Sea Basin Program, within the project M3-HABs (Project Reference Number: IIB/2.1/0096). Data analyses and writing of the manuscript were done with the financial support of the GDR Phycotox project.

Table of Figures

Table of tables

  (a)). Wind speed ranged between a minimum of 0 m/s (Dec 2015, BAT) and a maximum of 13 m/s (Feb 2015, NAQ) (Figure 3(b)). Maximum Wind speed recorded in the previous week (WSmax) ranged between 3 m/s (Dec 2015, BEY) and 17 m/s (Jan 2015, BAT). WSmax was generally lower in the autumn months. WSmax was also highest in the most northern site TRI, (Figure 3(c)). Total precipitation during the previous week reached a maximum of 125 mm/week (NAQ, Jan 2015),

  (a)). In TRI the autumn of 2014 water temperature remained high throughout autumn. High water temperatures also stood out at BAT in the summer of 2014 and spring of 2016. In each site, water temperature corresponding to the maximum of abundance of O. fattorussoi (TCmax) never exceeds the maximum temperature (Tmax) reached on site (Figure 5 (a)). A gradient of TCmax vs. latitude was observed, as TCmax increases from south to north (Figure 5(b)). The average measured salinity was 38.6 ± 1.3, ranging between a minimum of 34.5 (Jan 2015, BYB) and a maximum of 39.6 (Oct 2014, TRI). Salinity was higher in the most northern sites TRI and BAT as it rarely drops below 38.5; It was lower in NAQ (South) and BYB as it barely reaches 39.1 (Figure 4(b)).

  ; it was highest in NAQ and BYB and lowest in BAT and TRI (Figure 6(b)). Orthophosphates concentration ranged between a minimum of 0.01 µmol/L (BEY, Sep 2014) and a maximum of 3.53 µmol/L (NAQ, Aug 2015) (Table

Figure 1 -Figure 2 -

 12 Figure 1 -The position of Lebanon on the Eastern Mediterranean coast as well as the location of

Figure 3 -Figure 4 -Figure 7 -Figure 8 -

 3478 Figure 3 -Variation of climatological parameters: (a): Air temperature "Tair" (°C), (b): Wind speed

Figure 1 -Figure 2 -

 12 The position of Lebanon on the Eastern Mediterranean coast as well as the location of the sampling sites during the study (preliminary and regular sampling sites) Light micrographs of calcofluor stained Ostreopsis fattorussoi cells under UV light, showing the tabulation. Left: epitheca from TRI (October 2015). Right: hypotheca from BEY (October 2015). Apical pore Po (left image, under 2') and 1' plate forms are characteristic of O. fattorussoi (see description in Accoroni et al., 2016). DV: left (75 µm) and right (65 µm).

Figure 3 -Figure 1 -Figure 1 -

 311 Figure 1 -Position of Lebanon on the Eastern Mediterranean coast as well as the location of the sampling sites during the study (preliminary and regular sampling sites)

Figure 2 -

 2 Figure 2 -Variation of climatological parameters: (a): Air temperature "Tair" (°C), (b): Wind speed "WS" (m/s) and (c): Maximum wind speed reached during the previous week "WSmax" and (d): Precipitation in the previous week "Rain WB" (mm)

Figure 7 -

 7 Figure 7 -Variation of O. fattorussoi abundances (Cells/L) in planktonic sea water samples at 20 cm depth in the 5 sites during the sampling period

Table 1 -

 1 Description and geographical coordinates (in decimal degrees) of the five sampling sites

Table 2 -

 2 Descriptive statistics (average ± Standard deviation and Minimum-Maximum) of all variables in the 5 sites

Table 3 -

 3 Spearman rank correlation between O. fattorussoi abundances and environmental variables

Table 4 -

 4 Maximum benthic abundances (MaxB) (cells/g FW) and maximum planktonic abundances (MaxPl) (Cells/L) of Ostreopsis spp. reported in various areas in and outside the Mediterranean Sea.

Table 1 -

 1 Description and geographical coordinates (in decimal degrees) of the five sampling sites

	Site	Latitude Longitude	Site description	Sampling end date	Sampling frequency	Dominant macroalgae
	TRI	34.3675	35.736	Rocky	Jul 2016	Monthly*	E. elongata
	BAT	34.2515	35.6568	Rocky	Jul 2016	Weekly	E. elongata
	BYB	34.1144	35.6482	Sandy/ Rocky Dec 2015	Monthly	Variable
	BEY	33.902	35.4753	Rocky	Dec 2015	Monthly	E. elongata
	NAQ	33.1284	35.1458	Rocky	Dec 2015	Monthly	E. elongata

Table 3 -

 3 Spearman rank correlation between O. fattorussoi abundances and environmental variables

		All sites	TRI		BAT		BYB		BEY
		OstreoPl	OstreoB	OstreoPl	OstreoB	OstreoPl	OstreoB	OstreoPl	OstreoB	OstreoPl	OstreoB
	OstreoPl (Cells/ L)	1**		1**		1**		1**		1**
	OstreoB (Cells / g FW)	0.88**	1**	0.92**	1**	0.82**	1**	0.71**	1**	0.89**	1**
	Twater (°C)	0.49**	0.61**	0.8**	0.83**	0.43*	0.52**	0.38	0.52*	0.6**	0.8**
	Tair (°C)	0.41**	0.47**	0.67**	0.66**	0.38	0.43*	0.36	0.43	0.51 *	0.67**
	Salinity	0.25**	0.31**	0.52**	0.54**	-0.04	0.16	0.26	0.34	0.29	0.53*
	ws (m / s)	0.26**	0.19	0.42*	0.39*	0.46*	0.21	0.19	0.32	0.3	0.27
	WSmax (m / s)	-0.07	-0.12	-0.3	-0.35	0.27	0.22	0.05	0.21	-0.14	-0.17
	Rain WB (m / s)	-0.38** -0.38**	-0.29	-0.37	-0.32	-0.27	-0.39	-0.24	-0.72** -0.82**
	Orthophosphates (µmol/L)	0.13	0.1	0.33	0.26	0.36	0.22	-0.08	0.12	0.08	0.08
	Nitrites (µmol / L)	0.04	0.1	0.11	0.23	0.07	0.05	-0.12	-0.05	0.16	0.21
	Nitrates (µmol / L)	-0.06	-0.05	0.03	0.16	-0.09	-0.08	-0.34	-0.59*	-0.55	-0.53*

Table 4 -

 4 Maximum benthic abundances (MaxB in cells/g FW) and maximum planktonic abundances (MaxPl in Cells/L) of Ostreopsis spp. reported in various areas in and outside the Mediterranean Sea.

	Area		Species	Year	Timing	MaxB	MaxPl	R
		Italy (Ligurian Sea)	O. cf. ovata	2006	Jul	2.5x10 6	87x10 3	M
		Spain	O. cf. ovata & O.cf. siamensis	2008	Oct	7.24x10 6	6.44x10 3
		Spain	O. cf. ovata & O.cf. siamensis	2009	Jul	1.0x10 6	385x10 3
	NW MS	Monaco	O. cf. ovata ***	2007	Jul	1.56x10 6	20.9x10 3	M
		France	O. cf. ovata ***	2009	Jul	1.61x10 6	12x10 3
		France	O. cf. ovata ***	2008	Jul	5.1x10 5	104x10 3
		France	O. cf. ovata	2008	Jul	8.54x10 6	7x10 3	Co
		Italy (Conero Riviera)	O. cf. ovata	2010	Aug	1.2x10 6	102x10 3
	NA Sea	Italy (Conero Riviera) Italy (Conero Riviera)	O. cf. ovata O. cf. ovata	2011 2012	Aug Sep	1.9x10 6 1.9x10 6	39x10 3 77.9x10 3	A
		Croatia	O. cf. ovata **	2010	Oct	3.34x10 5	42.6x10 3 Pf
		Tunisia	O.cf. siamensis	2001	Aug-Sep	3.6x10 3	n.a.	Tu
	S MS	Tunisia Tunisia	O. cf. ovata O.cf. siamensis	NA 2008-09	Jul Jul & Jan*	n.a. 1 to 5	84 n.a.	Be M
		Algeria	Ostreopsis spp. **	2009	Jul	7.9x10 4	5.9x10 3	Ill
		Egypt	O. cf. ovata & Ostreopsis morph1.	2005	Jul	9x103	n.a.	Is
		Greece (Aegean Sea)	O. cf. ovata & O.cf. siamensis	2003	Sep	4.05x10 5	16x10 3	A
		Greece (Aegean Sea)	O. cf. ovata	2005	May	n.a.	0.6x10 3	Sp
	East MS	Lebanon	O.cf. siamensis	2001 2001	July Sep	n.a. n.a.	10.5x10 3 7.7x10 3	A
			O. fattorussoi	2014 2015	Jun Jun	8.8x10 5 2.64x10 5	1.7x10 3 18.10 3	Pr
		New Zealand	O. siamensis	2004	Feb	1.41x10 6	n.a.	Sh
	Non MS	Sea of Japan	O. ovata & O. siamensis	2010	Sep	52x10 3	n.a.	Se
		Mexico	Ostreopsis heptagona	2005	May-Jun	1x10 3	n.a.

O

Footnotes: *: Sampling limited to these two months; **: Associated with toxic events; ***: Sporadic cooccurrence of O. siamensis is possible; Bold = maximum abundance; MS: Mediterranean Sea; Non MS:

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