Sedigheh Mohammadzadeh

Sakineh Yeganeh
email: skyeganeh@gmail.com

Fatemeh Moradian

Sylvain Milla

Bahram Falahatkar

Spawning induction in Sterlet sturgeon (Acipenser ruthenus) with recombinant GnRH: Analysis of hormone profiles and spawning indices

Highlights

1. rGnRH had a positive effect in final maturation in Sterlet sturgeon.

2. rGnRH at the dose of 10 µg kg body weight -1 was efficient in spawning induction.

3. rGnRH can stimulate the secretion of sex-steroids in vivo.

4. rGnRH can be used as a specific homologue to treat reproductive disorders in fish.

Introduction

Sterlet sturgeons (Acipenser ruthenus) are one of the 27 sturgeon species that are endangered worldwide [START_REF] Muller | Attempts on artificial induction of sexual maturation of sterlet (Acipenser ruthenus) and identification of late spermatogenesis stage in hermaphroditic fish[END_REF]. They are promising candidates for freshwater aquaculture production and they have several indisputable advantages compared with the other sturgeon species. They require smaller space in captivity due to smaller size and they display a quicker sexual development [START_REF] Akhavan | Changes of vitellogenin and lipase in captive Sterlet sturgeon Acipenser ruthenus females during previtellogenesis to early atresia[END_REF][START_REF] Abdollahpour | Influence of thyroxine on spawning performance and larval development of Sterlet sturgeon Acipenser ruthenus[END_REF]. In comparison with lengthy gonadal cycle in the large migratory species, they are an ideal model to study the reproductive physiology of sturgeon due to earliest maturation [START_REF] Akhavan | Changes of vitellogenin and lipase in captive Sterlet sturgeon Acipenser ruthenus females during previtellogenesis to early atresia[END_REF]. However, the broodstock reproduction and the larval rearing in captivity are two major bottlenecks for its aquaculture development [START_REF] Abdollahpour | Influence of thyroxine on spawning performance and larval development of Sterlet sturgeon Acipenser ruthenus[END_REF]. In captivity, sturgeons do not spawn and this dysfunction may result from the combination of captivity-induced stress and absence of suitable natural spawning condition [START_REF] Alavi | Sperm biology and control of reproduction in sturgeon: (I) testicular development, sperm maturation and seminal plasma characteristics[END_REF]. Intramuscular administrations of pituitary extracts [START_REF] Linhart | Ionic composition and osmolality of paddlefish (Polyodon spathula, Acipenseriformes) seminal fluid[END_REF][START_REF] Piros | Biochemical characterization of Siberian sturgeon Acipenser baeri and sterlet, Acipenser ruthenus, milt plasma and spermatozoa[END_REF] and GnRHa [START_REF] Linhart | Spermiation of paddlefish (Polyodon spathula) stimulated with injection of LHRH analogue and carp pituitary extract[END_REF][START_REF] Williot | An analogue of GnRH is effective for induction of ovulation and spermiation in farmed Siberian sturgeon, Acipenser baerii Brandt[END_REF][START_REF] Van Eenennaam | Induced spawning, artificial fertilization and egg induction techniques for Green sturgeon[END_REF][START_REF] Nazari | Application of synthetic hormone LHRH-A 2 on the artificial propagation of Persian sturgeon Acipenser persicus[END_REF][START_REF] Roosta | Comparative study on hormones and biochemistry indices in plasma, ovarian fluid and oocytes of LHRHa2-induced female Stellate sturgeon (Acipenser stellatus)[END_REF] have been used to induce final maturation in both sexes. Various GnRH analogues with different half-lives exist and they are used with differential efficiency for inducing final sexual development in sturgeon broodstock.

GnRH is the main hypothalamic hormone regulating the synthesis and release of gonadotropins [START_REF] Nagahama | Regulation of oocyte maturation in fish[END_REF][START_REF] Gaillard | Characterization of Gonadotropin-Releasing Hormone (GnRH) Genes From Cartilaginous Fish: Evolutionary Perspectives[END_REF](. At the N-terminal extremity of the precursor structure of the GnRH protein, there is a signal peptide, followed by the decapeptide, which is a bioactive site of the protein and is almost entirely preserved in all fish species. Next, there is a proteolytic site, which is conserved in all vertebrates. Ultimately, at the C-terminal extremity, there is a GnRH-associated peptide (GAP) that it is composed of 60 amino acids approximately, which is less preserved in fish than other regions of the GnRH protein (Zmora et al., 2002). Previous studies indicated that GAP is involved in the stabilization of GnRH structure and that it is not related to the biological function of the protein [START_REF] Andersen | The salmon GnRH encoding gene in teleost fish[END_REF].

In synthetic GnRH analogues, the biological active site (decapeptide region) is used for the production of a hormone, because GAP region would not be involved in the biological activity of GnRH. In addition, the amino acid sequences of GAP are long, so it is not economical to produce by synthetic method. Apparently, a reliable, more effective, available and cheap product, useful to induce the final maturation, is required [START_REF] Aizen | Biologically active recombinant carp LH as a spawning inducing agent for Carp[END_REF]. Therefore, recombinant protein methods are used instead of synthetic production for managing the costs [START_REF] Mohammadzadeh | Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish[END_REF]. The potential for the generation of recombinant hormones has been achieved in recent years due to recent advances in construction cDNA encoding target protein. Different studies have shown in vivo and in vitro effects of GnRH analogues or recombinant proteins to treat the reproduction disorders in fish. In tench (Tinca tinca), treatment with GnRHa with or without a dopamine antagonist increased luteinizing hormone levels and induced spawning [START_REF] Podhorec | The effects of GnRHa with and without dopamine antagonist on reproductive hormone levels and ovum viability in tench Tinca tinca[END_REF]. Intramuscular administrations with different synthetic GnRH analogues and their combinations with an anti-dopaminergic compound were also used to induce final maturation in sterlet sturgeon [START_REF] Ronyai | Effect of different synthetic gonadotrop-releasing hormone analogues and their combinations with an anti-dopaminergic compound on the reproduction performance of sterlet (Acipenser ruthenus L.)[END_REF]. In the orange-spotted grouper (Epinephelus coioides) or common carp (Cyprinus carpio), treatments with recombinant luteinizing hormone (LH) altered the hormonal levels along the hypothalamus-pituitary-gonad axis [START_REF] Cui | Production of recombinant orange-spotted grouper (Epinephelus coioides) luteinizing hormone in insect cells by the baculovirus expression system and Its biological effect[END_REF][START_REF] Aizen | Biologically active recombinant carp LH as a spawning inducing agent for Carp[END_REF]). An injection of rGnRH recently increased the plasma levels of 17α, 20βdihydroxy-4pregnen-3-one, which led to final maturation induction in goldfish (Carassius auratus) [START_REF] Mohammadzadeh | Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish[END_REF]. Seemingly, producing rGnRH with suitable biological function could pave the way for introducing an effective hormone therapy in aquaculture and could be applied as specific analogues to treat reproductive disorders in some related species for developing their aquaculture production. Due to the positive features regarding rGnRH for inducing final maturation in breeding female fish [START_REF] Mohammadzadeh | Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish[END_REF], this study tested rGnRH as an effective agent to stimulate sex steroids, inducing final maturation and spawning in Sterlet sturgeon.

Material and methods

Production of recombinant GnRH

The amino acid sequence of pre pro cGnRH Beluga (Huso huso) (GenBank: EF534706.2) was selected without its signal peptide. Some amino acids (1, 6 and 7) were changed in the decapeptide sequence to increase the half-life and the stability of the peptide. The GAP region was considered for this sequence in order to increase the stability of recombinant protein in production, extraction, and purification procedures. This gene sequence was synthesized and then cloned on the expression vector pET28a. Recombinant vector pET28a/GnRH was transformed into expression host Escherichia coli BL21 (DE3). The supernatant of cultured recombinant bacteria was applied for the purification using Batch/Gravity-Flow column purification with TALON metal affinity resin. The solubilized protein was loaded on Co 2+ -CMA, TALON resin (Clontech) at 4 °C for 1 h on a platform shaker to allow the His-tagged protein to bind the resin. Then, the column was washed with 10 bed volumes by washing buffer (50 mM Tris, 300 mM NaCl, 10 mM Imidazole and 0.05% Triton x-100 at pH 8.0). Finally, the His-tagged protein was eluted with 5 bed volumes elution buffer (50 mM Tris, 150 mM Imidazole and 1% (v/v) Triton x-100, pH 7.5) at a flow rate of 1 mL min -1 and before molecular characterization [START_REF] Mohammadzadeh | Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish[END_REF]. rGnRH quantity was determined by Bradford's method (1976) with bovine serum albumin as a standard. The lyophilized peptide was dissolved in saline solution (Nacl 0.9 %, pH 7) and assayed with Bradford buffer.

Broodstock and holding system

This study was conducted at Dr. Yousefpour Marine Fishes Restocking and Genetic Conservation Center (Siahkal, Guilan, Iran). Sterlet sturgeons were originally obtained from Hungary and then reproduced two generations in captivity. In this center, Sterlet sturgeon breeders were kept in round concrete tanks (185 cm diameter, 35 cm depth, and 940.7 ± 0.2 L volume). Water was supplied by river with a constant flow rate of 13 ± 0.3 L min -1 with a natural photoperiod and thermal regime. During the trial early in spring in April (14 h light/ 10 h dark), dissolved oxygen and water temperature was 12.5 ± 0.7 mg L -1 and 14.5 ± 0.5 ºC respectively.

The breeders were fed with commercial sturgeon feed (Fara Daneh, Shahrekord, Iran) twice a day (9:00 and 19:00). The pelleted diet included 45% protein, 17% lipid, 4% fibre and was 8 mm in diameter.

Ovulation induction and experimental design

Female broodstocks were checked for maturity stage. To this aim, 400 ppm clove powder extract was applied for anesthetizing the fish [START_REF] Ghiasi | Physiological changes and reproductive performance of Sterlet sturgeon Acipenser ruthenus injected with thiamine[END_REF]. A steel probe (3-4 mm diameter and 3-6 cm groove length) was used to sample a gonad part. For this purpose, it was placed between the 3rd and 4th ventral scute [START_REF] Abdollahpour | Hormonal and physiological changes in Sterlet sturgeon Acipenser ruthenus treated with thyroxine[END_REF].

Germinal vesicle (GV) position was measured for selection of appropriate broodstock and injection time according Chapmman and Van Eenennaam (2007). Briefly, 20-30 oocytes were collected that were immediately well-kept in 10% neutral buffered formalin for 1 hour and they were boiled for 5 min and then cut through the vegetal pole (VP) to the animal pole (AP). A dissecting microscope (Olympus, Tokyo, Japan) was used to measure the distances form VP to AP and from AP to nucleus under. For calculating germinal vesicle migration (GVM), GVM was examined according to this formula: GVM = distance between nucleus and AP / distance between VP and AP (X100)

The gonad maturity of all sampled females proved to be in the pre-spawning stage IV (GV = 8.63 ± 1.74); thus, it was decided to perform the trial.

Based on this indicator, 24 seven year-old farmed females with mean weight (± SD) of 1200.9 ± 9.2 g, total length 60.3 ± 0.7 cm were selected for the spawning experiment. Female breeders were randomly distributed into eight tanks (185 cm diameter, 35 cm depth, 940.7 ± 0.2 L volume) with three fish per tank (four treatments with two replicates). Upon the optimum water temperature for spawning (15.5-16.5°C), the fish was injected with NaCl 0.9% (control); 2.5 µg kg body weight -1 LHRH-A2 (L2.5, pGlu-His-Trp-Ser-His-Gly-Ttp-Arg-Pro-Gly-NH2; San Shen Ningbo, Sheng, China,) [START_REF] Ghiasi | Physiological changes and reproductive performance of Sterlet sturgeon Acipenser ruthenus injected with thiamine[END_REF]; 2.5 µg kg body weight -1 rGnRH (rGn2.5) and 10 µg kg body weight -1 rGnRH (rGn10). The doses were chosen according to the common practices of sturgeon propagation in Iran which use LHRH analogues in the range of 2.5-50 µg kg body weight -1 [START_REF] Ghiasi | Physiological changes and reproductive performance of Sterlet sturgeon Acipenser ruthenus injected with thiamine[END_REF][START_REF] Sarameh | The effect of fish and rapeseed oils on growth performance, egg fatty acid composition and offspring quality of starlet sturgeon (Acipenser ruthenus)[END_REF]. Hormonal treatment for females was divided into 2 injections with 10 h interval (20% as priming dose and 80% as resolving dose), this procedure also stemming from the usual practices in Iran [START_REF] Abdollahpour | Influence of thyroxine on spawning performance and larval development of Sterlet sturgeon Acipenser ruthenus[END_REF].

Females ripping were checked based on Dettlaff curve and water temperature [START_REF] Dettlaff | Sturgeon Fishes, Developmental Biology and Aquaculture[END_REF]. The broodstock , s abdomen was tested for ovulation 18 hours after the second injection, and this manipulation was repeated at 2 h intervals up to 48 hours [START_REF] Abdollahpour | Influence of thyroxine on spawning performance and larval development of Sterlet sturgeon Acipenser ruthenus[END_REF].

The breeders spawned within 29-30 h after the first injection. Latency period was recorded as the lapse of time between the first hormone injection and ovulation. Ovulated eggs were manually stripped from the females using an incision made in the caudal section of the oviducts [START_REF] Sarameh | The effect of fish and rapeseed oils on growth performance, egg fatty acid composition and offspring quality of starlet sturgeon (Acipenser ruthenus)[END_REF]. Stripping of all fish was done until the flow of oocytes had considerably reduced or stopped [START_REF] Pourasadi | Minimally invasive surgical technique for egg collection from the Persian sturgeon Acipenser persicus[END_REF][START_REF] Abdollahpour | Hormonal and physiological changes in Sterlet sturgeon Acipenser ruthenus treated with thyroxine[END_REF]. Spawned females were disinfected with Chloramphenicol spray (Afagh, Tehran, Iran) and the fish were injected with 1 mL of oxytetracycline 10% (Nasr, Mashhad, Iran) as an antibiotic [START_REF] Falahatkar | Laparoscopy, a minimally-invasive technique for sex identification in cultured great sturgeon Huso huso[END_REF]. Finally, the fish were moved to a recovery tank. There was no mortality monitored between the hormone injection and the stripping. Eggs were weighed, and three sub-samples were kept to count the number of eggs per gram.

Sampling and analysis

First, fish were anesthetized with 400 ppm clove powder extract [START_REF] Abdollahpour | Hormonal and physiological changes in Sterlet sturgeon Acipenser ruthenus treated with thyroxine[END_REF].

Blood samples (3 fish into each tank) was collected from the behind of the anal fin with 5 ml heparinized syringe at the time of first injection, 10 h after the first injection and at ovulation time. Then the samples were transferred to the tubes, centrifuged (1,600 × g for 10 min) to separate plasma and stored at -20°C for later analysis.

Sex steroid concentration (ng mL -1 ) of testosterone (T, Cat. No: 3725-300), 17β estradiol (E2, Cat. No: 4925-300) and progesterone (P, Cat. No: 4825-300) were measured using enzymelinked immunosorbent assay (ELISA) using commercial kits (AccuBind ELISA Microwells, Monobind, Inc. Lake Forest, CA, USA) based on manufacturer's instruction. The intra-and inter-assay coefficients of variation were 5.8%, 9.8% (n=10), 6.4%, 9.5% (n=10) and 5.1%, 7.5% (n=10) for E2, T and P, respectively. Testosterone and progesterone were selected based upon their potential roles during the final stages of maturation in female sturgeon [START_REF] Semenkova | Sex steroid profiles in female and male Stellate sturgeon during final maturation induced by hormonal treatment[END_REF][START_REF] Skoblina | Stimulation of in vitro oocyte ovulation by progesterone and homologous pituitary gonadotropic hormone in sturgeons[END_REF][START_REF] Khara | Comparative analysis of steroid levels in blood serum of female Persian sturgeon, Acipenser persicus, induced by hormonal treatment[END_REF]) and the very low inter-individual variability of the plasma level during the reproductive season [START_REF] Barannikova | Steroids in the control of reproductive function in fish[END_REF].

Statistical analysis

Statistical analyses were performed using the SPSS software (version 16, Chicago, IL, USA). This research was done using a completely randomized design with different treatments. In order to check the data normality and homogeneity of variances Kolmogorov-Smirnov and Levene's test were applied respectively. One-way analysis of variance (ANOVA) was applied to analyze differences in reproduction performances between treatments. Sex steroid levels were analyzed by Two-way analysis of variance (ANOVA). Differences between several treatments were determined by Tukey's post-hoc tests. A Pearson correlation test was done to check the correlation between sex steroid hormones with each other [START_REF] Barannikova | Serum levels of testosterone, 11ketotestosterone and oestradiol-17β in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment[END_REF]. Values were reported as mean ± standard deviation (SD) in this paper.

Results

rGnRH quantity

After reading the absorbance of the samples at 595 nm with a spectrophotometer, the rGnRH concentration was estimated at 3.5 mg mL -1 .

Spawning performance

Reproductive response of Sterlet sturgeon broodstock injected with LHRH-A2 and rGnRH is shown in Table 1. 50% females injected with L2.5, 16.66% females injected with rGn2.5 and 66.66% females injected with rGn10 spawned, while none of the females of the C treatment spawned. The fish injected with rGn10 treatment exhibited a significantly higher number of eggs per gram (105.0 ± 3.5 eggs g -1 ) in comparison with the other treatments (Table 1; P < 0.001).

Hormone levels

E2 levels were not significantly different in C treatment among the different sampling times (Fig. 1; P = 0.983). The L2.5 or rGn10 treatments led to significantly lower E2 concentration 10 h after the first injection and at ovulation time compared to the baseline measures (Fig, 1; P = 0.002). By contrast, E2 levels increased in rGn2.5 treatment at ovulation time compared to the time of first injection (Fig. 1; P = 0.002).

T levels were significantly increased in L2.5 or rGn10 treatments 10 h after the first injection compared to the time of first injection (Fig. 2; P < 0.001). At ovulation time, a significant decline compared to 10 h after the first injection in T values was observed in the L2.5 or rGn10 treatments (Fig. 2; P < 0.001). There was a significant difference between treatments with the highest levels in the rGn10 treatment at both sampling times post-injection (Fig. 2; P < 0.001).

No significant differences were found in C (Fig. 2; P = 0.173) and rGn2.5 (Fig. 2; P = 0.961) treatments among the different sampling times.

No difference in P levels was found among treatments at the time of first injection (Fig. 3; P = 0.437). Ten h after the first injection and ovulation time, a surge in P levels was observed in plasma concentration in the L2.5 or rGn10 treatments, but no increases were detected in C or rGn2.5 treatments (Fig, 3; P < 0.001).

Regarding L2.5 and rGn10 treatments, a Pearson linear correlation test revealed negative correlation between plasma E2 and P (Fig, 4; Pearson correlation = -0.45; P = 0.055) and E2 was negatively correlated with T, but this correlation was not significant (Fig, 5; Pearson correlation = -0.45; P = 0.060). A significant positive correlation was also found between T and P levels at L2.5 and rGn10 (Fig, 6; Pearson correlation = 0.48; P = 0.042). [START_REF] Barannikova | Serum levels of testosterone, 11ketotestosterone and oestradiol-17β in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment[END_REF].

Discussion

In the current study, the biological function of rGnRH was confirmed in vivo by observing the induced changes of E2, P, and T blood levels and its ability to induce final maturation and spawning in the mature Sterlet sturgeon females. In the absence of a GtH (gonadotropin hormone) test for Sterlet sturgeon, the indirect impacts of rGnRH have been surveyed in the current study by estimating plasma levels of T, E2, and P; as peaks or drops in the circulating plasma levels of these steroids can be utilized as an indirect measurement of the advancement of final oocyte maturation. While it is generally admitted that E2 reaches a peak in fish undergoing vitellogenesis, T is raised during vitellogenesis and also during the early stages of final oocyte maturation and P is elevated during final oocyte maturation [START_REF] Morehead | Effect of treatment with LHRH analogue on oocyte maturation, plasma sex steroid levels and egg production in female striped trumpeter Latris lineata (Latrididae)[END_REF][START_REF] Semenkova | Sex steroid profiles in female and male Stellate sturgeon during final maturation induced by hormonal treatment[END_REF][START_REF] Barannikova | Serum levels of testosterone, 11ketotestosterone and oestradiol-17β in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment[END_REF][START_REF] Falahatkar | Long-term effects of intraperitoneal injection of estradiol-17β on the growth and physiology of juvenile stellate sturgeon Acipenser stellatus[END_REF].

In the present study, some females that received injection of recombinant GnRH and LHRH-A2 spawned, while none of the control fish spawned. This shows that both rGnRH and synthetic LHRH-A2, were able to induce the advancement of final oocyte maturation, hence spawned broodstock were most abundant in the rGn10 treatment. The induction effect of different GnRH analogues has been demonstrated in different species of sturgeon [START_REF] Nazari | Application of synthetic hormone LHRH-A 2 on the artificial propagation of Persian sturgeon Acipenser persicus[END_REF][START_REF] Amini | Evaluation of LHRH-a acute release implantation on final maturation and spawning in not-fully matured broodstocks of Persian sturgeon (Acipenser persicus Borodin, 1897)[END_REF][START_REF] Khara | Comparative analysis of steroid levels in blood serum of female Persian sturgeon, Acipenser persicus, induced by hormonal treatment[END_REF][START_REF] Roosta | Comparative study on hormones and biochemistry indices in plasma, ovarian fluid and oocytes of LHRHa2-induced female Stellate sturgeon (Acipenser stellatus)[END_REF]. [START_REF] Nazari | Application of synthetic hormone LHRH-A 2 on the artificial propagation of Persian sturgeon Acipenser persicus[END_REF] showed that using the LHRH-A2 at doses of 3.5, 7, 8, and 10 μg kg -1 BW can induce final maturation in Persian sturgeon (Acipenser persicus) broodstock and the doses of 3.5, 7, and 8 μg kg -1 yielded better results than the dose of 10 μg kg -1 . [START_REF] Amini | Evaluation of LHRH-a acute release implantation on final maturation and spawning in not-fully matured broodstocks of Persian sturgeon (Acipenser persicus Borodin, 1897)[END_REF] reported that the implantation of LHRH-A2 hormone at a dose of 15 µg kg -1 BW in premature Persian sturgeon broodstock had positive results in inducing final maturation. Also, [START_REF] Abdollahpour | Influence of thyroxine on spawning performance and larval development of Sterlet sturgeon Acipenser ruthenus[END_REF] showed that 4 µg kg -1 BW of LHRH-A2 induced the Sterlet sturgeon broodstock's final maturation. Thus, we confirm that GnRH analogues are quite effective to promote spawning in sturgeon species. Regarding the effectiveness of recombinant GnRH, no study is available, which makes it impossible to compare the present results by existing literature. Based on our observations, the number of spawned broodstock in rGn2.5 was less than those of the other hormonal treatments, which could be attributed to the inappropriate injected amount of this hormone for the induction of spawning in female Sterlet sturgeon broodstock. Similarly, low levels of injected GnRH analogues led to failed spawning in Japanese medaka (Oryzias latipes) and common carp broodstock [START_REF] Chan | Effect of synthetic luteinizing hormone-releasing hormone (LH-RH) on ovarian development in Japanese medaka, Oryzias latipes[END_REF][START_REF] Drori | Spawning induction in common carp, Cyprinus carpio, using pituitary extract or GnRH super active anagoge combined with metoclopramide: analysis of profile, progress of oocyte maturation and dependence on temperature[END_REF]. All together, the amount of required LHRHa seems to vary according to the species and the route of administration, and/or be due to the differences in the maturation level of female broodstock, their age, size, and sensitivity (Faridpak, 2008) and it might be also the case for recombinant GnRH.

In the current study, a positive effect of rGnRH treatment was displayed on the number of eggs per gram. It was consistent to previous research on Persian sturgeon, showing that increasing the LHRH-A2 levels from 4 to 6 µg kg -1 BW also increased the number of eggs per gram [START_REF] Mohammadi | Effect of different doses of synthetic hormone LHRH-A2 on serum sex hormones, ovulation percent and egg hatching rates of Persian sturgeon (Acipenser persicus)[END_REF]. The egg quality and quantity from induced spawning appear to be partially related to the interaction between the stage of maturity or oocyte diameter and GnRHa dose [START_REF] Ibarra | GnRHa-induced spawning of wild-caught spotted rose snapper Lutjanus guttatus[END_REF]. Doses higher than the optimal may result in reduced egg quality [START_REF] Garcia | Dose-dependent spawning response of mature female Sea Bass, Lates calcarifer (Bloch), to pelleted luteinizing hormone-releasing hormone analogue (LHRHa)[END_REF] while lower doses may result in reduced spawning frequency [START_REF] Ibarra | GnRHa-induced spawning of wild-caught spotted rose snapper Lutjanus guttatus[END_REF]. In the present study, egg production varies among rGnRH doses, suggesting that rGnRH 2.5 μg kg -1 has a lower efficiency than rGnRH 10 μg kg -1 to induce females' egg release.

In female broodstock, the level of E2 increases during the vitellogenesis and then decreases at the end of this stage [START_REF] Mojazi Amiri | Ovarian development and serum sex steroid 'and vitellogenin profiles in the female cultured sturgeon hybrid, the bester[END_REF][START_REF] Barannikova | Sex steroids in the serum of Caspian sturgeons and their specific cytosol binding in brain and gonads during the migratory cycle[END_REF]. [START_REF] Majumder | G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio[END_REF] confirmed that estrogens play an inhibitory role on the meiotic maturation of mature oocytes through activation of estrogen membrane receptors, necessitating a reduction in estrogen levels for final maturation. Significant reduction in E2 values from baseline 10 h after the first injection and at ovulation time was observed for both L2.5 and rGn10 treatments. A similar pattern for E2 level was found in common carp [START_REF] Levavi-Zermonsky | Changes in gonadotropin and ovarian-steroids associated with oocytes maturation during spawning induction in the carp[END_REF]) and tench injected with GnRHa [START_REF] Podhorec | The effects of GnRHa with and without dopamine antagonist on reproductive hormone levels and ovum viability in tench Tinca tinca[END_REF] and in Persian sturgeon injected with LHRH-A2 [START_REF] Khara | Comparative analysis of steroid levels in blood serum of female Persian sturgeon, Acipenser persicus, induced by hormonal treatment[END_REF]. Secretion patterns for this steroid correspond to the expected natural shift in the biosynthetic pathway from the production of mainly C 19 to C 21 steroids [START_REF] Podhorec | The effects of GnRHa with and without dopamine antagonist on reproductive hormone levels and ovum viability in tench Tinca tinca[END_REF]. The broodstock in the L2.5 or rGn10 treatments were in final maturation stage, hence we cannot rule out the fact that a reduction in E2 levels was required in these fish to achieve the final maturation coincidently with potential increase of the maturation inducing hormone. E2 levels increased in rGn2.5 treatment 10 h after the first injection and at ovulation time compared to the time of the first injection. A similar E2 secretion pattern after GnRH or LHRH treatment was reported in previous observations on Stellate sturgeon (Acipenser stellatus) and Sterlet sturgeon [START_REF] Bayunova | Sex steroids and cortical levels in the blood stellate sturgeon (Acipenser stellatus Pallas) during final maturation induced by LH-RH analogue[END_REF][START_REF] Yooneszadeh | Sequential of sex steroids and cortisol levels[END_REF][START_REF] Falahatkar | Effects of LHRH-A2 on sex steroids levels, stress indices, and some plasma biochemical parameters in female Sterlet sturgeon, Acipenser ruthenus, broodstock. Iran[END_REF]. The slight increase in plasma E2 could occur because of a high aromatase activity in the ovary upon GnRH administration [START_REF] Sharaf | Effect of GnRHa, pimozide and Ovaprim on ovulation and plasma sex steroid hormones in African catfish Clarias gariepinus[END_REF]. Thus, in the females exposed to rGn2.5, the increase of E2 after injection could be related with the lower efficiency to trigger ovulation than the females exposed to L2.5 or rGn10, which displayed a drop in E2.

In this study, T increased significantly in L2.5 or rGn10 treatments 10 h after the first injection but it decreased following the ovulation time. A similar T secretion pattern after GnRH and LHRH treatments was reported in Stellate and Sterlet sturgeon with more frequent blood sampling [START_REF] Semenkova | Sex steroid profiles in female and male Stellate sturgeon during final maturation induced by hormonal treatment[END_REF][START_REF] Barannikova | The Dynamics of Steroid Hormones in Blood under Hormonal Stimulation of Maturation in the Northern Dvina Sterlet (Acipenser ruthenus)[END_REF][START_REF] Falahatkar | Effects of LHRH-A2 on sex steroids levels, stress indices, and some plasma biochemical parameters in female Sterlet sturgeon, Acipenser ruthenus, broodstock. Iran[END_REF]. Increased T levels in response to GnRH injection and its decrease at ovulation time have been observed in many studies suggesting that GnRH stimulated the secretion of the pituitary GtH and, in turn, pituitary GtH stimulated the production of T by the vitellogenic follicles and mature oocytes (Morehead et al., 1993). In some teleosts, high levels of T led to accelerated maturation of oocytes, and such concentrations in the pre-ovulatory phase may affect the synchronicity of final oocyte maturation and ovulation [START_REF] Bayunova | Sex steroids and cortical levels in the blood stellate sturgeon (Acipenser stellatus Pallas) during final maturation induced by LH-RH analogue[END_REF]. In the present study, the highest levels of T were observed in L2.5 or rGn10 treatments at the beginning of the experiment, which were significantly higher than those of the other treatments at all sampling stages; these treatments also contained the highest number of spawned broodstock. T levels were significantly lower in rGn2.5 treatment than the other treatments at all sampling times. [START_REF] Ceapa | Plasma sex steroids and vitellogenin levels in stellate sturgeon (Acipenser stellatus Pallas) during spawning migration in the Danube River[END_REF] found that high T concentrations might play an important role in initiating and continuing reproductive behaviors in both males and females, so that broodstock with low T levels would not reach ovulation stage. We suggest that the elevation of T post-injection after L2.5 or rGn10 treatments contributed to the better spawning success with these hormonal therapies.

In the current study, P levels showed significant increases in L2.5 or rGn10 treatments 10 h after the first injection, but no increase occurred in C or rGn2.5 treatments. At the time of ovulation, P levels were significantly higher in L2.5 or rGn10 treatments than those of the other treatments.

These findings are in agreement with previous observations on sturgeon where the level of serum P was higher after LHRH-A injection [START_REF] Semenkova | Sex steroid profiles in female and male Stellate sturgeon during final maturation induced by hormonal treatment[END_REF][START_REF] Khara | Comparative analysis of steroid levels in blood serum of female Persian sturgeon, Acipenser persicus, induced by hormonal treatment[END_REF]. Elevation of P over a short period could indicate its indirect role in the final maturation of oocytes, as a precursor of the putative MIS (Maturation-Inducing-Steroid) namely 17α, 20β-progesterone and 17α, 20β, 21-thrihydroxy-4-pregnen-3-one at the time of ovulation [START_REF] Semenkova | Effect of progestins on germinal vesicle break down in sturgeon follicles in vitro[END_REF].

However, the direct role of P in the induction of final oocyte meiotic maturation in fish cannot be excluded in sturgeon [START_REF] Skoblina | Stimulation of in vitro oocyte ovulation by progesterone and homologous pituitary gonadotropic hormone in sturgeons[END_REF]. Nevertheless, the role of P in reproductive function of sturgeon remains unclear [START_REF] Barannikova | Steroids in the control of reproductive function in fish[END_REF][START_REF] Khara | Comparative analysis of steroid levels in blood serum of female Persian sturgeon, Acipenser persicus, induced by hormonal treatment[END_REF]. P is present in measurable concentrations in sturgeon and its level increases during the final maturation, but it does not demonstrate a distinct peak [START_REF] Semenkova | Sex steroid profiles in female and male Stellate sturgeon during final maturation induced by hormonal treatment[END_REF]. In our study, P levels increased in L2.5 or rGn10 treatments after hormone injection, coincidently with E2 decrease. A similar pattern after recombinant LH and LHRH treatment was reported in common carp [START_REF] Levavi-Zermonsky | Changes in gonadotropin and ovarian-steroids associated with oocytes maturation during spawning induction in the carp[END_REF][START_REF] Aizen | Biologically active recombinant carp LH as a spawning inducing agent for Carp[END_REF]. Indeed, [START_REF] Aizen | Biologically active recombinant carp LH as a spawning inducing agent for Carp[END_REF] showed that injection of recombinant LH to common carp broodstock led to increased P and decreased E2. Dropped E2 levels along with elevated P levels may confirm a shift in the steroidization pathway from estrogen formation towards P production. This process may be due to a decrease in 17-20 lyase activity and an increase in 20-β-hydroxysteroid dehydrogenase activity [START_REF] Aizen | Steroidogenic response of carp ovaries to piscine FSH and LH depends on the reproductive phase[END_REF][START_REF] Aizen | Biologically active recombinant carp LH as a spawning inducing agent for Carp[END_REF].

To date, few in vivo studies have reported the usage of recombinant hormones in fish. In the Japanese eel (Anguilla japonica), different in vitro methods proved biological function of recombinant gonadotropins, which showed low activities in gonads when tested in vivo [START_REF] Kazeto | Japanese eel follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): production of biologically active recombinant Fsh and Lh by Drosophila S2 cells and their differential actions on the reproductive biology[END_REF]. In immature Japanese eel, recombinant eel Follicle-Stimulating Hormone (FSH) induced testicular development and spermatopoiesis [START_REF] Kamei | In vivo gonadotropic effects of recombinant Japanese eel follicle-stimulating hormone[END_REF]. The gonadosomatic index significantly increased after injection with recombinant FSH in Manchurian trout (Brachymystax lenok), [START_REF] Ko | Biological activities of recombinant Manchurian trout FSH and LH: their receptor specificity, steroidogenic and vitellogenic potencies[END_REF]. Injection of recombinant LH induced ovulation and altered the levels of sex steroids in common carp [START_REF] Aizen | Biologically active recombinant carp LH as a spawning inducing agent for Carp[END_REF]. Based on the results of present research, it can be speculated that recombinant GnRH is capable of binding to its receptor on pituitary surface and inducing final maturation in Sterlet broodstock.

The present study is the first report on the use of recombinant GnRH in fish that was able to stimulate P secretion and induce spawning. Complementary research is necessary concerning the effects of recombinant GnRH on expression of genes involved in the reproductive system of different fish species at different stages of sexual maturation to determine the exact mechanisms of action of this hormone. Also, further optimization of effective dose is necessary to obtain 100% of ovulation in sturgeon and higher doses than 10 µg kg -1 BW can be tested in further experiments. Finally, testing the efficiency of recombinant GnRH to stimulate the sperm quality and quantity in males could also open new avenues of investigation for the optimization of sturgeon reproduction in captivity. Such new findings make it possible to further use recombinant GnRH as a suitable alternative for pituitary extract and LHRH-A2 hormone in sturgeon breeders and maybe other fish species.

in farmed stellet sturgeon (Acipenser stellatus Pallas) female brooders after induced ovulation by GnRH (Ova-Fact Ш). Iran. J. Fish. Sci. 3,[21][22][23][24][25][26][27][28]. Zmora, N., Gonzalea, M.D., Munoz, J.A., Madigou, T., Sanchez, E., Doste, S.Z., Zohar, Y., 2002. The GnRH system in the European sea bass (Dicentrarchus labrax). J. Endocrinol. 172, 105-116.

Figures legends

Fig. 1. Plasma 17β-estradiol levels in Sterlet sturgeon (Acipenser ruthenus) broodstock after injection of NaCl 0.9% (control); 2.5 µg kg body weight -1 LHRH-A2 (L2.5); 2.5 µg kg body weight -1 rGnRH (rGn2.5) and 10 µg kg body weight -1 rGnRH (rGn10). Mean ± SD; n = 3 for each tank. Different letters designate significant differences as determined by Tukey's post-hoc tests. LHRH-A2 (L2.5) and 10 µg kg body weight -1 rGnRH (rGn10).

Fig. 6. Positive correlation between plasma concentrations of testosterone and progesterone in

Sterlet sturgeon (Acipenser ruthenus) broodstock after injection of 2.5 µg kg body weight -1 LHRH-A2 (L2.5) and 10 µg kg body weight -1 rGnRH (rGn10). Different letters designate significant differences as determined by Tukey's post-hoc tests.

1 Germinal vesicle migration (%) = distance between nucleus and AP / distance between VP and AP (X100); 2 Spawning success (%): the number of females that ovulated after injection divided by the total number of injected females. 3 Latency period (h): time between the first hormone injection and ovulation.

.

Fig. 2 .

 2 Fig.2. Plasma Testosterone levels in Sterlet sturgeon (Acipenser ruthenus) broodstock after injection of NaCl 0.9% (control); 2.5 µg kg body weight -1 LHRH-A2 (L2.5); 2.5 µg kg body weight -1 rGnRH (rGn2.5) and 10 µg kg body weight -1 rGnRH (rGn10). Mean ± SD; n = 3 for

Fig. 3 .Fig. 4 .

 34 Fig. 3. Plasma Progesterone levels in Sterlet sturgeon (Acipenser ruthenus) broodstock after injection of NaCl 0.9% (control); 2.5 µg kg body weight -1 LHRH-A2 (L2.5); 2.5 µg kg body weight -1 rGnRH (rGn2.5) and 10 µg kg body weight -1 rGnRH (rGn10). Mean ± SD; n = 3 for

Fig. 5 .

 5 Fig. 5. Negative correlation between plasma concentrations of 17β-estradiol and testosterone in Sterlet sturgeon (Acipenser ruthenus) broodstock after injection of 2.5 µg kg body weight -1

  29

Fig

  Fig. 1.

Table 1 .

 1 Reproduction performance in Sterlet sturgeon (Acipenser ruthenus) broodstock after injection of NaCl 0.9%; 2.5 µg kg body weight -1 LHRH-A2; 2.5 µg kg body weight -1 rGnRH and 10 µg kg body weight -1 rGnRH (Mean ± SD).

	Treatment	No. of	Germinal vesicle	No. of	Spawning	Latency	Number of
		injected fish	migration 1	spawned fish	success (%) 2	period (h) 3	eggs /g
	0.9% NaCl	6	8.64 ± 2.23	0	-	-	-
	LHRH-A (2.5 µg kg-1 )	6	8.53 ± 2.23	3	50	29.5 ± 0.5	101.7 ± 4.7 ab
	rGnRH (2.5 µg kg -1 )	6	8.59 ± 0.94	1	16.66	30	96.3 b
	rGnRH (10 µg kg -1 )	6	8.63 ± 1.62	4	60.66	30 ± 0.5	105.0 ± 3. 5 a

Acknowledgements

The authors are thank the Fisheries Labs, Sari Agricultural Sciences and Natural Resources University and Dr. Yousefpour Marine Fishes Restocking and Genetic Conservation Center for their support. The Iran Biotechnology Development Council under project No. 970202 and Sari Agricultural Sciences and Natural Resources University funded this work.


José Luis Ulloa

Roberta Vastano
email: roberta.vastano@gmail.com

Nathalie George
email: nathalie.george@upmc.fr

Marcel Brass
email: marcel.brass@ugent.be

The impact of eye contact on the sense of agency

Keywords: Eye contact, Self-referential processing, Agency, Intentional binding

Recent research suggests that eye contact can lead to enhanced self-awareness. A related phe-nomenon, the sense of agency deals with the notion of the self as the origin of our actions. Possible links between eye contact and agency have been so far neglected. Here, we investigated whether an implicit sense of agency could be modulated by eye gaze. We asked participants to respond (button press) to a face stimulus: looking or not at the participant (experiment 1); or displaying distinct eye gaze before or after a mask (experiment 2). After each trial, participants estimated the time between their key press and the ensuing effects. We found enhanced inten-tional binding for conditions that involved direct compared to averted gaze. This study supports the idea that eye contact is an important cue that affects complex cognitive processes and suggests that modulating self-processing can impact the sense of agency.

Introduction

Eye contact is a powerful social cue and is fundamental in the development of social skills in humans [START_REF] Emery | The eyes have it: The neuroethology, function and evolution of social gaze[END_REF]. Eye contact plays an important role in social behavior; it is the customary prelude for social interaction [START_REF] George | Facing the gaze of others[END_REF], it is used for regulating turn taking in conversations, in expressing certain attitudes such as liking or loving, and in exercising social control [START_REF] Kleinke | Gaze and eye contact: A research review[END_REF]. The underlying mechanisms associated with the effects of eye gaze are now being extensively studied in psychology and neuroscience [START_REF] Carlin | The neural basis of eye gaze processing[END_REF][START_REF] Senju | Direct gaze captures visuospatial attention[END_REF]. Some theoretical developments suggest that important effects of perceiving eye contact derive from cognitive processes associated with the processing of the self. Eye contact is said to induce "attention toward the self" and to trigger a self-centered frame of reference for cognitive processing [START_REF] Conty | Watching Eyes effects: When others meet the self[END_REF]. The emergent literature on this topic gives support to these ideas. For instance, eye contact displays enhanced self-awareness, making participants better at reporting their own emotional responses [START_REF] Baltazar | Eye contact elicits bodily self-awareness in human adults[END_REF]. Given the nature of these effects, one could venture that cognitive processes that depend on perception of the self could be modulated by eye contact. The sense of agency (SoA), i.e. the experience that we are in control of our actions and their effects [START_REF] Gallagher | Philosophical conceptions of the self: Implications for cognitive science[END_REF][START_REF] Haggard | The experience of agency: Feelings, judgments, and responsibility[END_REF]) is a cognitive process closely related to the processing of the self. SoA involves performing actions, monitoring their consequences, and attributing (or not) these consequences to oneself [START_REF] David | The "sense of agency" and its underlying cognitive and neural mechanisms[END_REF]. There are now several studies investigating the factors that modulate SoA. It has been shown that SoA is modulated by basic sensorimotor processes (e.g. [START_REF] Chambon | Sense of control depends on fluency of action selection, not motor performance[END_REF] as well as high-level inferential factors associated with intentions and beliefs (see [START_REF] Synofzik | The experience of agency: An interplay between prediction and postdiction[END_REF] for an integrative view). Another aspect of SoA is its affective and social dimension [START_REF] Gentsch | Affective coding: The emotional dimension of agency[END_REF]. It has been shown for instance that eliciting sadness by invoking social exclusion memories [START_REF] Malik | Social exclusion reduces the sense of agency: Evidence from intentional binding[END_REF], evoking fear [START_REF] Christensen | I just lost it! Fear and anger reduce the sense of agency: A study using intentional binding[END_REF] or anger [START_REF] Christensen | I just lost it! Fear and anger reduce the sense of agency: A study using intentional binding[END_REF] in participants, reduces intentional binding, an implicit form of the SoA. Other aspects related to emotional processing also change SoA, for instance valence and arousal of stimuli. [START_REF] Yoshie | Effects of emotional valence on sense of agency require a predictive model[END_REF] showed that negatively-valenced outcomes lead to decreased temporal binding, while increased levels of arousal have been shown to increase intentional binding [START_REF] Wen | The influence of action-outcome delay and arousal on sense of agency and the intentional binding effect[END_REF]. In addition, it has been shown that social interaction can shape the SoA. [START_REF] Obhi | Sense of agency and intentional binding in joint action[END_REF] have shown that a person involved in a joint action with another subject show intentional binding over its own actions (and that of the other subject) and joint effects. Social cues, however, have been less investigated. Recently, a study investigated the effects of eliciting joint attention on SoA. This study manipulated a face display so that it looked it was engaged in joint attention with the participant or not [START_REF] Stephenson | Eyes that bind us: Gaze leading induces an implicit sense of agency[END_REF]. Participants were asked to reproduce the time between an object appearance (which triggered the participant's saccades) and the gaze onset of the face (the consequence of the participant's saccades). This study showed that making saccades which were followed by another person elicited a reduced intentional binding effect, and thus increased SoA on these outcomes. This effect was absent in experiments with passive gaze changes or gaze changes elicited in a non-social context [START_REF] Stephenson | Eyes that bind us: Gaze leading induces an implicit sense of agency[END_REF]. Here, unlike focusing on eye movements we were interested in understanding the influence of eye contact on SoA. Indeed, a crucial aspect of SoA is self-referential processing, and as mentioned above, current evidence indicates that eye contact elicits an enhanced processing of the self. Given this obvious relationship between self-related processes and SoA, it is remarkable that no study has been performed to investigate whether changes in self-referential processing affects SoA. In the current study we investigated whether enhanced self-processing, induced by eye contact, could modulates SoA. We adopted an intentional binding approach as a measure of SoA. The intentional binding effect is an implicit measure of agency, which denotes the subjective compression of the temporal interval between actions and their ensuing effects [START_REF] Haggard | Voluntary action and conscious awareness[END_REF]. This effect can be measured by asking participants to report the perceived duration of intervals between an action and its ensuing effect [START_REF] Cravo | Voluntary action and causality in temporal binding[END_REF][START_REF] Engbert | Who is causing what? The sense of agency is relational and efferent-triggered[END_REF][START_REF] Kühn | Feeling in control: Neural correlates of experience of agency[END_REF][START_REF] Vastano | The action congruency effect on the feelings of agency[END_REF]. The advantage of implicit measures versus explicit measures of agency (self-report of action-effect control) is that implicit measures do not rely on conscious reflection prone to subjective beliefs, contextual cues and differences in personality [START_REF] Synofzik | The experience of agency: An interplay between prediction and postdiction[END_REF].

In a first experiment, we asked participants to respond to the sight of face stimuli. The response of participants triggered a gaze shift in the face stimulus with gaze transitions going either from averted to direct gaze, or from direct to averted gaze. The gaze shift occurred after a random interval following participants' response. At the end of each trial we asked participants to estimate the time interval between their action and the ensuing gaze change (the action effect). We hypothesized that a situation where eye contact is induced (a transition from averted to direct gaze) would enhance self-processing and will produce a larger intentional binding effect (and thus enhanced SoA) relative to the condition where the action induces averted gaze. Next, we performed a second experiment to assess this effect for stimuli that displayed a single static gaze direction (either direct or averted gaze). In this second experiment, we masked the eyes of the face before or after the participants' action. Participants observed faces with visible eyes (with direct or averted gaze) that turned into faces with masked eyes (as an effect of their action); or they observed faces with masked eyes that turned into faces with visible eyes (with direct or averted gaze). Following our previous findings, we hypothesized that only those trials where eyes are directed to the participant would increase intentional binding, independently of whether direct gaze was presented before or after the participants' action. Finally, in order to control confounds related to changes in time estimations due to eye contact we performed a third experiment. In this experiment participants were asked to estimate time between sensory events (without performing actions). We do not find evidence for the idea that gaze direction improve the precision of time estimations. Thirty healthy volunteers (23 females, age: 18-21 years, SD: 0.9 years) were enrolled for the study. All participants were righthanded. All testing procedures were approved by the local ethical committee of Ghent University. All participants gave written informed consent and received academic credits for their participation.

Stimuli and procedure

Subjects were seated at a distance of ∼60 cm from the computer monitor (refresh rate: 60 Hz, dimensions: 53 × 30 cm, resolution: 1920 × 1080). Experimental stimuli consisted of images (680 × 808 pixels) of 18 neutral human faces (9 female, 9 male) displaying either direct or averted eye gaze (toward left or right). We used the Radboud Faces Database (RaFD; [START_REF] Langner | Presentation and validation of the Radboud Faces Database[END_REF]. Participants were presented with a fixation cross between 1500 and 3000 ms, followed by a face to which they had to respond with a key press at the time they chose (provided the experiment was completed in 30 min). The key press produced a gaze change after a variable interval (300, 500 or 700 ms). Gaze transitions went either from averted to direct gaze (Averted-Direct) or from direct to averted gaze (Direct-Averted). The face remained on screen for 500 ms after the gaze change and then it was followed by a Visual Analogue Scale (VAS; Fig. 1). Participants were asked to estimate the time interval between their action (key press) and the ensuing gaze shift, by positioning the mouse pointer along the VAS from 100 ms to 900 ms (the scale displayed a marker every 100 ms just for visualization). They had a maximum of 5000 ms to give their answer. Finally, after a variable inter-trial interval ranging from 1000 to 2000 ms, the next trial started. Participants were told that the interval between their action and the gaze shift was always random between 100 ms and 900 ms. The experiment consisted of one block of 216 randomized trials: 108 trials for Averted-Direct transitions (54 trials from leftward to direct gaze, and 54 trials from rightward to direct gaze) and 108 trials for Direct-Averted transitions (54 trials from direct to leftward gaze, and 54 trials from direct to rightward gaze). The 54 trials of each transition were composed of 18 trials for each action-effect time interval (300, 500, and 700 ms). Before starting the actual experiment, participants were trained to discriminate between 100 and 900 ms (the endpoints of the VAS used in the interval-time estimation task). They observed a visual stimulus (a dot) that changed color after their action with an interval of 100 or 900 ms, and they were asked to evaluate whether the time elapsed between the two color changes was either 100 or 900 ms. At the end of their judgment, they received a feedback (correct or incorrect response). Thirty randomized trials (15 for each interval) were presented with a variable inter-trial interval between 1000 and 2000 ms. After this initial training, participants get familiarized with the main experiment (16 trials). The tasks were implemented in E-prime 2.0 Professional software (Psychology Software Tools, Pittsburgh, PA).

Data analysis

We computed the time-judgment errors as the difference between the estimated intervals and the actual intervals. For the analyses, we first ran a paired t-test on reaction times (RTs) with Gaze transition (2 levels: Averted-Direct, Direct-Averted) as factor to investigate whether there was a difference in reaction time when seeing the averted gaze or the eye contact first. Most importantly, we performed a repeated measures analysis of variance (ANOVA) on the time-judgment errors with Gaze transition (2 levels: Averted-Direct, Direct-Averted) and Interval (3 levels: 300, 500, and 700 ms) as within-subject factors. We tested for sphericity violations and Greenhouse-Geisser corrections were applied when necessary; we report corrected degrees of freedom in these cases. Significant effects found in the ANOVA were followed by Newman-Keuls corrected post hoc tests. Alpha level was fixed at 0.05 for all statistical tests.

2.1.4. Results 2.1.4.1. Reaction times. The paired t-test on RTs revealed no difference for Gaze transition (means: 608 and 607 ms for Averted-Direct and Direct-Averted transitions, respectively, t(29) = -0.63, p = 0.52) indicating that participants responded equally fast to the face with initial averted gaze as to the face with initial direct eye.

2.1.4.2. Time-judgement errors. The ANOVA on time-judgment errors revealed a main effect of Interval (F(1.13, 32.95) = 40.31; p < 0.001; partial eta-squared, η 2 p = 0.58) with larger time-judgement errors for longer intervals and an interaction between Gaze transition and Interval (F(2,58) = 4.18; p = 0.019; η 2 p = 0.12). Post-hoc comparisons showed that Averted-Direct transitions led to significantly greater time estimation errors (mean ± SD: -21 ms ± 67 ms) than Direct-Averted transitions (-6 ms ± 70 ms; p = 0.003; Fig. 2) for the interval 300 ms only. No other comparison was statistically significant (p > 0.1).

Discussion

In the first experiment, we observed a larger intentional binding effect indicated by larger negative time-judgement error when participants perform key presses that produce a display of direct gaze as compared to those that produced an averted gaze. This effect was restricted to the short time interval. This result suggests that eye contact has an impact on SoA. However, the specific interpretation of this result is difficult because the first experiment confounds a number of factors. It is not clear whether the influence on intentional binding is caused by eye contact in the first frame (before the action) or the second frame or by the transition from the first to the second image. In addition, it remains an open question whether inducing the direct gaze alone, or in conjunction with averted gaze in this dynamic transition is crucial for the effect to occur. Therefore, we carried out a second experiment in which we controlled for these confounding factors by displaying the face with averted or direct gaze either in the beginning of the trial or as an effect of the key press, at the end. When the eye gaze was presented at the beginning of the trial, the eyes were masked at the end. When the eye gaze was presented at the end, the eyes were masked in the beginning of the trial.

Experiment 2 2.2.1. Participants

Twenty-seven healthy volunteers (25 females, 18-22 years, SD: 0.9 years) who did not participate in the first experiment were enrolled for the study. All participants were right-handed. All testing procedures were approved by the local ethical committee of Ghent University. All participants gave written informed consent and received academic credits for their participation.

Stimuli and procedure

This experiment was conducted under similar conditions as Experiment 1. Experimental stimuli consisted of 10 neutral human faces (5 female, 5 male) displaying either direct gaze, averted gaze (toward the left or the right), or masked eyes in the form of black sunglasses. In this experiment, gaze transitions could go from masked to visible eyes or from visible to masked eyes (Fig. 3). The visible eyes displayed Direct or Averted gaze, and the display of the visible eyes could occur Before or After the participant's action (Visibility order). The experiment consisted of one block of 192 randomized trials: 96 trials started with visible eyes and ended with masked eyes, and 96 trials started with masked eyes and ended with visible eyes. From the 96 trials for each eye visibility order condition, 48 trials comprised direct gaze, and 48 trials comprised averted gaze (24 averted gaze to the left; 24 averted gaze to the right). Finally, from the 48 trials for each gaze and visibility order condition, there were 16 trials for each action-effect time Interval (300, 500, and 700 ms). Before starting the actual experiment, participants performed a similar short training and a task familiarization as in Experiment 1.

Fig. 3. The sequence of events for the experiment 2. Subjects responded with a button press to the sight of the face. Upon the participant's response and a variable interval there was a change in the face (the presence or absence of the black sunglasses). Following the eye gaze change, a Visual Analogue Scale was displayed.

Data analysis

We computed the time-judgment errors as in Experiment 1. We performed a repeated-measures ANOVA on RTs with Gaze condition (2 levels: Averted, Direct) and Visibility order condition (2 levels: Before, After) as within-subject factors. We performed a repeated-measures ANOVA on the time-judgment errors, with Gaze condition (2 levels: Averted, Direct), Visibility order (2 levels: Before, After) and Interval (3 levels: 300, 500, and 700 ms) as within-subject factors. We tested for sphericity violations and Greenhouse-Geisser correction were applied when necessary; we report corrected degrees of freedom in these cases. Significant effects found in the ANOVA were followed by Newman-Keuls corrected post hoc tests. Alpha level was fixed at 0.05 for all statistical tests. 2.2.4. Results 2.2.4.1. Reaction times. The ANOVA on RTs revealed no main effect nor interaction (all p > 0.1) indicating that as in Experiment 1, reaction times were not influenced by the gaze manipulation in the first frame.

2.2.4.2. Time-judgement errors. The ANOVA on time-judgment errors revealed a main effect of Interval (F(1.14, 29.84) = 53.4; p < 0.001; η 2 p = 0.67) with larger time-judgement errors for longer intervals and an interaction between Gaze and Interval (F (2,52) = 8.15; p < 0.001; η 2 p = 0.24). Since the Visibility order did not show any main effect or interaction, post-hoc tests were performed collapsing across this factor. Post-hoc comparisons showed that Direct gaze led to significantly greater time estimation errors (mean ± SD: -113 ± 93 ms) than Averted gaze (-90 ms ± 93 ms; p < 0.001; Fig. 4) for the interval 500 ms only. No other comparison was statistically significant (p > 0.1;Fig. 5).

Discussion

The second experiment investigated if the effect of eye gaze could be observed when gaze stimuli were displayed in the beginning or at the end of the trial only. We observed that conditions involving a direct gaze (regardless of whether it was presented before or after the action) produced enhanced intentional binding, with greater underestimation of time interval between the key press action and its effect for Direct relative to Averted gaze. This effect, however, was restricted to the 500 ms condition. Thus, eye contact elicits enhanced intentional binding irrespective of whether the eye contact precedes or follows the key press, but in a restricted time frame. Finally, we conducted an additional experiment to control confounds related to changes in time estimations due to eye contact. In this experiment participants were asked to estimate time between sensory events (without performing actions). Thirty healthy volunteers (19 females, age: 18-25 years, SD: 1.47 years) were enrolled for the study. All participants were righthanded. All testing procedures were approved by the ethical committee of the Faculty of Psychology at the University of Talca. All participants gave written informed consent and received monetary compensation for their participation.

Stimuli and procedure

The experiment was conducted under similar conditions as experiment 2 using the same visual stimuli (to visualize a trial see Fig. 1). However, in this experiment the participants do not responded with a button press to the sight of the face. Instead, they only witnessed the transitions between visual displays (the same previous time intervals of 300, 500 and 700 ms were used). As in the previous experiment gaze transitions could go from masked to visible eyes or from visible to masked eyes. The visible eyes displayed Direct or Averted gaze, and the display of the visible eyes could occur Before or After the visual change. Upon these events participants were asked to estimate, in a Visual Analogue Scale, the changes in the face (the presence or absence of the black sunglasses). The experiment consisted of the same number of trials as in the previous experiment and the participants also performed a short training and a task familiarization.

Data analysis

We computed the time-judgment errors as in Experiment 2. We performed a repeated-measures ANOVA on the time-judgment errors, with Gaze condition (2 levels: Averted, Direct), Visibility order (2 levels: Before, After) and Interval (3 levels: 300, 500, and 700 ms) as within-subject factors. We tested for sphericity violations and Greenhouse-Geisser corrections were applied when necessary; we report corrected degrees of freedom in these cases. Significant effects found in the ANOVA were followed by Newman-Keuls corrected post hoc tests. Alpha level was fixed at 0.05 for all statistical tests. 2.3.4. Results 2.3.4.1. Time-judgement errors. The ANOVA on time-judgment errors revealed a main effect of Interval (F(1.26, 36.62) = 92.1; p < 0.001; η 2 p = 0.76) and a main effect of Visibility order (F(1, 29) = 4.24; p = 0.048; η 2 p = 0.12). There was also an interaction between Interval and Visibility order (F(1.66,48.3) = 6.9; p = 0.003; η 2 p = 0.19). Post-hoc tests revealed that this interaction reflect a reduction of time-judgement errors when gaze was displayed before (as compared to after) the visual transition, only for the 500 ms Interval (t(65.2) = 3.2, p = 0.026). We do not observe any main effect or interaction of gaze (p > 0.1).

Discussion

In a third experiment we investigated whether we could find effects of eye gaze direction on a time estimation task involving only sensory events. We observed a main effect of interval with larger time-judgement errors for longer intervals. We also observed that time-judgement errors were overall reduced compared to the previous experiments where participants performed actions. We found that the presence of eyes after relative to before the visual change induced increased time-judgement errors. This effect might be attributed to expectation of a meaningful event such as the imminent presence of eye gaze (regardless of gaze direction). Importantly, we do not observe any main effect or interaction with gaze. This experiment suggest that our previous findings involving modulations of intentional binding by gaze direction cannot be explained only by changes in time estimation. This experiment also suggest that the effects of gaze in intentional binding occur under the presence of voluntary actions only.

General discussion

In the present study, we investigated whether eye contact can impact an implicit measure of SoA, namely intentional binding. We observe that eye contact elicits larger intentional binding when the action of participants induced a shift from averted to direct gaze compared to the reverse shift (Experiment 1). In Experiment 2 we further investigated this effect by manipulating eye gaze independently of its occurrence (relative to the action effect) in the time course of a trial. This second experiment shows that eye contact leads to enhanced intentional binding independent of whether direct gaze is presented before the action or as a result of the action. In a control experiment, similar to Experiment 2, we ask participants to estimate time intervals for sensory events only and we found no effects of eye gaze on time estimations. Overall, our results suggest that gaze direction impacts the intentional binding effect. Interestingly, the effect of eye contact on intentional binding was restricted to specific time intervals. In Experiment 1 we observe an effect in the shortest time interval (300 ms). In Experiment 2 we observe the effect for the time interval of 500 ms.

Eye contact is a specific form of social stimulus that has a wide range of social functions [START_REF] Emery | The eyes have it: The neuroethology, function and evolution of social gaze[END_REF]. Importantly, it has been demonstrated to increase self-referential processing [START_REF] Carver | Self-focusing effects of dispositional self-consciousness, mirror presence, and audience presence[END_REF], for a review see [START_REF] Conty | Watching Eyes effects: When others meet the self[END_REF]. In fact, experimental research on the self has been often carried out with paradigms where the attention of participants has been turned to themselves. For instance, by placing someone in front of a mirror [START_REF] Ainley | Looking into myself: Changes in interoceptive sensitivity during mirror self-observation[END_REF][START_REF] Carver | Self-focusing effects of dispositional self-consciousness, mirror presence, and audience presence[END_REF], using a camera turned to the participants [START_REF] Davis | Use of first person pronouns as a function of increased objective self-awareness and performance feedback[END_REF] or by including the presence of someone [START_REF] Carver | Self-focusing effects of dispositional self-consciousness, mirror presence, and audience presence[END_REF]. All these manipulations involve being observed either by oneself or by someone else. More recent studies have confirmed the self-referential power of eye contact [START_REF] Baltazar | Eye contact elicits bodily self-awareness in human adults[END_REF][START_REF] Hietanen | Genuine eye contact elicits self-referential processing[END_REF][START_REF] Pönkänen | Does it make a difference if I have an eye contact with you or with your picture? An ERP study[END_REF]. This emerging set of studies suggests that eye contact can increase self-processing and consequently elicits increased self-awareness. Eye contact would induce attention toward the self and trigger a self-centered frame of reference for cognitive processing [START_REF] Conty | Watching Eyes effects: When others meet the self[END_REF]. In the context of our study, we speculated that direct gaze, because of its influence on self-referential processing, would impact on SoA. Interestingly, the very concept of SoA is related to the self-concept. Experiencing agency only makes sense if one is the agent of an action. Given this central role of the self for SoA it is surprising that little research has been carried out to relate the self-concept to SoA. Our study, thus, adds evidence to the notion that eye contact can affect other self-related processes such as the sense of agency.

Currently it is acknowledged that the sense of agency is modulated by predictive processes as well by postdictive inferences (see [START_REF] Synofzik | The experience of agency: An interplay between prediction and postdiction[END_REF] for a review). While the former takes the form of sensorimotor processes that inform fluency of actions [START_REF] Wolpert | An internal model for sensorimotor integration[END_REF][START_REF] Frith | Abnormalities in the awareness and control of action[END_REF], the latter describe inferential processes where agency is constructed after actions are performed [START_REF] Wegner | The mind's best trick: How we experience conscious will[END_REF]. In our study, we observed that the effects of eye contact occur both before and after the action (experiment 2) and thus, we can't favor any of these two mechanisms. Since eye contact induces a generalized state of self-referential processing, one possibility is that the effects of eye contact on the SoA might be related to changes in arousal. It is well known that eye contact can increase arousal [START_REF] Nichols | Eye gaze and the GSR[END_REF][START_REF] Strom | Staring and participants' sex: Physiological and subjective reactions[END_REF], and this effect could potentially induce self-awareness. The idea that eye contact effects in our experiments could take place through arousal is convergent with previous findings showing larger intentional binding with increased levels of arousal [START_REF] Wen | The influence of action-outcome delay and arousal on sense of agency and the intentional binding effect[END_REF]. We must however recognize that, as noted by [START_REF] Conty | Watching Eyes effects: When others meet the self[END_REF], arousal cannot explain all the effects of eye contact, among them, increased self-awareness. In fact, the study of [START_REF] Baltazar | Eye contact elicits bodily self-awareness in human adults[END_REF] demonstrated effects associated with eye contact which were independent of arousal. Furthermore, in Experiment 1 eye contact was presented in both conditions. The only difference was that in one condition eye contact was induced by the action and in the other it was preceding the action. Hence, an interpretation based only on arousal for the influence of eye contact on SoA is unlikely.

Our findings might also be related to attentional consequences of being exposed to relevant psychosocial stimuli. Higher attentional allocation for some stimuli (like direct gaze) might have induced participants to better estimate time intervals. This could reduce time-judgements errors (and thus increase intentional effects) for conditions involving direct gaze. However, there is previous literature showing differences between time estimation and intentional binding that argues against this confound. There is an ongoing debate about the crucial components of intentional binding. Some arguing that voluntary actions are essential, while other arguing the causal relationship between action and effect is the more important ingredient (for a review see [START_REF] Moore | Intentional binding and the sense of agency: A review[END_REF]. Causal inferences are based on properties of an action-outcome relationship such as contiguity. Contiguity refers to the temporal proximity of the action and the outcome. The closer together in time these events appear the stronger the inferred causal relation [START_REF] Hume | A treatise of human nature[END_REF]. Thus, causal estimations would directly reflect estimations of time without the involvement of actions. [START_REF] Cravo | Voluntary action and causality in temporal binding[END_REF] used a Michotte paradigm where participants controlled the launching of a stimulus. Participants were then asked to estimate time and causality between collisions of two circles. This study shows that measures of intentional binding (time estimations for voluntary actions) were dissociated of causality estimations. In our study we provide further evidence against the idea of a general effect of gaze on time perception by running a third experiment. In this experiment participants were asked to judge time intervals (and not perform any action) between two visual events. We found no effect of gaze on time-judgements errors. There was only a main effect of interval which was modulated by order. This control experiment renders it very unlikely that our effects of eye gaze on intentional binding are related changes in time estimation. More importantly, our results suggest that the action component is crucial for the modulation of eye gaze on SoA. Thus, considering the complete set of experiments we found that modulations of intentional binding by gaze perception arises for trials involving action execution and direct gaze. It is worth of notice that in our control experiment time-judgement errors were overall reduced relative to the two previous experiments. By definition, voluntary movements should elicit increased time-judgements errors as reflect of intentional binding. In the control experiment there was no action involved, but some time-judgement errors may arise due the presence of highly temporally-correlated events. Our findings of reduced time-judgements errors in the control experiment relative to our previous experiments converge with previous literature. It has been shown that an increase of time-judgements errors might occur but is attenuated for highly temporally-correlated events when no voluntary action is performed [START_REF] Buehner | Awareness of voluntary and involuntary causal actions and their outcomes[END_REF]. Altogether, changes in time estimations cannot explain intentional binding effects. In order to produce intentional binding both causality and intentional actions must be present. Our findings suggest that gaze perception has a strong impact on agency, but that this effect depend upon action execution.

In Experiment 1 intentional binding was increased for the Averted-Direct compared to the Direct-Averted transition. Since both transition conditions include a direct gaze display, one could have expected no difference between these conditions. However, it is possible that the Direct-Averted transition, which involved a first display of direct gaze, might have elicited self-referential processing, but when replaced by a display with averted gaze, this self-referential processing was overridden by the reorienting effect of averted gaze. There is indeed ample evidence of the attention orienting effects of averted eye gaze (e.g. [START_REF] Driver | Gaze perception triggers reflexive visuospatial orienting[END_REF]Frischen, Bayliss, & Tipper, 2007 for review). This hypothesis is in line with Experiment 2 where direct gaze induced increased intentional binding irrespective of its position regarding the transition. When direct gaze is followed by a mask it produces increased intentional binding relative to averted gaze. For trials with direct gaze, the effect of eye contact is not overridden because this stimulus is not followed by a reorienting cue such as averted gaze.

We observed a strong effect of interval on intentional binding for both experiments. It has been previously recognized that tasks involving time estimation or reproduction are associated with underestimation of time intervals, with these effects increasing proportionally with interval length [START_REF] Caspar | The relationship between human agency and embodiment[END_REF][START_REF] Humphreys | Magnitude estimation reveals temporal binding at super-second intervals[END_REF][START_REF] Kühn | Feeling in control: Neural correlates of experience of agency[END_REF]. Some authors suggest that this results from a slowdown of an "internal clock" which predicts action outcomes, and which increases with interval length [START_REF] Humphreys | Magnitude estimation reveals temporal binding at super-second intervals[END_REF], 2010;[START_REF] Wenke | How voluntary actions modulate time perception[END_REF]. Here, we used the distinct intervals to create variability in the responses, but we found that intentional binding occurs for specific action-outcome intervals in the experiments 1 and 2. Experiment 1 showed increased intentional binding due to eye contact for 300 ms-intervals only, while in experiment 2 this occurred for the 500-ms interval only. We had not a-priori hypotheses about when the time-judgement error should be the largest. In consequence, we can only speculate why it was maximal for the 300 ms interval in Experiment 1 and 500 ms in Experiment 2. The lack of effects in the 500 and 700 ms range in Experiment 1 may be related to the fact that in our paradigm the participant anticipated the display of eye contact for Averted-Direct transitions and this expectation was fulfilled the most when eye contact was the fastest (for the 300 ms interval), inducing then a larger intentional binding effect. Another possibility might be related to natural properties of eye gaze changes. When exploring our environment typical saccades range between 20 and 250 ms [START_REF] Rayner | Eye movements in reading and information processing: 20 years of research[END_REF]. It is reasonable to think that we may be more sensitive to eye gaze shifts that occur within the range of natural eye movements. In the second experiment, the stimuli were somewhat different. Participants were confronted with visual stimulus changes rather than gaze changes. Differences in the time intervals where we found the effects might be related to the stimulus differences in the two experiments. Using a design where we varied gaze and order independently makes difficult to predict whether eye contact would be displayed, and thus there might have not been expectations for changes in the shortest interval of 300 ms. Another possibility might be related to the display of eyes masked by sunglasses rendering the stimuli ambiguous: the gaze could be either averted or direct. This could have distracted the participants and result in an overestimation of the time when action-outcome interval was the shortest. However, this does not explain why the changes were seen at the 500 ms interval only in the experiment 2. The nature of this experiment was quite distinct. In this experiment the transition involved a change in relatively central portion of the image rather than a change restricted to the eye gaze direction. There was no dynamic eye gaze shifts, but rather changes of images depicting static eye gaze displays. These displays were devoid of dynamic gaze information. One could putatively say that differences arose due to distinct neural systems involved in the processing of static and dynamic aspects of the face [START_REF] Pitcher | Differential selectivity for dynamic versus static information in face-selective cortical regions[END_REF]. The nature of the changes might have elicited effects that are different from the effects elicited by natural gaze changes to which were are sensitive almost from birth. However, as outlined above, these constitute post-hoc interpretations and we would need to corroborate these effects testing other time intervals and additional manipulations of eye gaze. We believe that the effects we found are purely related to short action-outcome intervals, especially when task ambiguity is reduced as in the first experiment; this would also explain why in both experiments the 700 ms interval was not influenced by gaze direction. Unfortunately, no study to date directly investigated the effects of different time intervals on intentional binding, since completely random delays have been used [START_REF] Poonian | Implicit agency in observed actions: Evidence for N1 suppression of tones caused by self-made and observed actions[END_REF], or the effects of the intervals were not discussed, or they were excluded from the statistical analysis [START_REF] Caspar | The relationship between human agency and embodiment[END_REF][START_REF] Caspar | The sense of agency as tracking control[END_REF][START_REF] Sidarus | Difficult action decisions reduce the sense of agency: A study using the Eriksen flanker task[END_REF]. The effect of different actionoutcome intervals when using time estimation and/or reproduction tasks to test the intentional binding needs to be further investigated to better understand how timing impacts the SoA.

It is interesting to note a previous study that use eye gaze to modulate the sense of agency. The study of [START_REF] Stephenson | Eyes that bind us: Gaze leading induces an implicit sense of agency[END_REF] asked participants to reproduce time intervals between the appearance of objects and observed eye gaze shifts. Thus, in this study it is leading and making someone to engage in joint attention that produced the intentional binding between gaze actions and subsequent sensory effects (the gaze movements of the perceived face). In our experiment there were no effects derived from ocular actions, but merely from manual actions. In our case, we hypothesized that visual stimulation with a direct gaze display would evoke self-related processing. In line with this idea, it is noticeable that the first image in a trial of Stephenson's experiment was a face looking straight ahead. In the context of joint attention the communicative context is established by an indicator of communicative intent: a verbal cue or eye contact [START_REF] Emery | The eyes have it: The neuroethology, function and evolution of social gaze[END_REF]. Thus, the occurrence of eye contact sets the communicative stance of behaviors involving eye gaze. However, a control experiment in the study of Stephenson et al. found that the straight gaze was not mandatory to find the effect; what was critical was rather the fact of directing other's eye movements toward an object. In this respect, our effects are of a different nature. Altogether, there is converging evidence in support of a link between either performing or perceiving eye gaze and the sense of agency. These effects might, nevertheless, involve distinct mechanisms.

We note that our conclusions remain speculative and are limited by the fact that we did not directly measure self-awareness (e.g. questionnaires) or arousal (e.g. galvanic skin responses). We base our premises in the cumulative evidence that eye contact increases 1 0 self-awareness and arousal. A follow-up study should aim at empirically testing how arousal and/or self-awareness are related to the eye gaze manipulation. Our findings are limited to intentional binding effects and it could be argued that they are unrelated to explicit measures of agency. While intentional binding was intended as a proxy of the SoA (for a review see [START_REF] Moore | Intentional binding and the sense of agency: A review[END_REF], some studies have found dissociations between intentional binding and explicit measures of SoA (e.g. [START_REF] Saito | Discrepancy between explicit judgement of agency and implicit feeling of agency: Implications for sense of agency and its disorders[END_REF]. It has also been shown that with increasing intervals between action and outcome, explicit SoA is reduced while implicit SoA increases [START_REF] Ebert | Time warp: Authorship shapes the perceived timing of actions and events[END_REF]. Since these two measures have been shown to be dissociated, we cannot predict the extent to which eye gaze may affect explicit measures of SoA and whether this would map into the same timing specificity as in our present findings. This will be interesting to test in future studies. At the same time, it is important to note that explicit measures of SoA are prone to subjective beliefs and contextual cues [START_REF] Synofzik | The experience of agency: An interplay between prediction and postdiction[END_REF]. Therefore, regarding the features of our study, involving socially relevant stimuli, explicit measures would likely have been more sensitive to individual differences in personality and beliefs. Future studies will need to take these variables into account in order to fully uncover the mechanism through which eye contact modulates intentional binding.

Studies of the impact of eye gaze behavior and the sense of agency might have potential implications for interactions in social contexts. For instance, when we perform actions in social situations, other's eye contact can act as a cue that intensify our feeling of agency and thus of responsibility over outcomes. In this framework, it could be interesting to investigate the impact of emotional expressions when combined with eye contact on the sense of agency.

Fig. 1 .

 1 Fig. 1. The sequence of events for the experiment 1. Subjects responded with a button press to the sight of the face. Upon the participant's response and a variable interval there was an eye gaze change in the face. Following the eye gaze change, a Visual Analogue Scale was displayed.

Fig. 2 .

 2 Fig. 2. Time-judgements errors in the experiment 1 as a function of Gaze transitions (Direct-Averted/Averted-Direct) and Intervals (300/500/700). Error bars represent standard errors of the mean.

Fig. 4 .

 4 Fig. 4. Time-judgements errors in the experiment 2 as a function of Gaze (Averted/Direct), Intervals (300/500/700) and Visibility order (Before/ After). Error bars represent standard errors of the mean.

Fig. 5 .

 5 Fig. 5. Time-judgements errors in the experiment 3 as a function of Gaze (Averted/Direct), Intervals (300/500/700) and Visibility order (Before/ After). Error bars represent standard errors of the mean.

Funding

This work was supported by CONICYT (grant PFCHA/POSTDOCTORADO BECAS CHILE/2014-74150065) to JLU, by the French program "Investissement d'Avenir" of the Agence Nationale de la Recherche to the Centre MEG-EEG and ICM (grant numbers ANR-10-IAIHU-06 and ANR-11-INBS-0006) to NG, and by a Ghent University-Special Research Fund (grant BOF16/ZAPV2/053) to MB. None of these funding bodies had a role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.

Declaration of Competing Interest

None.