Printzi Aliki

Kourkouta Chara

Fragkoulis Stefanos

Anastasia Dimitriadi

Geladakis George

Orfanakis Michail

Mazurais David

Zambonino Infante

Koumoundouros George

Balancing between Artemia and microdiet usage for normal skeletal development in zebrafish ( Danio rerio )

Keywords: feeding, fish larvae, skeletal abnormalities, welfare

Targeting in zebrafish fast growth, high survival rates and improved reproductive performance has led over the last years in variable feeding regimes between different facilities. Despite its significance on fish function and welfare, normal skeletal development has rarely been evaluated in establishing the best feeding practices for zebrafish. The aim of this study was to establish a protocol for normal skeletal development, growth and survival of zebrafish larvae through live feed-to-microdiet transition at an appropriate rate. Four feeding regimes including feeding exclusively on Artemia nauplii (A) or dry microdiet (D), and feeding on both Artemia and microdiet at two different transition rates (slow (B) or fast (C)) were applied from 5 to 24 dpf (days post-fertilization). Results demonstrated a significant effect of feeding regimes on the incidence of skeletal abnormalities (gill cover, fins and vertebral column, p < .05) in zebrafish larvae. The A and B experimental groups presented the highest (88 ± 3 and 84 ± 17%, respectively), but the C and D the lowest (18 ± 14 and 11 ± 2%, respectively), rates of normal fish (fish without any abnormality). Similarly, growth rate was comparatively elevated in A and B groups. No significant differences were observed in fish survival between A, B and C groups. However, D group presented a significantly lower survival rate. To our knowledge, this is the first study to show that the live feed-to-microdiet transition rate influences larval growth, survival and abnormality rates in a nonhomogenous pattern.

Introduction

Skeletal abnormalities have been frequently reported to develop in reared fish, as a result of unfavorable environmental conditions and often under the control of genotype [START_REF] Boglione | Skeletal anomalies in reared E uropean fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF][START_REF] Fragkoulis | Saddleback syndrome in European sea bass Dicentrarchus labrax (Linnaeus, 1758): anatomy, ontogeny and correlation with lateral-line, anal and pelvic fin abnormalities[END_REF][START_REF] Fragkoulis | Recovery of haemal lordosis in Gilthead seabream (Sparus aurata L.)[END_REF]. They develop mainly during the embryonic and larval period, up to the end of skeleton ontogeny and rarely in the following developmental stages. In addition to their significant effects on fish morphology, they may also influence fish survival, growth, swimming mode or susceptibility to diseases [START_REF] Boglione | Skeletal anomalies in reared E uropean fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF][START_REF] Koumoundouros | Morpho-anatomical abnormalities in Mediterranean marine aquaculture[END_REF]. Hence, the presence of skeletal abnormalities has been suggested as an index of animal welfare, as well as of the appropriateness of husbandry practices applied during the early life [START_REF] Boglione | Skeletal anomalies in reared E uropean fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF][START_REF] Huntingford | Current issues in fish welfare[END_REF][START_REF] Sloman | Ethical considerations in fish research[END_REF].

Zebrafish is a valuable model species for developmental biology and genetics, fish biology, ecotoxicology, neurophysiology and biomedicine [START_REF] Bhagat | Zebrafish: An emerging model to study microplastic and nanoplastic toxicity[END_REF][START_REF] Lieschke | Animal models of human disease: zebrafish swim into view[END_REF][START_REF] Patterson | Zebrafish pigment pattern formation: Insights into the development and evolution of adult form[END_REF][START_REF] Spence | The behaviour and ecology of the zebrafish, Danio rerio[END_REF], including human skeletal pathology with respect to genetic disorders [START_REF] Boswell | Understanding idiopathic scoliosis: A new zebrafish school of thought[END_REF][START_REF] Gistelinck | Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies[END_REF]. Despite the significance of husbandry conditions for the normal skeleton development of this species, existing relevant literature is scarce [START_REF] Martini | Plasticity of the skeleton and skeletal deformities in zebrafish (Danio rerio) linked to rearing density[END_REF][START_REF] Martins | Early transition to microdiets improves growth, reproductive performance and reduces skeletal anomalies in zebrafish (Danio rerio)[END_REF][START_REF] Printzi | Exercise-induced lordosis in zebrafish Danio rerio (Hamilton, 1822)[END_REF]. Understudying the importance of nutrition for fish biological performance, as well as for zebrafish maintenance cost and easiness, different studies target on establishing larval feeding protocols for fast growth, high survival rates and improved reproduction capacity [START_REF] Carvalho | Rearing zebrafish (Danio rerio) larvae without live food: evaluation of a commercial, a practical and a purified starter diet on larval performance[END_REF][START_REF] Delomas | Improved protocol for rapid zebrafish growth without reducing reproductive performance[END_REF][START_REF] Gomez-Requeni | A reference growth curve for nutritional experiments in zebrafish (Danio rerio) and changes in whole body proteome during development[END_REF][START_REF] Kaushik | Growth and body composition of zebrafish (Danio rerio) larvae fed a compound feed from first feeding onward: toward implications on nutrient requirements[END_REF][START_REF] Lawrence | Successful replacement of Artemia salina nauplii with marine rotifers (Brachionus plicatilis) in the diet of preadult zebrafish (Danio rerio)[END_REF][START_REF] Önal | Characterization of two microparticle types for delivery of food to altricial fish larvae. Characterization of two microparticle types for delivery of food to altricial fish larvae[END_REF], but rarely for normal skeletal development [START_REF] Martins | Early transition to microdiets improves growth, reproductive performance and reduces skeletal anomalies in zebrafish (Danio rerio)[END_REF].

Live food is traditionally used in zebrafish larval rearing either exclusively or partially during first days of exogenous feeding. Existing protocols involve the use of cultured paramecia (Paramecium sp.), rotifers (Brachionus sp.) or Artemia nauplii (Artemia sp.), in different combinations and variable rates of transition to dry commercial microdiets [START_REF] Best | A novel method for rearing first-feeding larval zebrafish: polyculture with Type L saltwater rotifers (Brachionus plicatilis)[END_REF][START_REF] Lawrence | Successful replacement of Artemia salina nauplii with marine rotifers (Brachionus plicatilis) in the diet of preadult zebrafish (Danio rerio)[END_REF][START_REF] Markovich | Determination of the proper feeding density of rotifers Brachionus plicatilis for larval zebrafish Danio rerio[END_REF][START_REF] Westerfield | The Zebrafish Book: A guide for the laboratory use of zebrafish (Danio Rerio[END_REF]. Despite the extensive use of live feed in zebrafish larval rearing, the shift to dry microdiets has been considered as a critical step for research on nutritional preferences of fish larvae, as well as for the establishment of standard larval rearing protocols with low nutritional variability and high biosecurity level [START_REF] Cahu | Substitution of live food by formulated diets in marine fish larvae[END_REF][START_REF] Watts | The vital relationship between nutrition and health in zebrafish[END_REF]. Currently, a variety of microdiets has been developed for the larval rearing of zebrafish, with associated feeding protocols rarely been tested with respect to the morphological quality of fish. To our knowledge, in the only existing relevant study, [START_REF] Martins | Early transition to microdiets improves growth, reproductive performance and reduces skeletal anomalies in zebrafish (Danio rerio)[END_REF] demonstrated that the early transition from Artemia nauplii to microdiets significantly reduces vertebral abnormalities in zebrafish (from 90% to ca 50%).

In the present paper we examined the effect of live-feed to microdiet transition rate on the prevalence of skeletal abnormalities, growth and survival rate of zebrafish larvae. We tested Artemia nauplii AF grade, which have been shown suitable live feed for first feeding zebrafish larvae [START_REF] Martins | Early transition to microdiets improves growth, reproductive performance and reduces skeletal anomalies in zebrafish (Danio rerio)[END_REF] and one commercial zebrafish diet.

Materials and methods

Maintainance of fish populations

The effect of four different larval feeding regimes on zebrafish skeleton was tested during the early larval period, from first feeding to 7-10 mm standard length (SL, 24 days post fertilization, dpf), where most of the skeletal features have been developed [START_REF] Bird | Developmental morphology of the axial skeleton of the zebrafish, Danio rerio (Ostariophysi: Cyprinidae)[END_REF][START_REF] Parichy | Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish[END_REF]. Feeding regimes comprised of exclusive feeding on Artemia nauplii (A) or dry microdiet (D), as well as combined use of Artemia and microdiet of two different transition rates (slow or fast, B, C) (Fig. 1, Table 1). At the end of the experimental period all groups were examined for the presence of skeletal abnormalities, survival and growth rates. All treatments were applied in triplicate.

For each replicate trial, approximately one thousand embryos were collected and divided into four cubic net pens (4.5 L volume each), of 100 μm mesh size, allowing the free water circulation in the pen, but not the escape of Artemia nauplii. Pens were placed into one common aquarium of 40 L volume, equipped with a closed recirculation system. After swimbladder inflation (5 dpf), fish larvae were fed 5 times daily, with newly hatched Artemia nauplii (Artemia AF, INVE, Dendermonde, Belgium) and/or commercial dry microdiets (Zebrafeed<100 μm, 100-200 μm, 200-400 μm, Sparos Lda, Olhao, Portugal) (Table 1). All experimental treatments were reared under common abiotic conditions, with temperature, oxygen, pH and conductivity being monitored daily (Table 2). Nitrogen compounds were recorded on a weekly basis (Table 2).

Fertilized eggs were obtained from a common broodstock of 250 wild type zebrafish (ZF WT2 F13; Wageningen Agricultural University, Wageningen, The Netherlands), which were kept in a 100 L aquarium (with a closed recirculation system), at 28.0 °C (±0.5 °C), 450-600 μS/cm conductivity, 7.0-7.5 pH, 6.5-7.0 mg/L oxygen concentration and 14/10 h light/dark photoperiod. Breeders were fed twice per day on commercial dry diet (Zebrafeed 400-600, Sparos Lda, Olhao, Portugal), and twice per week on newly hatched Artemia nauplii (Artemia AF, INVE, Dendermonde, Belgium).

Skeletal abnormalities, fish survival and fish growth

At the end of the trials (24 dpf), all fish were counted and mortality rates were estimated. To estimate the rate of skeletal abnormalities and measure fish body size, a random sample of 52-61 individuals was taken from each population and replicate. Fish were euthanized using an overdose of phenoxyethanol (2-phenoxyethanol, 0.3-0.5 ml/L), fixed in phosphate buffered formalin and stained for bone and cartilage [START_REF] Walker | A two-color acid-free cartilage and bone stain for zebrafish larvae[END_REF]. Stained fish were photographed under a stereoscope (Olympus SZ61) and measured for standard length (SL, from tip of snout to posterior tip of the caudal peduncle), by means of tpsDig2 software [START_REF] Rohlf | tpsDig v2. 16. Free software available[END_REF]. In the case of C group (first replicate), sample size was reduced to 30, due to accidental fish loss and thus limited availability. The terminology used to describe cranial, vertebral and fin abnormalities followed [START_REF] Koumoundouros | Morpho-anatomical abnormalities in Mediterranean marine aquaculture[END_REF]. Multiple abnormality scores were included in the data when an individual presented more than one abnormality types. Differences in abnormality rates between the different treatments were tested by means of G-test [START_REF] Rohlf | Biometry: the principles and practice of statistics in biological research[END_REF]. Kruskal-Wallis and Mann-Whitney tests were used to test the differences in survival rate between the different treatments. ANOVA and a posteriori Bonferroni tests were used to test the differences in body size between the different treatments (IBM SPSS Statistics for Windows, v26, Armonk, NY). In all cases, the significance level was se to 5% (a= 0.05).

Feed proximate analysis

Chemical analyses of feeds were performed in duplicate for each sample (Table 3): ash (7 h at 550 °C), crude fat [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF], and crude protein (Kjedhal method, N×6.25). Phospholipid and neutral lipid contents in the diets were assayed by the [START_REF] Juaneda | Rapid and convenient separation of phospholipids and non phosphorus lipids from rat heart using silica cartridges[END_REF] method.

Results

A variety of different abnormality types was observed on the fins, vertebral column and gill-cover of the examined samples. Gill-cover abnormalities were characterized by an inside folding of the operculum and twisted branchiostegal rays (Fig. 2a, 2a'). Fin abnormalities consisted of missing caudal-fin rays and abnormalities of the caudal-fin supporting skeleton (Fig. 2b), as well as of abnormal formation of anal or dorsal-fin pterygiophores and their associated rays (Fig. 2c). Observed vertebral abnormalities involved pre-haemal kyphosis (Fig. 2d), scoliosis of the caudal peduncle (Fig. 2f), or miscellaneous abnormalities (shape and size deformations, missing elements) of vertebrae and their processes (e.g. Fig. 2e).

Larval feeding protocol significantly affected the incidence of skeletal abnormalities, with the A and B experimental groups presenting the highest (88±3 and 84±17% respectively) and C and D the lowest (18±14 and 11±2% respectively) rates of normal fish (i.e., fish without any abnormality, Fig. 3a). Exclusive feeding of zebrafish larvae on Artemia nauplii (group A) resulted in complete absence of abnormalities in some anatomical areas (i.e. caudal-fin, dorsal fin and gill-cover abnormalities, Fig. 3b,3d,3f,3h), or to negligible abnormality rates in other (Fig. 3c, 3e, 3g). Compared with group A, the slow transition from Artemia to microdiets (group B) did not significantly increase the different abnormality rates (Fig. 3b-3g), except of gill-cover abnormalities which presented a small increase to 12±17% (Fig. 3h). The incidence of fin abnormalities, pre-haemal kyphosis and gill-cover abnormalities significantly increased as Artemia was further replaced with microdiet (groups C and D,Fig. 3b,3c,3e,3h). Caudal-peduncle scoliosis and miscellaneous vertebral abnormalities presented a different response, with comparatively elevated rates at intermediate levels of Artemia replacement (group C, Fig. 3f,3g).

Larval feeding protocol significantly influenced fish survival at the end of the trials (24 dpf), with group B presenting the highest (89±5%) and group D the lowest survival rate (55±10%, Fig. 4a,4b). As it was indicated by the larval size (SL) at the end of the trials, feeding regimes significantly affected zebrafish growth rate. Groups A and B presented significantly larger larvae than groups C and D (Fig. 4c).

Discussion

Despite the great advances of the last years in microdiet preparation technology and in fish larval nutrition, the rate of transition from live feed to microdiets is still a critical factor for the successful rearing of many species, with significant effects on fish survival, growth and skeletal development [START_REF] Kestemont | Effect of weaning age and diet on pikeperch larviculture[END_REF][START_REF] Kolkovski | Microdiets as alternatives to live feeds for fish larvae in aquaculture: Improving the efficiency of feed particle utilization[END_REF][START_REF] Łączyńska | Effect of age, size and digestive tract development on weaning effectiveness in crucian carp, Carassius carassius (Linnaeus, 1758)[END_REF]. In the present paper we examined the effect of Artemia to microdiets transition rate on the development, survival and growth of zebrafish larvae.

Our results showed that differences in survival or growth rates between the regimes with Artemia (groups A, B and C) were insignificant or very small, respectively. When skeletal abnormalities were taken into consideration, the results of the present study showed that the fast transition to dry feed (groups C and D) has significant negative effects on the skeletal development of the larvae.

No data exist on the effects of different commercial dry feeds on zebrafish skeletal development and thus, we are unable to discuss the effectiveness of microdiets to live feed replacement during early zebrafish feeding. In the only relevant study, [START_REF] Martins | Early transition to microdiets improves growth, reproductive performance and reduces skeletal anomalies in zebrafish (Danio rerio)[END_REF] were the first to show significant differences in the abnormality rates between different zebrafish larval feeding protocols, involving exclusively Artemia or the combined use of Artemia and microdiets (experimental or commercial).

Reported rates concerned only vertebral abnormalities and were comparatively elevated in the fish fed exclusively Artemia (90% vs 48-51% in the other two groups) [START_REF] Martins | Early transition to microdiets improves growth, reproductive performance and reduces skeletal anomalies in zebrafish (Danio rerio)[END_REF]. In the present study, the Artemia group (A) presented the lowest rate of vertebral abnormalities (3.5±3%), as well as of all types of abnormalities recorded (12±3%, vertebral, fins, skull). Observed differences between the two studies could be attributed to the effect of other rearing parameters (e.g. use of static water conditions during the early larval period by [START_REF] Martins | Early transition to microdiets improves growth, reproductive performance and reduces skeletal anomalies in zebrafish (Danio rerio)[END_REF], and/or to the genetic background of fish, all of which are well known to have a significant role in the development of skeletal abnormalities [START_REF] Boglione | Skeletal anomalies in reared E uropean fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF][START_REF] Fragkoulis | Recovery of haemal lordosis in Gilthead seabream (Sparus aurata L.)[END_REF][START_REF] Fragkoulis | Saddleback syndrome in European sea bass Dicentrarchus labrax (Linnaeus, 1758): anatomy, ontogeny and correlation with lateral-line, anal and pelvic fin abnormalities[END_REF]. In the present study, the combined use of net pens and a common recirculation system ensured high water-quality conditions, common for all treatments of each replicate throughout the entire experimental period (Table 2).

In most fish species, first feeding larvae are characterized by limited prey-capture capabilities and an immature but rapidly developing digestive system [START_REF] Zambonino-Infante | Ontogeny and physiology of the digestive system of marine[END_REF]. Thus, successful replacement of planktonic live preys by artificial dry feeds is based on a variety of feed characteristics (e.g. size, sinking rate, color, leaching rate, biochemical composition and ingredients bioavailability, digestibility etc) and their suitability to match the preferences of developing larvae [START_REF] Cahu | Substitution of live food by formulated diets in marine fish larvae[END_REF][START_REF] Kolkovski | Microdiets as alternatives to live feeds for fish larvae in aquaculture: Improving the efficiency of feed particle utilization[END_REF][START_REF] Lazo | Ed.). in larval red drum (Sciaenops ocellatus[END_REF][START_REF] Yúfera | Effect of nutrition on marine fish development and quality[END_REF]. Similar to our results, previous studies on other fish species have also shown that the early shift from Artemia to compound diet may result to increased rates of skeletal abnormalities [START_REF] Kestemont | Effect of weaning age and diet on pikeperch larviculture[END_REF][START_REF] Łączyńska | Effect of age, size and digestive tract development on weaning effectiveness in crucian carp, Carassius carassius (Linnaeus, 1758)[END_REF]. This effect could be linked with the difficulty of early larvae to ingest, digest or absorb dry feed, and/or with possible nutritional imbalances of live feed with respect to critical components for bone development (e.g. vitamins, fatty acids, amino acid profile, reviewed by Zambonino-Infante & Cahu, 2010).

Skeletal abnormalities are common in zebrafish laboratory stocks [START_REF] Murray | Water quality and idiopathic diseases of laboratory zebrafish[END_REF]. In their recent review, with 12-years data from 752 cases (involving 10121 fish), Kent et al. (2020) indicated vertebral (lordosis, kyphosis, scoliosis, platyspondyly) and opercular abnormalities as of the most important and common non-infectious diseases in zebrafish research facilities. In the present paper, a variety of skeletal abnormalities was shown to develop in the zebrafish skull (i.e. gill-cover abnormalities), fins (e.g. abnormal caudal-fin) and vertebral column (e.g. pre-haemal kyphosis, scoliosis of the caudal peduncle). The majority of these abnormalities has been reported to develop in other fish species too, at occasionally high rates [START_REF] Koumoundouros | Morpho-anatomical abnormalities in Mediterranean marine aquaculture[END_REF]. Gill-cover abnormalities are mainly induced by the inward folding of the opercle, subopercle and branchiostegal rays, during the early larval stage [START_REF] Koumoundouros | The opercular complex deformity in intensive gilthead sea bream (Sparus aurata L.) larviculture. Moment of apparition and description[END_REF][START_REF] Verhaegen | Deformities in larval gilthead sea bream (Sparus aurata): A qualitative and quantitative analysis using geometric morphometrics[END_REF]. Caudal-fin abnormalities are expressed in a variety of phenotypes, involving lateral bending, duplication, partial lack or stricture [START_REF] Boglione | Skeletal anomalies in reared E uropean fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF][START_REF] Koumoundouros | Morpho-anatomical abnormalities in Mediterranean marine aquaculture[END_REF]. In European sea bass, pre-haemal kyphosis was shown to be associated with abnormalities of the branchiostegal rays, and induce high mortality rates due to significant compression of the neural tube [START_REF] Koumoundouros | Kyphosis in reared sea bass (Dicentrarchus labrax L.): ontogeny and effects on mortality[END_REF]. To our knowledge, zebrafish is the only species developing caudal-peduncle scoliosis at high rates (present study, [START_REF] Martins | Early transition to microdiets improves growth, reproductive performance and reduces skeletal anomalies in zebrafish (Danio rerio)[END_REF].

In the present study, the response pattern of abnormality rate against larval feeding regime was not uniform for all the reported abnormalities. The rate of scoliosis and miscellaneous vertebral abnormalities was elevated in C group, whereas the rate of rest abnormalities was elevated in D group. As different skeletal elements develop during different ontogenetic periods, which are often characterized by different larval nutritional preferences, the various types of skeletal abnormalities may present variable response patterns against the same factor [START_REF] Koumoundouros | Morpho-anatomical abnormalities in Mediterranean marine aquaculture[END_REF][START_REF] Mazurais | Optimal levels of dietary vitamin A for reduced deformity incidence during development of European sea bass larvae (Dicentrarchus labrax) depend on malformation type[END_REF]. Our results on the elevated rates of scoliosis and miscellaneous vertebral abnormalities in C group, might be related with a difficulty of the early larvae (10 dpf) to adapt to a rapid shift in their diet (vs the slower shift in D group).

A variety of behavioral, morphological, functional and physiological indices have been suggested for the evaluation of fish welfare, with emphasis given on the juvenile and adult phases [START_REF] Huntingford | Current issues in fish welfare[END_REF][START_REF] Sloman | Ethical considerations in fish research[END_REF]. Despite the high susceptibility of fish larvae to a variety of factors causing skeletal abnormalities, the value of the latter as an index for larval welfare has been underestimated. Our results clearly suggest that the rate of skeletal abnormalities is a valuable welfare index, representative of the conformity of husbandry conditions with the preferences of fish during the embryonic and larval stages. Given the effects of skeletal abnormalities on fish growth, survival and function [START_REF] Chatain | Abnormal swimbladder development and lordosis in sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus)[END_REF][START_REF] Georgakopoulou | Effect of temperature on the development of skeletal deformities in Gilthead seabream (Sparus aurata Linnaeus, 1758)[END_REF][START_REF] Koumoundouros | Kyphosis in reared sea bass (Dicentrarchus labrax L.): ontogeny and effects on mortality[END_REF][START_REF] Loizides | A new type of lordosis and vertebral body compression in Gilthead sea bream, Sparus aurata L.: aetiology, anatomy and consequences for survival[END_REF][START_REF] Paperna | Swimbladder and skeletal deformations in hatchery bred Spams aurata[END_REF], once developed, they negatively affect the wellbeing of fish during the following juvenile and adult stages. frequencies are given in Table 1.

2. 4

 4 Ethical statement All the experimental procedures involving zebrafish were performed in accordance with Greek (PD 56/2013) and EU (Directive 63/2010) legislation for animal experimentation and welfare. All protocols were approved by the Animal Care Committee of the Biology Department of the University of Crete (Permit number: 81843/20).

Figure 1 .

 1 Figure 1. Experimental design. Fish were reared under common abiotic conditions, divided in four feeding regimes including Artemia nauplii (A), dry feed (D) and cofeeding of both (B, C). dpf, days post fertilization. Feed quantities and feeding

Figure 2 .

 2 Figure 2. Main types of skeletal abnormalities developed in zebrafish larvae. (a-a') side and top view of an abnormal gill cover. Arrows show the inside folded operculum. (b) incomplete formation of caudal-fin. (c) abnormal anal fin, with lacking pterygiophores (arrow). (d) pre-haemal kyphosis. (e) abnormal vertebrae with missing haemal processes. (f) scoliosis of the caudal peduncle. (BrR) branchiostegal rays. (Dr) distal radial. (HS) haemal process. (Hy) hypural. (NS) neural process. (Op) operculum. (Prx) proximal pterygiophore. (Sb) swimbladder. (V) vertebral centrum. Scale bars = 0.5 mm.

Figure 3 .

 3 Figure 3. Effect of larval feeding protocol (A-D) on the frequency of skeletal abnormalities in zebrafish larvae. (a) Normal fish (fish without any abnormality). (b) Caudal-fin abnormalities. (c) Anal-fin abnormalities. (d) Dorsal-fin abnormalities. (e) Pre-haemal kyphosis. (f) Scoliosis of the caudal peduncle. (g) Miscellaneous vertebral abnormalities. (h) Abnormal gill-cover. Different letters indicate significant differences (p<0.05). Error bars equal to 1 SE.

Figure 4 .

 4 Figure 4. Effect of larval feeding protocol (A-D) on the survival and growth of zebrafish larvae. (a) Survival rate of the different protocols and replicates (Rep1-Rep3). (b) Mean survival rate in the different protocols. (c) Mean standard length (SL) of zebrafish larvae in the different protocols and replicates (Rep1-Rep3), at 24 days post-fertilization. Different letters in (b) and (c) indicate significant differences (p<0.05). # indicates the accidental loss of larvae in C group of the first replicate (Rep1). This group was excluded from the mean estimation in graph (b). Error bars equal to 1 SE.

Table 2 .

 2 Mean values (± SD) of abiotic parameters throughout the three replicates.Oxygen was measured in each net pen separately, whereas rest abiotic parameters were measured in the water outside the pens.

		Nutritional
	Parameters		Replicate І	Replicate ІІ	Replicate ІІІ
		Treatment
	Temperature ( o C)	A-D	28.1±0.2	28.0±0.2	28.1±0.3
	pH	A-D	7.7±0.2	7.5±0.2	7.8±0.2
	Conductivity (μS/cm)	A-D	509±31	516±77	531±55
	Ammonia (mg/L)	A-D	<0.01	<0.01	<0.01
	Nitrate (mg/L)	A-D	15±14	9±13	4±6
	Nitrite (mg/L)	A-D	0.030±0.040	0.014±0.020	0.003±0.008
		A	6.9±0.5	6.8±0.7	6.7±0.3
		B	6.9±0.5	6.8±0.6	6.6±0.4
	O2 concentration (mg/L)
		C	6.7±0.4	6.6±0.7	6.5±0.3
		D	7.0±0.4	6.7±0.8	6.7±0.4

Table 3 .

 3 Chemical composition (%) of the experimental diets tested (mean values).

	Composition (%) Artemia nauplii	Dry feed
	Dry matter	94.8	91.0
	Ash	6.54	12.7
	Total Lipids	19.2	13.0
	Neutral Lipids	9.2	4.0
	Phospholipids	6.6	6.3
	Proteins	61.7	67.7

fish larvae. In: Cyrino, J.E.P., Bureau, D.P. & Kapoor, B.G., (eds), Feeding and Digestive Functions of . Science Publishers, Enfield, NH.

Author contributions

A.P., C.K., S.F., A.D., G.G., M.O. contributed to fish sampling and husbandry, A.P., C.K., S.F., G.K. performed analysis of skeletal abnormalities, growth and survival, D.M., J.Z-I. supervised diet analysis, A.P., G.K. analysed the data, A.P., D.M., J.Z-I., G.K. wrote the paper, G.K. conceived the study. All authors reviewed the manuscript.

Conflict of interest

The authors have no conflict of interest to declare.


Anais Médieu
email: anais.medieu@gmail.com

Fany Sardenne

Anne Lorrain

Nathalie Bodin

Chloé Pazart

Herve Le Delliou

David Point

Médieu Anaïs

Sardenne Fany

Lorrain Anne

Pazart Chloé

Le Delliou Hervé

Point David

Anaïs Médieu

Lipid-free tuna muscle samples are suitable for total mercury analysis

Keywords: methylmercury, fat content, delipidation, yellowfin, bigeye, skipjack

Introduction

Mercury (Hg) is a widespread heavy metal of particular concern to wildlife and human health.

In oceans, it is naturally converted into methylmercury (MeHg), its organic and highest neurotoxic form, well known for its persistence and unique bioaccumulation properties in food webs [START_REF] Hintelmann | Organomercurials. Their Formation and Pathways in the Environment[END_REF]. Consumption of contaminated seafood is considered as the main route of human exposure to MeHg. Top predators like tunas are known to display relatively high MeHg concentrations, sometimes exceeding food safety guidelines (1 µg g -1 fresh tissue) (WHO and UNEP Chemicals, 2008) depending on the oceanic basin and the tuna species. Yet, tunas are also among the most popular marine species consumed worldwide, particularly tropical species that account for more than 90% of the global tuna fishery (FAO, 2018). In terms of food and nutrition security, they provide a major source of proteins, essential fatty acids, vitamins and minerals [START_REF] Di Bella | Trace elements in Thunnus Thynnus from Mediterranean Sea: benefit-risk assessment for consumer[END_REF][START_REF] Sirot | A risk-benefit analysis approach to seafood intake to determine optimal consumption[END_REF].

Knowing their economic importance, nutritional benefits and potential impact on human health, tropical tunas have been studied broadly but at relatively small spatial scale to document their Hg exposure and characterize the processes driving Hg biomagnification along food webs [START_REF] Bodin | Trace elements in oceanic pelagic communities in the western Indian Ocean[END_REF][START_REF] Chouvelon | Chemical contaminants (trace metals, persistent organic pollutants) in albacore tuna from western Indian and south-eastern Atlantic Oceans: Trophic influence and potential as tracers of populations[END_REF][START_REF] Houssard | A Model of Mercury Distribution in Tuna from the Western and Central Pacific Ocean: Influence of Physiology, Ecology and Environmental Factors[END_REF][START_REF] J O U R N A L P R E -P R O O F Kojadinovic | Mercury content in commercial pelagic fish and its risk assessment in the Western Indian Ocean[END_REF][START_REF] Nicklisch | Mercury levels of yellowfin tuna (Thunnus albacares) are associated with capture location[END_REF]. Complex regional interplay between physical (e.g. light intensity), geochemical (e.g. redox status), physiological (e.g. organism's length and age), and ecological factors (e.g. tuna's foraging depth) have been identified to govern Hg concentrations in these top predators [START_REF] Choy | The influence of depth on mercury levels in pelagic fishes and their prey[END_REF][START_REF] Houssard | A Model of Mercury Distribution in Tuna from the Western and Central Pacific Ocean: Influence of Physiology, Ecology and Environmental Factors[END_REF][START_REF] J O U R N A L P R E -P R O O F Kojadinovic | Mercury content in commercial pelagic fish and its risk assessment in the Western Indian Ocean[END_REF][START_REF] Médieu | Stable mercury concentrations of tropical tuna in the south western Pacific ocean: An 18-year monitoring study[END_REF][START_REF] Wang | How closely do mercury trends in fish and other aquatic wildlife track those in the atmosphere? -Implications for evaluating the effectiveness of the Minamata Convention[END_REF] Pelagic food web structuration and functioning have been broadly investigated, mainly through the use of carbon and nitrogen stable isotopes data (δ 13 C and δ 15 N values) [START_REF] Fry | Stable isotope ecology[END_REF]. Recently, collaborative and global studies relying upon δ 13 C and δ 15 N values enabled identifying broad-scale patterns of trophic structure, movements and trophodynamics of tunas in relation to environmental conditions [START_REF] Logan | Global patterns and inferences of tuna movements and trophodynamics[END_REF][START_REF] Lorrain | Trends in tuna carbon isotopes suggest global changes in pelagic phytoplankton communities[END_REF][START_REF] Pethybridge | A global meta-analysis of marine predator nitrogen stable isotopes: Relationships between trophic structure and environmental conditions[END_REF]. Based on individual records with associated metadata (i.e. fish length, fishing date and position) [START_REF] Bodin | Global data set for nitrogen and carbon stable isotopes of tunas[END_REF], these collaborative and global studies also represent a gold mine of already collected and preserved samples to characterize spatial and/or temporal Hg trends in tunas.

An issue with global modern and historical datasets is that samples from different laboratories are not always processed the same way. To account for the influence of lipids on δ 13 C values [START_REF] Deniro | Mechanism of carbon isotope fractionation associated with lipid synthesis[END_REF] while making a single analysis for both δ 13 C and δ 15 N values, δ 13 C values are either produced from i) bulk tissue and a mathematical correction [START_REF] Sardenne | Methods of lipid-normalization for multi-tissue stable isotope analyses in tropical tuna: Lipid-normalization for multi-tissue SIA in tuna[END_REF], or from ii) lipid-free tissue, with lipids removed through various methods and solvents selected not to alter δ 15 N values. In the latter case, manual or automated (high temperature and pressure) methods are generally applied with solvents of low polarity such as dichloromethane and cyclohexane [START_REF] Bodin | ASE extraction method for simultaneous carbon and nitrogen stable isotope analysis in soft tissues of aquatic organisms[END_REF][START_REF] Ménard | Isotopic evidence of distinct feeding ecologies and movement patterns in two migratory predators (yellowfin tuna and swordfish) of the western Indian Ocean[END_REF]. While these methods do not affect δ 15 N values and provide valuable data on lipid content, nothing is known regarding their effect on Hg content, restricting the development of global studies on tuna's Hg concentration with preserved samples prepared for tuna foraging ecology research.

Here, we investigated i) the efficiency and repeatability of two common lipid extraction methods (manual and automated) and two neutral solvents (dichloromethane and cyclohexane) on lipid content determination (experiment A), and ii) the influence of the most J o u r n a l P r e -p r o o f efficient solvent for lipid extraction on total Hg concentrations (experiment B). Experiments were carried out in three tropical tuna species, i.e. bigeye (Thunnus obesus), yellowfin (T. albacares) and skipjack (Katsuwonus pelamis), and on white muscle tissues, the commonly used tissue in studies investigating both trophic ecology and Hg bioaccumulation and the final storage for MeHg in fish. .

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Material & methods

Sample collection

All bigeye, yellowfin, and skipjack tuna samples (n total = 33) were collected in the western Indian Ocean during the unloading of commercial vessels (purse seine) at Victoria port (Seychelles). To test for efficiency and repeatability of different lipid extraction protocols (experiment A), we used three individuals, one per tuna species (n exp A = 3). To test for the effect of lipid extraction on Hg concentrations (experiment B), we used 10 other individuals per tuna species (n exp B = 30), all collected from the same fishing set. For these 30 individuals, fork length (FL) was measured to the lowest cm and ranged respectively for bigeye, yellowfin and skipjack from 44 to 91 cm (62 ± 17 cm), 35 to 129 cm (70 ± 35 cm), and 34 to 64 cm (45 ± 10 cm) (Supplementary Information Appendix S1). For both experiments, white muscle samples of around 20 g (wet weight) were collected in the front dorsal position, stored frozen at -20°C, freeze-dried for 48 hours with an Alpha 1-4 LD freeze-dryer (Christ, Coueron, France) and finally ground to a fine homogeneous powder using a MM400 grinder (Retsch, Eragny sur Oise, France) prior to lipid extraction and total Hg analyses.

Lipid extraction protocols

Two lipid extraction methods (manual and automated) and neutral solvents (dichloromethane and cyclohexane) were first tested on five replicates of three individuals, one per tropical tuna species, to identify the most efficient and repeatable method and solvent in extracting lipids (experiment A, n exp A = 3, Fig. 1A). The use of one single individual per tuna species allowed overcoming the potential bias related to inter-individual variability when estimating both efficiency and repeatability of the lipid extraction methods, as recommended and done in J o u r n a l P r e -p r o o f other studies testing for different lipid extraction methods (AOCS, 2017;JCGM, 2012;Sardenne et al., 2019a). All lipid extractions were done at LEMAR (Plouzané, France).

Manual extraction:

Following the method of [START_REF] Ménard | Isotopic evidence of distinct feeding ecologies and movement patterns in two migratory predators (yellowfin tuna and swordfish) of the western Indian Ocean[END_REF], 100 ± 4 mg of each powdered and dried aliquot were mixed with 10 mL of solvent for 1 hour using a rotary shaker. The mixture of the powder and the solvent was then separated by centrifugation at 2500 rpm for 10 min at 10°C. The lipid-free powders were stored in a dry-room until total Hg analysis, while the lipid extracts were collected in a pre-weighted vial and evaporated to dryness under N 2 flow with a N-evap 111 nitrogen evaporator (OA-SYS, Berlin, USA).

Automated extraction:

150 ± 10 mg of homogenized dried aliquot were extracted with 20 mL of solvent at 100°C under 1400 psi for 10 min using a ASE 350 Accelerated Solvent Extractor (Dionex, Voisins de Bretonneux, France), following the method of [START_REF] Bodin | ASE extraction method for simultaneous carbon and nitrogen stable isotope analysis in soft tissues of aquatic organisms[END_REF]. The lipid-free powders were stored in a dry-room until total Hg analysis, while the lipid extracts were collected in a pre-weighted vial and evaporated to dryness under N 2 flow with a N-evap 111 nitrogen evaporator (OA-SYS, Berlin, USA).

Total lipid content:

For both extraction methods, lipid residuals were weighted on a Mettler Toledo MX5 analytical micro-balance (Mettler Toledo, Colombus, Ohio) to the nearest 0.001 mg to determine the total lipid content (TLC, %) of the samples, expressed on a dry weight basis (dw).

Total mercury analysis

The potential impact of lipid extraction methods on Hg concentrations was assessed on 10 individuals per tuna species, with Hg concentrations determined on both bulk and lipid-free powders (Experiment B, n exp B = 30, Fig. 1B). Total Hg concentrations were measured on powdered, dried and homogenized tissue by thermal decomposition, gold amalgamation and J o u r n a l P r e -p r o o f atomic adsorption (AMA 254, Altec, Czech Republic, Száková et al., 2004) at GET (Toulouse, France). Blanks and two biological materials, TORT-3 (lobster hepatopancreas; Hg = 292 ± 22 ng g -1 dw) and BCR-464 (tuna fish; Hg = 5240 ± 100 ng g -1 dw), covering a wide range of Hg concentrations, were routinely used every 5-10 samples in each analytical batch to check Hg measurement accuracy. Recovery rates (defined as the ratios of observed value by certified value) were calculated for the two certified reference materials (TORT-3: 100 ± 3%; and BCR-464: 100 ± 2%) and variability was below 4%. Hg concentrations are expressed in µg g -1 (equivalent to part per million, ppm), on a dw basis.

Statistical analysis

All statistical analyses were performed with the statistical open source R software 3.6.1 (R Core Team, 2018). We first evaluated the efficiency of both solvents (i.e. dichloromethane and cyclohexane) and methods (i.e. manual and automated) in extracting lipids with an analysis of variance (ANOVA) on TLC values (experiment A). The higher the TLC values, the higher the efficiency. Repeatability of each extraction experiment was assessed per tropical tuna species using the coefficient of variation (CV, defined as the ratio of standard deviation by mean) of TLC values measured on the five replicate samples. The lower the CV, the higher the repeatability. Then, we assessed the influence of manual and automated extraction methods on total Hg concentrations with two separated paired t-test on ΔHg values (with ΔHg = Hg bulk -Hg lipid-free ) (experiment B). For these two tests, we considered all individuals (n exp B = 30 pairs of observations), no matter the species, as ΔHg values do not significantly differ between species (ANOVA, p>0.05 for both methods). TLC, ΔHg values, and residuals of ANOVA were tested for normality with a Shapiro test. Observations were considered as outliers according to the "1.5 rule", i.e. if they were less that Q1 -1.5*IQR, or greater than Q3 + 1.5*IQR, where Q1, Q3 and IQR are the lower quantile, upper quantile and inter-quantile range (defined as Q3-Q1), respectively.

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Results & discussion

Efficiency and repeatability of lipid extraction methods and solvents: TLC values

measured in white muscle varied between 0.4 and 1.7% dw (Supplementary Information Appendix S1). These values are similar to levels reported in same tropical tuna species from the Indian (~3%, [START_REF] Sardenne | Trophic niches of sympatric tropical tuna in the Western Indian Ocean inferred by stable isotopes and neutral fatty acids[END_REF], Atlantic (~6%, Sardenne et al., 2019) and Pacific (~2%, [START_REF] Peng | Amino Acid and Fatty Acid Composition of the Muscle Tissue of Yellowfin Tuna (Thunnus Albacares) and Bigeye Tuna (Thunnus Obesus)[END_REF] Oceans, which confirms tropical tunas are lean species.

For the three tropical tuna species, TLC values were significantly higher when lipids were extracted with dichloromethane than with cyclohexane (Fig. 2; ANOVA, p<0.01).

Dichloromethane and cyclohexane are both non-polar solvents recommended for routine analysis of lipids/fatty acids and/or prior δ 13 C and δ 15 N analysis [START_REF] Bodin | ASE extraction method for simultaneous carbon and nitrogen stable isotope analysis in soft tissues of aquatic organisms[END_REF][START_REF] Ménard | Isotopic evidence of distinct feeding ecologies and movement patterns in two migratory predators (yellowfin tuna and swordfish) of the western Indian Ocean[END_REF]. Here, the higher efficiency of dichloromethane may result from its slightly higher Snyder polarity index (3.1) compared to cyclohexane (0.2) and therefore to the fact that dichloromethane, although a neutral solvent is likely to extract some membrane polar lipids (e.g. phospholipids and sphingolipids) too, increasing TLC values. Nevertheless, this might be of minor importance regarding the protein fraction as this solvent has been shown to have negligible effects on the δ 15 N values across tissues and tuna species [START_REF] Sardenne | Methods of lipid-normalization for multi-tissue stable isotope analyses in tropical tuna: Lipid-normalization for multi-tissue SIA in tuna[END_REF].

Regarding differences between manual and automated methods, TLC values were significantly higher for bigeye and skipjack replicates for the automated extraction (Fig. 2; ANOVA, p<0.05). For bigeye replicates, interaction between solvent and method was also significant, showing that the efficiency of the extraction method depends on the solvent used, with automated extraction with dichloromethane being the most efficient protocol to extract lipids (Fig. 2, ANOVA p<0.05). Conversely, TLC values did not differ significantly between the two extraction methods for yellowfin replicates (Fig. 2; ANOVA, p>0.05). The higher J o u r n a l P r e -p r o o f efficiency of the automated method may be explained by its combined conditions of elevated temperature and pressure that maintain solvents near their supercritical region where they have high extraction properties [START_REF] Ramos | Current use of pressurised liquid extraction and subcritical water extraction in environmental analysis[END_REF]. This suggests that lipid extraction with automated method would be preferred over manual method. On the other hand, this automated method is twice more costly in polluting solvent, and its cleaning steps are longer compared to the manual method. Considering the low difference in extraction efficiency and the fact that is was significant only for the skipjack in-house reference sample, both manual and automated methods are relevant to extract lipids from lean tuna muscle tissues.

The repeatability of the different experiments varied between the protocols between 4 and 27%, but remained overall lower than 18% (Supplementary Information Appendix 2).

Acceptable repeatability often ranges between 5 and 10%, but for lean tissues like white muscle of tropical tunas, repeatability down 20-30% is considered acceptable too, as found in other studies [START_REF] Bodin | ASE extraction method for simultaneous carbon and nitrogen stable isotope analysis in soft tissues of aquatic organisms[END_REF][START_REF] Dodds | Microscale recovery of total lipids from fish tissue by accelerated solvent extraction[END_REF]. As expected, automated method showed generally lower CV values, as ASE 350 has been developed to reduce handling steps and to ensure increased reproducibility. This would be another argument to prefer the automated method; yet given the pros and cons arguments listed above, both manual and automated methods remain acceptable for the lipid extraction from white muscle tissue of tropical tunas.

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Influence of lipid extraction on total Hg concentrations:

Bulk total Hg concentrations (mean ± sd, min-max, dw) varied between tuna species (Kruskal-Wallis, p<0.05), with significantly higher levels in bigeye (0.81 ± 0.47 µg g -1 , 0.20-1.37 µg g -1 ; Dunn's test, p<0.05) than yellowfin (0.38 ± 0.27 µg g -1 , 0.12-1.03 µg g -1 ) and skipjack (0.30 ± 0.17 µg g -1 , 0.12-0.61 µg g -1 ) (Supplementary Information Appendix S1). Those levels and differences are similar to those reported previously in the western Indian Ocean or in both north and south Pacific Oceans for similar fish size ranges [START_REF] Bodin | Trace elements in oceanic pelagic communities in the western Indian Ocean[END_REF][START_REF] Choy | The influence of depth on mercury levels in pelagic fishes and their prey[END_REF][START_REF] Médieu | Stable mercury concentrations of tropical tuna in the south western Pacific ocean: An 18-year monitoring study[END_REF]. The highest Hg concentrations in bigeye are likely to result from confounding effects: a higher trophic position, a deeper vertical habitat giving access to mesopelagic species with higher Hg concentrations, and a longer lifespan compared to the two other tropical tunas [START_REF] Choy | The influence of depth on mercury levels in pelagic fishes and their prey[END_REF][START_REF] Olson | Chapter Four: Bioenergetics, Trophic Ecology, and Niche Separation of Tunas[END_REF].

Manual and automated lipid extractions with dichloromethane did not significantly affect the Hg concentrations in white muscle samples of the three tuna species (Fig. 3; paired t-test, p>0.05). This agrees with [START_REF] Cipro | Influence of Delipidation on Hg Analyses in Biological Tissues: A Case Study for an Antarctic Ecosystem[END_REF] where no difference of Hg levels was found J o u r n a l P r e -p r o o f between bulk and lipid-free muscle samples of two marine fish (i.e. Notothenia coriiceps and N. rossii). This may be related to the particular chemical properties of MeHg in fish flesh and the polarity of the solvent used for lipid extraction. In tuna white muscle, most of total Hg (>91%) is in its methylated form [START_REF] Bloom | On the Chemical Form of Mercury in Edible Fish and Marine Invertebrate Tissue[END_REF][START_REF] Houssard | A Model of Mercury Distribution in Tuna from the Western and Central Pacific Ocean: Influence of Physiology, Ecology and Environmental Factors[END_REF], which is known to bioaccumulate in the protein fraction of the muscle, mostly by forming complexes with the amino acid cysteine [START_REF] Amlund | Accumulation and elimination of methylmercury in Atlantic cod (Gadus morhua L.) following dietary exposure[END_REF][START_REF] Leaner | Methylmercury uptake and distribution kinetics in Sheepshead Minnows, Cyprinodon Variegatus, after exposure to Ch3Hg-spiked food[END_REF][START_REF] Manceau | In Vivo Formation of HgSe Nanoparticles and Hg-Tetraselenolate Complex from Methylmercury in Seabirds-Implications for the Hg-Se Antagonism[END_REF].

On the other hand, we used dichloromethane, a neutral solvent that does not affect the protein fraction by limiting the removal of proteins bound to membrane lipids [START_REF] Bodin | ASE extraction method for simultaneous carbon and nitrogen stable isotope analysis in soft tissues of aquatic organisms[END_REF]. Thus, by preserving the Hg-protein fraction in muscle tissues during lipid extraction, we assumed to have preserved MeHg concentrations of the samples. The absence of a significant effect of lipid extraction on Hg concentrations may be due also to the low lipid content of tropical tuna species (<6%), as measured in our experiment and showed in other studies [START_REF] Peng | Amino Acid and Fatty Acid Composition of the Muscle Tissue of Yellowfin Tuna (Thunnus Albacares) and Bigeye Tuna (Thunnus Obesus)[END_REF]Sardenne et al., 2019b[START_REF] Sardenne | Trophic niches of sympatric tropical tuna in the Western Indian Ocean inferred by stable isotopes and neutral fatty acids[END_REF], and therefore to an unchanged mass balance in the samples. Finally, when discussing the effects of the different protocols tested, it is worthwhile mentioning that high temperature and pressure conditions of the automated extraction did not significantly affect total Hg concentrations neither, suggesting no thermal and pressure degradation of the total Hg pool in the samples. This confirms that MeHg is bound up tightly to the protein fraction in fish muscle tissues, where it is known to remain for a long time [START_REF] Leaner | Methylmercury uptake and distribution kinetics in Sheepshead Minnows, Cyprinodon Variegatus, after exposure to Ch3Hg-spiked food[END_REF]..

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Conclusion

This study reveals the higher efficiency of dichloromethane compared to cyclohexane to extract lipids in tropical tuna white muscle tissue, which may be attributed to its slightly higher polarity index. Automated method appeared more efficient than manual method to extract lipids, especially when used with dichloromethane, which may be attributed the combined conditions of elevated temperature and pressure. Repeatability of all experiments were in acceptable ranges considering that we were working on lean samples.

We show no significant effect of lipid extraction with dichloromethane on total Hg concentrations in tropical tuna white muscle tissue. This may be related to i) the affinity of MeHg (i.e. the most abundant Hg chemical form in tuna muscles) to proteins, ii) the relative low lipid content in white muscle tissue of tropical tuna species, and iii) the non-polar characteristics of dichloromethane, known to efficiently extract storage lipids without affecting the protein fraction. This suggests that white muscle samples that have undergone lipid extraction with dichloromethane can be used equivalently to bulk samples to investigate J o u r n a l P r e -p r o o f Hg bioaccumulation in these pelagic top predators from the western Indian Ocean. Knowing that tropical tunas in the Atlantic, Indian and Pacific Oceans all have relatively low lipid content in white muscle tissue (<6%, [START_REF] Peng | Amino Acid and Fatty Acid Composition of the Muscle Tissue of Yellowfin Tuna (Thunnus Albacares) and Bigeye Tuna (Thunnus Obesus)[END_REF]Sardenne et al., 2019[START_REF] Sardenne | Trophic niches of sympatric tropical tuna in the Western Indian Ocean inferred by stable isotopes and neutral fatty acids[END_REF], this suggests that our results might be valid at a global scale, but further studies are needed to confirm this statement. As lipid content can vary between tissues, species and oceanic basins because of differences in diet and/or in energy allocation strategies distributed among metabolic functions (i.e. growth, reproduction and maintenance), we recommend using bulk samples to infer precisely Hg concentrations in marine fish other than tropical tunas. Further studies would be particularly needed for tissues and species with high lipid content (e.g. swordfish white muscle tissue, between 20-40% dw, [START_REF] Young | Discrimination of prey species of juvenile swordfish Xiphias gladius (Linnaeus, 1758) using signature fatty acid analyses[END_REF].

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  . Nevertheless, key global aspects remain poorly understood, in particular biogeochemical methylation/demethylation mechanisms controlling MeHg bioavailability at the base of the food web, as well as factors driving both fate and accumulation through the food web. Global studies combining Hg concentrations and ecological tracers are therefore needed to clarify these points, especially in the context of the J o u r n a l P r e -p r o o f UNEP Minamata Convention since monitoring studies in marine biota have become essential the better characterize Hg cycle and fate in oceans.

Figure 1 :

 1 Figure 1: Outline of the two experiments testing for A) the efficiency and repeatability of each lipid extraction protocol (manual and automated method, and dichloromethane and cyclohexane solvent) using five replicates of the same individual per tropical tuna species ; and for B) the influence of manual and automated method with

Figure 2 :

 2 Figure 2: Boxplots of total lipid content (TLC, % dry weight) associated to the four lipid extraction protocols and measured in five replicates of each single individual per tuna species. Thick bar is the median value, points are outliers of five replicates, and the box contains 50% of the data.

Figure 3 :

 3 Figure 3: Boxplots of the differences of Hg concentrations (µg g -1 , dry weight) between bulk and lipid-free samples. Box colours display lipid extraction methods (manual and automated). Thick bar is the median value, points are outliers, and the box contains 50% of the data.

Acknowledgments

We thank the fishermen and crews of the SAPMER fishing company who gave the tuna samples. We are grateful to Jean-Marie Munaron from LEMAR (Plouzané, France) for his help with lipid extractions and the access to his lab.

Funding: Funding was provided by ANR-17-CE34-0010, MERTOX from the French Agence Nationale de la Recherche.

Conflicts of interest/Competing interests:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Keywords: Microalgae, biofilm growth, L/D cycles, modeling

Biofilm-based systems have attracted more attention recently as an alternative to the standard suspended-cells process for microalgae cultivation. It has also been shown that cells can be protected from photoinhibition under intense sunlight when submitted to alternately light and dark cycles. However, the biological response to light variation is not well understood. Therefore, in this work, a mechanistic model is developed based on Han's model to describe photosynthesis and respiration dynamics that cover different cycle times and a wide range of light fractions (Light/Dark fractions). Keeping the same average light intensity, increasing the light frequency enhances growth thanks to reduced photoinhibition. Increasing light fraction improves the growth rate as both the peak light intensity and the dark period decrease. Respiration variation in intermittent light regimes is for the first time considered in this model, giving a better explanation of biofilm growth experimental data. Understanding the model response will guide strategies for growth enhancement, and further optimize biofilm reactor design and operation and eventually benefit biomass productivity.

Microalgal biofilm behavior under light/dark cycles

Yan Gao * J. Ignacio Fierro U. * * * Patrick Perré * * Filipa Lopes * Olivier Bernard * * *

INTRODUCTION

Microalgae are regarded as one of the most promising resources for producing food, animal feed, pharmaceuticals, and long-term biofuels [START_REF] Mata | Microalgae for biodiesel production and other applications: a review[END_REF][START_REF] Bhattacharya | Microalgaea green multi-product biorefinery for future industrial prospects[END_REF]. They are mainly cultivated in suspension-based systems [START_REF] Costa | Open pond systems for microalgal culture[END_REF], with an actual yield significantly lower than the theoretical ones.

The high economical cost of cultivation is due, in a large fraction, to harvesting [START_REF] Milledge | A review of the harvesting of micro-algae for biofuel production[END_REF]. This has motivated a recent interest in biofilm-based microalgae technology [START_REF] Mantzorou | Microalgal biofilms: A further step over current microalgal cultivation techniques[END_REF]. Unlike planktonic cells suspended in the medium, microalgae biofilms are thin slimy layers of microalgae that attach and grow on solid surfaces [START_REF] Mantzorou | Microalgal biofilms: A further step over current microalgal cultivation techniques[END_REF]. One of the most interesting advantage of biofilmbased technologies is that harvesting is easy to proceed just by scraping off the biofilm from the surface.

⋆ We thank the financial support of China Scholarship Council and ANR PhotoBiofilm Explorer (Project-ANR-20-CE43-0008) /LaSIPS Greenbelt. We thank also the Département de la Marne, Grand Reims, Région Grand Est and the European Union along with the European Regional Development Fund (ERDF Champagne Ardenne 2014-2020) for their financial support of the Chair of Biotechnology of CentraleSupélec. This work benefited from the ITN Digitalgaesation project funded by the European Union's Horizon 2020 research, and innovation program under the Marie Sk lodowska-Curie grant agreement No. 955520.

Various biofilm reactors with different configurations were developed, as summarized by [START_REF] Wang | Biofilm based attached cultivation technology for microalgal biorefineries-a review[END_REF]. Biofilm reactor design and operation are related to the light regimes received by the attached microalgal cells. Especially, in revolving biofilm systems partially submerged in liquid, cells are submitted to dark periods which may mitigate photoinhibition caused by the constant exposure to intense sunlight. Here, we focus on the process by which the cells in the biofilm grow in periodic light-dark cycles. The light fraction, which is an operating factor, refers to the ratio between the time of exposure to sunlight to the cycle time (T) [START_REF] Gross | Evaluating algal growth performance and water use efficiency of pilot-scale revolving algal biofilm (rab) culture systems[END_REF]. The growth rate (µ) of the biofilm under periodic light regimes depends on the cycle time (T), the light fraction (or duty cycle, ε) and the light intensity (I peak ) during the illumination period.

The impact of fluctuating light on growth has been mainly investigated in planktonic cultures. It was demonstrated that photoinhibition can be reduced by decreasing the cycle time, reaching a flashing regime tending to continuous light of averaged light intensity, which is called full light integration in literature [START_REF] Sforza | Adjusted light and dark cycles can optimize photosynthetic efficiency in algae growing in photobioreactors[END_REF]. All the photons are then used when the flash time is short enough (e.g., in milliseconds scale, [START_REF] Vejrazka | Microalgal photosynthesis under flashing light[END_REF]). This phenomenon corresponds to when photosynthesis rate enhancement is maximal while inhibition is minimal so that the average growth rate is µ S (I peak • ε), where µ S is the growth rate at steady state. Inappropriate L/D cycles, induce growth penalty [START_REF] Graham | A penalty on photosynthetic growth in fluctuating light[END_REF]. By comparison, the socalled no light integration describes that photoinhibition is maximal, with no improvement of photosynthesis rate, due to long light/dark periods (so that the average growth rate is µ S (I peak ) • ε) [START_REF] Graham | A penalty on photosynthetic growth in fluctuating light[END_REF].

By contrast, only a few studies have focused on biofilms subjected to fluctuating light regimes [START_REF] Toninelli | Scenedesmus dimorphus biofilm: Photoefficiency and biomass production under intermittent lighting[END_REF][START_REF] Martín-Girela | Flashing light effects on co2 absorption by microalgae grown on a biofilm photobioreactor[END_REF]. For instance, [START_REF] Toninelli | Scenedesmus dimorphus biofilm: Photoefficiency and biomass production under intermittent lighting[END_REF] studied biofilms of Scenedesmus dimorphus exposed to fluctuating light with T ranging from 0.2 s to 24 h and with ε of 0.33 and 0.5. They observed an increase in the photoefficiency when decreasing T. In biofilms, unlike planktonic cultures in high density reactors, the light regime is entirely controlled by the reactor design and operation. It is therefore identical for all cells which experience the same T (related to rotation speed) and the same ε (related to the reactor design). Only the light intensity changes according to biofilm depth, due to the light attenuation within the biofilm [START_REF] Huang | Comparison of chlorella vulgaris biomass productivity cultivated in biofilm and suspension from the aspect of light transmission and microalgae affinity to carbon dioxide[END_REF].

The biological response of biofilm cells to intermittent light is not fully understood yet [START_REF] Toninelli | Scenedesmus dimorphus biofilm: Photoefficiency and biomass production under intermittent lighting[END_REF].

A detailed insight into photosynthesis dynamics response and quantitative and predictive models are still required [START_REF] Vejrazka | Microalgal photosynthesis under flashing light[END_REF]. In addition, the models presented in the literature have ignored respiration variation in intermittent light regimes, biasing the estimations of net growth and productivity [START_REF] Beardall | Studies on enhanced post-illumination respiration in microalgae[END_REF].

Therefore, to simulate biofilm growth and understand the effect of intermittent light regimes, we propose here a model considering the biofilm thickness and including respiration variation in light/dark periods. We compute the average growth rate in periodic light regimes. The model calibration and validation were guided by experiments performed with Chlorella vulgaris biofilms subjected to four continuous light regimes and six light/dark regimes. Finally, the model's ability to predict various light regime is discussed.

MODEL DEVELOPMENT

This model is developed based on the mechanistic model of [START_REF] Han | A mechanistic model of algal photoinhibition induced by photodamage to photosystem-ii[END_REF] to integrate the effect of photoinhibition on photosynthesis. It is based on the assumption that the reaction centers can be categorized in three states [START_REF] Megard | Kinetics of oxygenic photosynthesis in planktonic algae[END_REF]: open and ready for catching photons A, closed while processing photons B, or inhibited when some key proteins (D1) get damaged due to an excess of energy resulting from too many harvested photons C. The states A, B and C represent the proportion of the three states of photosynthetic units in each of these states. Therefore, it follows that:

A + B + C = 1
(1) The dynamics of the three states of the model is given by:

                 Ȧ = -σIA + B τ , Ḃ = σIA - B τ + k r C -k d σIB, Ċ = -k r C + k d σIB.
(2)

Here σ (µmol -1 •m 2 ) is the cross-section of PSII; τ (s) represents the turnover time of the electron transfer chain. k d (dimensionless) is the damage rate and k r (s -1 ) is the repair rate. The system (2) can be reduced using (1) into a two-dimensional system:

   Ȧ = -σI + 1 τ A + 1 -C τ , (3a)
Ċ = -(k r + k d σI) C + k d σI(1 -A).
(3b) The gross growth rate, which is the rate of carbon fixation from the Calvin cycle, can then be written as: µ = kσIA.

(4) Where k (s • s -1) is a scaling factor, linking the rate of photons capture to the microalgae growth rate.

In this paper, we consider piece-wise constant light/dark cycles with period T . The biofilm is exposed to a light intensity I peak for a time of T L = εT and then the biofilm remains in darkness for a time T D = (1 -ε)T . We denote by t 2i-1 the moment when dark period starts; by t 2i the moment when light period starts; and by t 2i+1 the moment when a cycle finishes. This light pattern is presented in Fig. 1.

Light/dark periods shorter than seconds

When the light changes rapidly, the dynamics of C is too slow to change, then we reduce the dynamics (3a) and (3b) into one equation assuming that C remains constant [START_REF] Hartmann | The effect of photosynthesis time scales on microalgae productivity[END_REF] and we denote this value as C. The dynamic of state A during the light phase is then deduced from Equation (3a) as a function of I:

A(t) = 1 - C τ σI + 1 1 -e -(σI+ 1 τ )(t-t2i) + A(t 2i )e -(σI+ 1 τ )(t-t2i) , (5)
where

A(t 2i ) = (1 -C)g, g = (1 -x + 1-y σIτ +1 x) 1 -xy , x = e -T (1-ε) τ , y = e -(σI+ 1 τ )T ε . (6)
The average value of A in the light phase (t ∈ [t 2i , t 2i+1 ]) (light period integrated, noted as ĀL ) is expressed by:

ĀL = 1 T ε t2i+1 t2i A(t) dt = (1 -C)h, (7)
where

h = 1 σIτ + 1 + 1 σIτ + 1 -g • 1 T ε σI + 1 τ • (y -1),
To get C, the asymptotic value of C, we do a cycle time integration at two sides of Equation (3b):

t2i+1 t2i-1 Ċdt = t2i+1 t2i-1 -k r C + k d σI -k d σIA -k d σIC dt, (8) Since in periodic regime C(t 2i-1 ) = C(t 2i+1 ), we get: 0 = -k r CT + k d σIT ε + k d σICT ε - t2i+1 t2i k d σIA(t) dt, C = k d σI -k d σIh kr ε -k d σIh + k d σI . ( 9
)
The average gross growth rate (light period integrated, noted as μL ) in light phase (t ∈ [t 2i , t 2i+1 ]) is then:

μL (I) = 1 T ε • t2i+1 t2i kσIA(t) dt = kσI ĀL . ( 10
)
2.2 Light/dark periods longer than seconds

As studied by [START_REF] Bara | Model development and light effect on a rotating algal biofilm[END_REF], for light/dark periods a range of order longer than the time constant characterizing its dynamics, A reaches its pseudo steady state quickly in the light phase and then in the dark phase. State A very rapidly converges to the following function of C:

A = 1 -C 1 + τ σI . (11)
Then the dynamics of C in Equation (3b) can be rewritten in:

Ċ = -αC + β, (12) where α
= k d τ (σI) 2 1 + τ σI + k r , β = k d τ (σI) 2 1 + τ σI . ( 13
)
The evolution of C deduced from Equation (12) in light phase (t ∈ [t 2i , t 2i+1 ]) can be expressed by:

C(t) = β α 1 -e -α(t-t2i) + C(t 2i )e -α(t-t2i) , (14)
where

C(t 2i ) = β α • (1 -e -αT ε ) e krT (1-ε) -e -αT ε . ( 15
)
The average value of C during light phase (t ∈ [t 2i , t 2i+1 ]) (noted as CL ) is:

CL = 1 T ε • t2i+1 t2i C(t) dt, = β α + β α 2 T ε - C(t 2i ) αT ε • e -αT ε -1 . ( 16
)
The growth rate in the light phase (noted as µ L ) is deduced from Equation (4) and Equation ( 11):

µ L = γ(1 -C), (17)
where

γ = kσI 1 + τ σI . ( 18
)
In periodic regime, the average gross growth rate in light phase (t ∈ [t 2i , t 2i+1 ]) (light period integrated, noted as μL ) can then be obtained:

μL (I) = 1 T ε • t2i+1 t2i γ(1 -C) dt = γ(1 -CL ). ( 19
)
where CL is given by Equation ( 16).

Respiration variation

In this model, we assume that respiration (noted as R) varies in light and dark phases, following a dynamics described in Equation ( 20):

Ṙ = k R (χ R (I) -R), (20)
where k R is the evolution rate (s -1 ) of the respiration rate (s -1 ), and

χ R (I) = R L , if I > I min , R D , if I ≤ I min , (21)
R L and R D are two constants, representing the highest respiration rate in light (R L , s -1 ), and the lowest respiration rate relieved in dark phase (R D , s -1 ), respectively. We assume that the actinic light I min is much lower than the values of I experimented during the light phases. Since in periodic regime R(t 2i-1 ) = R(t 2i+1 ), the cycle time average respiration R is deduced by integrating Equation ( 20):

1

T t2i+1 t2i-1 Ṙ dt = 0 = k R T ( t2i t2i-1 R L dt + t2i+1 t2i R D dt) - k R T t2i+1 t2i-1 R dt R = 1 T t2i+1 t2i-1 R dt = R L ε + R D (1 -ε) (22)

The net growth rate in biofilm

In a square-wave L/D cycle, the cycle time average photosynthetic growth rate (called the gross growth rate in the following) is μL (I) • ε.

When the light penetrates the biofilm, its intensity is attenuated following the Lambert-Beer law, meaning that, at each depth z the light intensity is

I(z) = I 0 e -kzz . ( 23
)
where k z is the light extinction coefficient. Note that k z = 1 z ln I0 I(z) . The Beer Lambert equation can be rewritten as dI = -k z Idz. The net growth rate (photosynthetic minus respiration) (noted as μ(I 0 )) of the film averaged over time is obtained by the integration of the local growth, as obtained by the local light intensity, along the film depth:

μ(I 0 ) = 1 h h 0 μL (I(z))ε dz - R = 1 ln I0 I(h) I0 I(h) μL (I)ε I dI -R.
(24)

MODEL RECALIBRATION AND VALIDATION

We aim at determining a vector of model parameter p = (k, k r , k d , τ, σ, R L ) that minimizes the error between the simulated state vector µ sim and the measured state vector µ m . For this purpose, the likelihood function of the error has to be maximized with respect to p. Assuming that the error is distributed according to a Gaussian white noise with a diagonal covariance matrix, this is equivalent to minimize the optimization criterion Ψ M LE (p):

Ψ M LE (p) = K k=1 J j=1 µ m k j -µ sim k j σ k 2 , ( 25
)
Where σ k is the standard deviation of measured growth rates of each light regimes. K is the total number of light regimes tested, and J is the number of the replicates of each tested light regime. k and j are the order of light regimes and replicates, respectively. The Matlab Nelder-Mead simplex direct search minimization algorithm (fminsearch) was used and initialized with the first parameter guess (Han model parameters values from [START_REF] Wu | A model integrating fluid dynamics in photosynthesis and photoinhibition processes[END_REF]) respiration rate from [START_REF] Grenier | The impact of light supply to moving photosynthetic biofilms[END_REF]), as shown in Table 1.

The net specific growth rate is computed from the experimental light transmittance as the slope of the regression between ln(ln light input light output ) and time t. 4. RESULTS

Cycle time effect

Fig. 2A displays the net growth rate dynamic response to cycle times of the intermittent light regimes. With the same average light intensity, the shorter cycle time leads to higher growth rate compared to longer time scale. This is in agreement with the work of [START_REF] Hartmann | The effect of photosynthesis time scales on microalgae productivity[END_REF].

For cycle time larger than 15 min light integration is no more observed, and the maximum level of photoinhibition is induced, as presented in Fig. 2C. In the range of seconds to tens of seconds, an intermediate regime is reached, which is not represented in [START_REF] Hartmann | The effect of photosynthesis time scales on microalgae productivity[END_REF]. Full light integration is obtained for cycle time shorter than 100 ms. In these conditions, the inhibition rate decreases to the same level of the one for the equivalent constant average light (see Fig. 2C). In the end, a higher growth rate with flashing light is obtained due to the lower average respiration rate than the one triggered by constant light. D1 proteins and increasing in the light phase. Compared to light regimes with the same duty cycle of 1/3 (310 µmol • m -2 •s -1 (D/L = 10s/5s; 60s/30s; 2min/1min), light regime with cycle time of 3 min induces higher maximum inhibition level than 90 s and 15 s. As only the C proportion in the light phase matters for growth, the average C in the light phase with different duty cycles is presented in Fig. 2C. This explains the response of growth rate to different light regimes in Fig. 2B. Light fraction effect on the net growth rate is presented in Fig. 4A for the various light regimes studied. With cycle time from 15 s to 3 min, the growth rate is positively impacted by the light fraction. As cycle times of 15 s and 25 s are the second plateau range (Fig. 2), the improvement of growth rate under 310 µmol • m -2 •s -1 (D/L = 10s/5s) compared with that at 500 µmol • m -2 •s -1 (D/L = 20s/5s) is due to effect of light fraction. The underlying reason is presented in Fig. 4B. With different cycle time scales (T > 1 ms), average C in the light phase decreases with light fraction, thus leading to a higher growth rate (see Fig. 4A). The inhibition level at different cycle time magnitudes are more significant when the light fraction is close to 0, which means intense but short light peaks. By contrast, a light fraction close to 1 indicates that the light regime is close to the continuous light regime with the same average light intensity. It is worth noting that there is not much difference in inhibition level between the different light regimes with larger light fractions. Especially, C remains the same at different duty cycles when cycle time is 1 ms, corresponding to the full light integration plateau in Fig. 2B and Fig. 2C.

DISCUSSION

The proposed model, improving Han's model [START_REF] Han | Photosynthesis-irradiance response at physiological level: A mechanistic model[END_REF] by including respiration dynamics, reproduces the experimental biofilm growth behavior at different cycle time scales and light fractions. On the contrary to the existing models [START_REF] Takache | Investigation of light/dark cycles effects on the photosynthetic growth of chlamydomonas reinhardtii in conditions representative of photobioreactor cultivation[END_REF], this model can simulate the non-linear partial light integration with short cycle time orders and give important leads for growth rate optimization. The point is that we computed the average growth rates in any regime as a function of the light/dark signal. This made the model calibration straightforward, since it was not necessary to integrate slow-fast differential equations on very long time scale, making minimization procedures more efficient. The model fits well with observations with a p value of 0.0093 < 0.05.

The photosynthetic efficiency computation, and the associated inhibition level, were so far determined only for duty cycles ε = 0.5 [START_REF] Hartmann | The effect of photosynthesis time scales on microalgae productivity[END_REF]. The cycle time effect on biofilm growth rate presented in Fig. 2 is consistent with the observations of [START_REF] Toninelli | Scenedesmus dimorphus biofilm: Photoefficiency and biomass production under intermittent lighting[END_REF]. Fig. 2B shows that full light integration phenomenon occurs in tens of milliseconds scale, in agreement with the study of [START_REF] Vejrazka | Microalgal photosynthesis under flashing light[END_REF]. From our model, this time order is the same for different light fractions. The difference between the net growth rate obtained for light/dark regimes and continuous light (ε of 1, grey line) is only 3 % with ε of 2/3 (red line) for T around 100 s. This is invisible from experimental data, which will be considered in both cases as full light integration. But the difference is more significant with smaller ε at the same time magnitude. This is coherent with the work of [START_REF] Toninelli | Scenedesmus dimorphus biofilm: Photoefficiency and biomass production under intermittent lighting[END_REF] wherefull light integration was observed at cycle time of seconds with the light fraction of 0.5, but was not detected with the light fraction of 0.33 even cycle time shortened down to sub-seconds citepToninelli2016.

No light integration is predicted at tens of minutes, which is supported by [START_REF] Grenier | The impact of light supply to moving photosynthetic biofilms[END_REF] focusing in Chlorella autotrophica biofilms . Interestingly, this time order seems longer than that published (seconds or hundreds of seconds) for planktonic cultures [START_REF] Xue | Growth of spirulina platensis enhanced under intermittent illumination[END_REF][START_REF] Graham | A penalty on photosynthetic growth in fluctuating light[END_REF]. More importantly, the characteristic inflexion point (second plateau in Fig. 2) occurs in different cycle time orders compared with planktonic Chlamydomonas reinhardtii cultures at 10-20 ms [START_REF] Vejrazka | Microalgal photosynthesis under flashing light[END_REF].

CONCLUSION AND PERSPECTIVES

Light/dark regimes have been broadly studied in the literature to identify light regimes which optimize microalgae growth. So far, the analytical computation of the solutions of the Han model were not available for duty cycles different from 0.5 or 1. Considering light induced respiration phenomenon deeply affects the model outcomes, and it seems that this feature is specific to photosynthetic biofilms. The model is still sketchy, and further work would be necessary to better understand the respiration dynamics in response to fluctuating light.

Fig. 1 .

 1 Fig. 1. Graphic description of the light pattern.

  Different light regimes were used to recalibrate the model. Continuous light with intensities of 100, 200, 310, 500 µmol • m -2 •s -1 were considered. D/L represents the dark phase duration/light phase duration. Four intermittent light regimes with peak light intensity of 310 µmol • m -2 •s -1 (D/L = 10s/5s; 60s/30s; 2min/1min), 500 µmol • m -2 •s -1 (D/L = 20s/5s) were also used in the recalibration procedure. The model was validated with two intermittent light regimes with peak light intensity of 310 µmol • m -2 •s -1 (D/L = 8s/4s) and 400 µmol • m -2 •s -1 (D/L = 5s/5s). The validated parameter values are shown in

Fig. 3 AFig. 2 .

 32 Fig.3shows the evolution of the inhibited state with respect to the light regimes. With continuous illumination, the inhibition level remains constant with time, and it increases with increasing light intensity. Interestingly, even

Fig. 3 .

 3 Fig. 3. C evolution in one photoperiod for each light regime studied 4.2 Light fraction effect

Fig. 4 .

 4 Fig. 4. With the same average light intensity of 100 µmol • m -2 •s -1 : (A), growth rate with respect to light fraction. Growth rate of fluctuating light regimes are fitted in the figures and compared with constant light regimes. Light regimes 310 µmol • m -2 •s -1 (D/L = 10s/5s; 60s/30s; 2min/1min) share the same light fraction: 1/3, but the latter two light regimes are shifted slightly on the right side to better distinguish from each other. Experimental data are shown with mean and standard deviation. (B), The average C in light phase with respect to light fraction at different cycle time scales.

Table 1 ,

 1 except the value of R D which is set at 9.15 • 10 -8 s -1 .

Table 1 .

 1 Validated biological parameters of Han model and respiration rate for biofilm growth rate simulation

	Parameters	First guess	Calibrated value	Unit