Aquaculture Reports 17 (2020) 100380

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Aquaculture Reports

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Thicklip (Chelon labrosus) and flathead ( cephalus) grey mullets fry T production in Tunisian aquaculture Raouf Besbesa, Amina Besbes Benseddika,*, Lambros Kokokirisb, Thomas Changeuxc, Ahlem Hamzaa, Fathi Kammouna, Hechmi Missaouia a Institut National des Sciences et Technologie de la Mer (INSTM). Centre of Monastir, Monastir, Tunisia b International Hellenic University, Department of Nutritional Sciences and Dietetics, Sindos University Campus, Thessaloniki, Greece c Institut Méditerranéen d’Océanologie (MIO), Marseille, France

ARTICLE INFO ABSTRACT

Keywords: For several years Tunisia has opted to breed freshwater in reservoirs and artificial lakes, created for irri- Teleosts gation, as a strategy of providing high quality aquatic protein to the interior regions and providing work op- Mugilidae portunities for local communities. The aim of this study was to summarize the main results accumulated by the last 20 year efforts made by the members of the National Institute for Marine Science and Technology (INSTM)- Aquaculture Laboratory to develop thicklip ( labrosus) and flathead (Mugil cephalus) grey mullets fry production from captive broodstocks intended for stocking inland reservoirs (artificial lakes) and grow-out purposes Administration of human chorionic gonadotropin (hCG) at a priming dose of 10.000 IU kg−1 female bw, followed by 10.000 IU hCG and (100 or 200 μg kg−1 female bw of thicklip or flathead grey re- spectively) as resolving dose resulted at the highest egg production for both . Mean fecundity was 494.655 eggs kg-1 bw for and 418.945 eggs kg-1 bw for . Fertilization rate of eggs produced was high (mean rate 83 % and 63 % for thicklip and flathead grey mullet respectively) and larvae hatched from those eggs at high rates (77 % and 88 %, respectively). The green method was more efficient than clear method to produce thicklip and flathead grey mullet fry, (i.e larvae had higher growth rate and better survival rates). The results of this study demonstrate the possibility to control reproduction and produce grey mullet fry in captivity. However, further research is needed to optimize the production protocol of eggs and fry of both grey mullet species.

1. Introduction Grey mullet fry stocking enhancement of inland waters for captures or and for grow out purposes (extensive and semi-extensive Among grey mullets the thicklip (Chelon labrosus) and the flathead rearing) still relies on fry collections from the wild in many countries, grey mullet (Mugil cephalus) are retained as potential candidates in the i.e Japan, Taiwan, Hawaii (Milne, 1972; Shehadeh et al., 1973), Phi- diversification of Tunisian aquaculture products. These fish, which are lippines (Pillay, 1990), Italy (Ravagnan, 1978; Cataudella et al., 1988a, eurythermal, and omnivorous have a relatively low dietary b; Crosetti and Cataudella, 1994), Spain (Arias et al., 1984), Portugal protein requirement. They are highly appreciated by the Tunisian (Vale et al., 1998), France (Labourg, 1976) Israel (Bar-llan, 1975) and consumer and are excellent candidates for continental aquaculture. Egypt (Ishak et al., 1982). However, fry availability is irregular com- They also constitute highly appreciated products for their organoleptic monly linked with problems of recruitment and overfishing (Crosetti, qualities (Romdhane et al., 2019). The (Boutargue or Pou- 2015). Besides, fry collections impose a risk compromising natural targa), mainly made from the of fully ripe female flathead grey stocks and causing a potential conflict of interest with the inshore mullets, a rare and luxurious Mediterranean specialty, sometimes called . Stocking Tunisian inland waters with grey mullets fry caught “Mediterranean caviar” has a high market value exceeding 200 euros along the coast, remained the main practise for over thirty years (Raïs per kilogram. and Turki., 1989). However, to ensure sustainability of the grey mullet

⁎ Corresponding author at: Institut National des Sciences et Technologie de la Mer. Centre de Monastir (INSTM), Route de Khniss, BP. 59 – 5000 Monastir, Tunisia. E-mail addresses: [email protected] (R. Besbes), [email protected] (A.B. Benseddik), [email protected] (L. Kokokiris), [email protected] (T. Changeux), [email protected] (A. Hamza), [email protected] (F. Kammoun), [email protected] (H. Missaoui). https://doi.org/10.1016/j.aqrep.2020.100380

Available online 05 June 2020 2352-5134/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). R. Besbes, et al. Aquaculture Reports 17 (2020) 100380

Table 1 Biochemical composition of food delivered to thicklip and flathead grey mullet broodstocks.

Food Moisture (%) Protein (%) Fat (%) Ash (%) Carbohydrates (%) Energy* (kcal/100 g) Feeding rate %

Commercial Dry Food 8.84 48.85 22.93 8.76 19.46 447.52 52 Local Semi-Dry Food 47.12 53.81 7.85 9.78 28.56 354.10 28 Fresh 74 68 6 12.8 13.20 417.48 20

* Digestible Energy (Kcal per 100 g of dry food) is calculated from the following respective metabolic energy density of 4.85 kcal/g of protein, 8 kcal/g of lipid and 3.1 kcal/g of carbohydrates.

Table 2 Protocol for feeding thicklip and flathead grey mullet larvae with live prey in three production systems (green water, clear water and mesocosm).

Larvae rearing system Volume (L) Initial density (larvae/L) Days post hatch (dph) Food Sequences

Green Water 550 100 7−25 Rotifers: Brachionus plicatilis Open circulation 15−35 Artemia: A. salina (nauplius, A0) Type AF-EG) 25−57 Artemia: A. salina (metanauplius, A1) Clear Water 400 100 5−23 Rotifera: Brachionus plicatilis Closed circulation 17−37 Artemia: A. salina (nauplius, A0) (Type AF -EG) 25−57 Artemia: A. salina (metanauplius, A1) Semi-extensif 20.000 2,5 5−23 Endogenous food + Rotifers: B. plicatilis Mesocosme 14−32 Artemia: A. salina (nauplius, A0) (Type AF -EG) 22−55 Artemia: A. salina (metanauplius, A1)

Fig. 1. Caligus pageti, copepod ectoparasite, detached from thicklip (C. labrosus) and flathead (M. cephalus) grey mullet brooders held in captivity. a: female, b:male individual. Bar scale: 1000 μm. aquaculture and to meet all the needs for fingerlings (estimated at flathead (M. cephalus) grey mullets to define methods of fry production several millions a year) the control of reproduction of captive brood- intended for stocking inland reservoirs and grow-out purposes. This stocks at a large scale should be achieved for a regular and sufficient study consists a synopsis of our effort to establish captive broodstocks to production (Besbes et al., 1999). induce spawning but also to test fry production of thicklip and flathead Considering that grey mullets represent an important fish produc- grey mullets, under various production protocols. tion with great economic importance in Tunisia and that breeding ap- pears to be essential for the diversification of the tunisian aquaculture 2. Materials and methods product and vital for the sustainability of the mullet fisheries (Romdhane et al., 2019), many efforts have been made at the hatchery 2.1. Spawning induction of the National Institute of Marine Sciences and Technology (INSTM, Center of Monastir) focused on the thicklip (C. labrosus) and the Adult thicklip and flathead grey mullet obtained from various

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Fig. 2. Chelon labrosus. a: taking an ovarian biopsy in vivo using a fine polypropylene cannula, introduced into the genital of an anaesthetized female. b: Macroscopic examination of oocytes of the ovarian biopsy to determine the stage of oogenesis of the most advanced oocytes. c: vitellogenic oocyte at mean diameter of 600 μm. d: vi- tellogenic oocyte at 900 μm in diameter, entering the final oocyte maturation stage (FOM), after the resolving dose of hormonal injection to induce spawning of female brooder. Notice, the distinction of follicular zone, the formation of lipid globule and the nucleus migration to the germinal pole.

Tunisian including that of Hergla (Governorate of Sousse) using and resolving injection of hCG (10,000 IU) and LHRH (100 μg kw−1), the bordigue trap placed along the channel connecting the and (d) priming injection of hCG (10,000 IU kg−1) and resolving injection the sea. They acclimatised in captivity and kept at a density of 4 kg m−3 of hCG (10,000 IU), (e) priming injection of LHRH (200 μg kg−1) and in concrete ponds (12 m)3 filled with filtered seawater under a seawater resolving injection of LHRH (200 μg kg−1). renewal rate of 20 % per hour. Broodstock feed consisted of commercial Each spawning tank was equipped with a surface drainage system food (Skretting, broodstock diet), semi-dry foods produced locally which allowed collection of pelagic eggs into a tank egg collector (mesh (Besbes and Geurbej, 1996) and fresh sardines (Table 1). Oxygen, size: of 500 μm). Spawning was monitored daily by inspection of tank temperature and water quality (NH3-N and NO2-N) were measured once egg collectors and when spawning was observed, eggs were transferred per week. into a 10-L bucket using a dip net and their number (fecundity) was Females were monitored for their stage of reproductive develop- estimated by counting the total number of eggs in a subsample of ment each year between February and April for thicklip grey mullet and 10 mL, after vigorous agitation. Fertilization success was evaluated at between July to September for flathead grey mullet. After females an- the same time by calculating the number of viable eggs with respect to aesthetized (0.3 mL 2-phenoxyethanol L−1) a wet mount of ovarian the total number of eggs spawned. biopsy obtained by inserting of a fine Pipelle de Cornier catheter into the genital pore. Fresh ovarian samples examined under a stereoscope, 2.2. Eggs incubation and hatching to measure the mean diameter of the largest, most advanced oocytes (n = 10) and evaluate the stage of oogenesis. Only females at late vi- Eggs were incubated at a density of 1500−3000 eggs L−1 in tank tellogenesis stage, i.e. with oocytes of a mean diameter greater than incubators (40 L) supplied with filtered (5 μm) and UV sterilized sea- 550 μm were used for spawning induction trials. Males were examined water (T = 18.6; pH = 8.45; S = 37.7ppt). The incubators were for maturation by the release of sperm upon application of gentle ab- equipped with a central inlet from the bottom and with another one dominal pressure. Females and males at a ratio 1:2 were kept un- tangential from the top, which allowed suspension of eggs and pre- disturbed to . vention of clogging. Spawning experiments were carried out in circular tanks (1 m3) The hatching rate is calculated from the ratio of the number of supplied with ambient filtered (5 μm) seawater (salinity = 37.0 ±2 hatched larvae collected to the total number of eggs incubated. ppt and pH = 8.02 ± 0.2). Five groups of female and male breeders established at a ratio 1:2 female to male. Females treated with hCG (Human Chorionic Gonadotropin; BBT Biotech Germany) and LHRH 2.3. Larvae and fingerlings production (Luteinizing Hormone-Releasing; Sigma-Aldrich US) hormones by in- tramuscular injections of approximately 5,000–10,000 IU hCG kg−1 Three fish larvae production systems used to test their efficiency in and 100–200 μg kg-1 at priming and resolving doses as following: (a) producing thicklip and flathead grey mullet larvae: (1)-an intensive priming injection of HCG (5000 IU kg−1) and resolving injection of hCG system in clear water, (2)-an intensive system in green water and (3)-a (5000 IU kg−1) and LHRH (200 μg kg−1), (b) priming injection of hCG semi-extensive system or mesocosm enclosure. Rearing trials carried (10,000 IU kg−1) and resolving injection of hCG at 10.000 IU kg−1 and out under equivalent thermal conditions corresponding to the natural LHRH at 100 μg kg−1, (c) priming injection of hCG (10,000 IU kg−1) conditions of the site. Intensive rearing (clear-water or green-water system) was done in

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indoor tanks (cylindroconical, from 0.55 to 5 m3) but the semi-ex- tensive (mesocosm) in outdoor ponds (20 m3) under a greenhouse production facility. In the "clear water" system, seawater was recycled through a series of filters (sand: 30 μm then cartridge: 2 μm), a biolo- gical filter and a ramp (U.V.). The hourly flow was set at 25 %andthe 74 (2) 77 (7) 53 (4) 0 (0) 13 (4) 43 (33) supply of fresh water was set at 10–15 %. The "green-water" system was applied in the presence of microalgae, Chlorella minutiossima at den- sities ranging from 10,000 to 80,000 cells ml−1. When the larvae en- tered the trophic phase, the water was partially renewed (between 10 and 30 %). Mesocosm system was filled with filtered seawater (mesh size: 360 μm) to eliminate competitors and predators of larvae. At the beginning, water turnover was at 0.3 L min−1 to ensure skimming of 55 (14) 83 (11) 28 (11) 0 (0) 20 (3) 37 (31) the air-water interface using appropriate equipment. Diffusers main- tained a slight stirring of the medium to avoid water column stratifi- cation. Larvae stocked at density of 1.5 larvae/L (30,000 larvae per mesocosm enclosure of 20 m3 (Zouiten et al., 2008)). Rotifers and Artemia, were successively delivered according to the size of larvae (Table 2) as soon as they opened mouth. The first se- quence was composed of Rotifers (Brachionus plicatilis) with (Chlorella minutissima) and yeast (Saccharomyces cerevisiae) enriched with DHA- Protein Selco (INVE), until 23–25 dph. Depending on the size of larva, 456.330 (57.623) 494.655 (26.113) 0 (0) 5.204 (3.355) second sequence was composed of small Artemia (Artemia salina) AF Type, then larger Artemia EG types enriched using DC-Super Selco (INVE) from 14–17 to 34–37 dph. From weaning onwards, commercial compound food of increasing size was successively delivered according to the size of fingerlings (Replace II, 100−300 μm, Rich-SA, LANZY W3, 300−500 μm, INVE, NRD 3/5, 300−500 μm, INVE, PERLA 5/8, 500−800 μm, Trouvit). Water temperature, pH, dissolved oxygen and nitrogen (N-NH4, N- 825.000 (35.355) 890.000 (14.142) 225.000 (106.066) 137.363 (77.704) 0 (0) 10.000 (7.071) 390.000 (413.474) 218.710 (229.817) NO2, N-NO3) were monitored daily. In all production systems, the seawater turnover was adjusted to keep ammonia concentration below 0.3 mg L−1 (Ounaïs-Guscheman, 1989). Specific Growth Rate (SGR) was used to address fish growthin terms of length (mm) or weight (in mg) using the equation: SGR = (ln (final weight in mg) - ln(initial weight in mg) x100) / t (in days).To study growth, 15–30 larvae or juveniles weighed and measured every four or five days during the first 40 days period for both the thicklipand flathead grey mullets. However, the monitoring of growth ofthe thicklip grey mullet continued by weighting and measuring total length of 15–30 larvae each 10–15 days intervals up to 60 dph. Samples were treated (prepared) in a way described in (Ben Khemis et al., 2006). 1.720 (141) Female bw (g) Latency Period (hrs) Nb of eggs per female Nb of eggs per kg female Fertilization rate (%) Hatching rate (%) 1.873 (151) 72 (0) 1.781 (131) 72 (0) 3. Results

Broodfish were infected by Caligus pageti (Copepoda, Siphonostomatoida, Caligidae), which caused host fish suffering from abrasions and skin sloughing followed by severe losses (Fig. 1). Mor- talities were systematically recorded twice a year: from March to April and from October to November when the water temperature was around 17 °C and favoured infection. The parasite could be detached with thin clamps, but chemical eradication was more easily applied and efficient than detaching from host broodfish. The treatment developed bw female) in INSTM consisted of a 20 min bath in formaldehyde (37 %) at a −1 concentration of 0.25 ppt, with a high oxygenation using liquid oxygen (minimum of 140–160% saturation). Oogenesis and spermatogenesis normally occurred in captivity and timing was synchronised to gametogenesis of wild populations. Male thicklip and flathead grey mullets were spermiating earlier than fe- 10.000 IU hCG 10.000 IU hCG 10.000 IU hCG 10.000 hCG +200 LHRHa 1.818 (152) 72 (0) 10.000 IU hCG 10.000 hCG +100 LHRHa 1.803 (124) 72 (0) Priming injection Resolving injection 5.000 IU hCG 5.000 IU hCG+200 LHRHa 1690 (184) 72 (0) Hormone dose (kg 200 μg LHRHa 200 μg LHRHa males However female broodfish had ovaries with oocytes arrested at secondary vitellogenesis stage, with largest oocytes having a diameter around 650 μm (Fig. 2). Females were failed to ovulate and spawn and various hormones were used to induce spawning. All therapies con- sisted of a two-step injection protocol with administration of various hormones (i.e gonadotropins and LHRH) and doses at priming and re- 4 3 2 1 Chelon labrosus 5 Total

Table 3 Egg production data after various experimental trials to induce spawning of thicklip grey mullet ( Chelon labrosus ) brooders in INSTM (Centre of Monastir, Tunisia). solving injections 24 h apart.

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Fig. 3. Chelon labrosus. Egg development stages. a: fertilized egg, b: 4 lipid globules, c,d,e: blastula stage, f: gastrula stage, g,h,i: formation of embryo: Ø 1150 μm.

3.1. The thicklip grey mullet ( ± 3.3) % in length and 13.4 ( ± 5.8) % in weight (Fig. 5a,b). Growth rate was retarded during weaning (30–60 dph), as a result of changes in Administration of hCG alone even at two-step injections (i.e priming feeding behaviour and dieting habits, thus SGR reached 2.5 ( ± 0.1) % 10,000 IU and resolving 10,000 IU kg−1 bw) wasn't effective in indu- in length and 7.19 ( ± 3.8) % in weight (Fig. 5a,b). cing spawning (Table 3). However, administration of hCG in combi- The overall larvae growth obtained during 60 dph with the green nation with LHRH was effective at any doses used. Actually, adminis- method described by the equation y = 3.14 e 0.033x in length tration of hCG at a priming dose of 10.000 IU, followed by a resolving (R2 = 0.97) and y = 0.436 e0.093x in weight (R2 = 0.97, Figs. 6 and 7). dose of 10,000 IU hCG and 100 μg kg-1 bw female resulted at the However, growth obtained using the clear method described by a lower highest egg production. Mean fecundity was 494,655 eggs kg−1 bw rate both in length (y = 3.090 e0.028x,R2 = 0.98) and weight (Table 3). Similarly, spawned eggs produced from those females ferti- (y = 0.432 e 0.087x,R2 = 0.98, Figs. 6, 7). lized at the highest rate (83 %) and larvae from those eggs hatched at the highest rate (77 %, Table 3). 3.2. The flathead grey mullet Mean temperature during spawning was 15 ± 2 °C. Spawned eggs had an average diameter of 1.15 mm (n = 23) and more than two oil Similarly, to results obtained from spawning trials of the thicklip globules. The incubation of fertilised eggs lasted 72 h (on average at grey mullet, the two-step injection was crucial. Administration of hCG 15 °C). The embryonic and larval development stages are shown in at a priming dose of 10,000 IU, followed by a resolving dose of 10,000 Figs. 3 and 4. The mean larvae length at hatching was 3.4 mm and the IU hCG and 200 μg kg−1 female bw resulted at the highest egg pro- mouth opened at 5 dph at the size of 4.1 ( ± 0.2) mm. The swim duction (mean relative fecundity: 418.945 eggs kg−1 bw) with the bladder inflation started approximately 10 dph and on 17 dph itwas highest fertilization (63 %) and hatching rates (88 %, Table 4). possible to estimate the rate of larvae with a functional . Spawned eggs of the flathead grey mullet were around 0.95 mmin The metamorphosis began 20 dph and increased gradually up to 30 dpf diameter. The incubation lasted 22 h. Some of the embryonic and the finalizing at a length of 7.5 ( ± 2) mm and at a weight of11.5(±2) larval development stages of the flathead grey mullet are shown in mg. Figs. 8 and 9. The larvae length at hatching was 2.5 mm and the mouth Mean growth was very slow the first two weeks post hatching opened at 2 dph at a total length (TL) of 3.2 ( ± 0.2) mm. The larvae (Fig. 5a,b). SGR in terms of length and weight were quite low until 15 sized around 2.5 mm at hatching but they reached 3.2 mm at 1 dph. The dph. Growth accelerated from 15 to 30 dph and mean SGR reached 7.49 larvae stagnated around 3.2 mm so that their length reached only 3.5

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Fig. 5. Specific growth rate (%) of thicklip (Chelon labrosus) and flathead grey mullet (Mugil cephalus) larvae in terms of length (a) and body weight (b) during the period of first 25 days post hatching (d0-d25 dph).

bw; equivalent to 7 pituitary glands) or hCG (10,000 IU kg−1 bw) as priming and LHRHa (200 μg kg−1) as resolving doses was efficient in Fig. 4. Chelon labrosus. Larval development. a: newly hatched larva, b: 3-day inducing spawning of the flathead grey mullet (Mousa et al., 2018). old larva, c: 5-day old larva: mouth opening, d: 14-day old larva. Bar scale: Similarly, administration of CPE (20−70 mg kg-1) or hCG (10.000 IU 500 μm. -1 −1 kg ) and LHRHa (200 μg kg ) at two step injections was efficient in inducing spawning of the flathead grey mullet (Lee et al., 1988). As far ( ± 0.2) mm at 5 dph. The yolk sac was completely absorbed the 6 dph as the hormonal treatments tested in this study, the highest spawning and the oil globule was completely disappeared the 7 dph. The pig- performance (i.e. in terms of egg production, fertilization and hatching mentation started the 4 dph and two days after (i.e. 6 dph) the dorsal rate) of both grey mullets achieved using hCG (5000 IU kg-1) as a and the lateral parts were the most intensely pigmented. The 6 dph the priming injection followed by resolving injections of hCG (5000 IU caudal fin was formed and rudimentary pectoral fins were appeared. At kg−1) and LHRHa (100 μg kg-1 bw). However, unless all the above 8 dph larvae started to swim actively and measured 4.2 mm. The me- treatments were physiologically effective they required extremely high tamorphosis began 18 dph at a length of 6.3 ( ± 2) mm and increased doses of hormones (i.e. hCG from 5 to 10,000 IU kg-1, CPE 200 mg kg-1, gradually up to 28 dpf finalizing at a length of 10.2 ( ±2)mm LHRHa 100–200 μg kg-1). However, one-step administration of dopa- Growth was accelerating and reached the highest values from 5 to mine antagonist (DA) (i.e. domperidone, metaclopramide, etc) with −1 −1 10 dph (13.6 % bw day ) and from 15 to 20 dph (6.6 % TL day , GnRHa even at low doses inhibited dopaminergic action and was very Fig. 5a,b). Overall growth obtained with the green method for a period potent in inducing spawning of flathead and thicklip grey mullet in 0.047x of 25 dph described by the equation y = 2.719 e in length captivity. Aizen et al. (2005) treated female flathead grey mullet with 2 084x 2 (R = 0.95) and y = 0.242 e 0. in weight (R = 0.96) (Figs. 10 and GnRHa (loaded on slow release implants at 10 mg kg−1 bw) in combi- 11). nation with dopamine antagonists (DA), i.e. metaclopramide (15 mg kg−1) or domperidone (5 mg kg−1), they achieved high rates of 4. Discussion ovulation and spawning success, without any priming dose of GtHs (hCG, CPE). Similarly, Kokokiris et al. (2015) reported that adminis- −1 −1 4.1. Spawning induction tration of metaclopramide (15 mg kg ) or domperidone (5 mg kg ) and GnRHa in the form of weekly injections of 10 μg kg−1 female bw or −1 Injections of gonadotropins (GtHs) in the form of pituitary via slow release implants (50 μg kg bw) were effective in inducing extracts (CPE) or human chorionic gonadotropin (hCG) in a two-step spawning of captive thicklip grey mullet. Additionally, administration −1 injection protocol combined with LHRHa analogues were the most of metaclopramide (20 mg kg ) and a high dose of GnRHa −1 widely adopted methods to induce spawning of thicklip and flathead (200 μg kg ) was effective to induce high rates of ovulation ofwild- grey mullet in captivity. Crosetti and Cordisco (2004) treated success- caught flathead grey mullet (Vazirzadeh and Ezhdehakoshpour, 2014) -1 fully female thicklip grey mullet with 7 CPE as priming dose followed when CPE (20 mg kg ) was administered as a priming injection. Thus, by 50 μg kg−1 bw as resolving dose. Administration of CPE (20 mg kg−1 unless the two-step injection protocol of hCG and hCG + LHRHa was

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Fig. 6. Length growth of thicklip grey mullet (Chelon labrosus) larvae in "green water” and "clear water”.

Fig. 7. Weight growth of thicklip grey mullet (Chelon labrosus) larvae in "green water” and "clear water”. effective to induce spawning of grey mullets in this study, theadmin- (0,2 ppt) allowed the total eradication of Caligus bombavensis (Ben istration of a dopamine antagonist in combination to administration of Jemaa-Najar, 1995). Also, a 20-min immersion of thicklip (or and hCG and LHRH could further improve the spawning perfromance of flathead) grey mullet in freshwater was effective in eliminating co- grey mullets. Such an enhancement of spawning performance could pepod parasites (Devauchelle, 1980). reduce the doses of hCG and LHRHa needed, thus reducing the cost of For the transport to any distance, quality eggs should be packaged spawning induction method. in 15 L plastic bags filled with 2/3 of filtered and sterilized sea water, In fact, three years of acclimatisation to intensive rearing conditions supersaturated with oxygen (300–400 % saturation). Plastic bags (i.e. maintenance in concrete ponds, fed artificial food) was the should be kept inside isothermal coolers in darkness. Egg density during minimum time required for grey mullet to reach sexual maturity, being transport should be around 30 g of eggs L−1 (Besbes et al., 2001). Eggs responsive to any spawning induction treatment. These results are in transport is recommended during early hours of embryonic develop- accordance to Devauchelle (Mousa, 2010) and Nash and Koningsberger ment when mortality is expected to be lower. Transport of eggs is more (Glubokov et al., 1994), who found than under extensive rearing con- safe compared to larvae transport since they are protected by an outer ditions, maturation is possible only after at least two years of accli- hard shell. matization. Larval rearing was tested successfully by three techniques (green The parasitic copepod, Caligus pageti was detached from the body water, clear water and mesocosm) that were fairly well suited for surface and the gills cavity of both grey mullet broodfish. Parasites of breeding grey mullets larvae with comparable results concerning sur- the Caligus species can occur in high numbers in marine and brackish vival rates. Larvae growth in "green waters" and "mesocosms" were waters grey mullets (Devauchelle, 1980) acting as disease causing significantly higher compared to clear water technique. Even so,the agent, reducing growth and even market value but also causing severe mesocosm method is less recommended for mass production because of losses when broodfish remain untreated for long time (Nash and its low yield, i.e. around 1000 weanling fry per m3 (Chatterji et al., Koningsberg, 1981; Noor El-Deen et al., 2012; Ben Hassine, 1983). In 1982). Meanwhile, intensive systems of rearing (green water and clear the laboratory, the treatment of flathead grey mullet with formaldehyde water) are more productive, with average production rates around

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16,000 fry per m3 (Besbes et al., 2012). Independently of the technique used, the growth of thicklip grey mullet larvae is very slow during the first two weeks post hatching despite the presence of yolk andlipid reserves and the sufficient abundance of live prey in the rearing en- vironment; it seems that such a slow growth is a peculiarity of thick lip 4 (3) 45(14) 88(4) 0 (0) 30 (7) 33 (34) grey mullet larvae. In fact, comparable growth results were reported by Boglione et al. (1992). The final survival rate obtained was considered satisfactory foreach of the production methods used: 13 % in clear water compared to 18 % in green water, 25 % in mesocosms at 60 dph. These results seem to indicate a greater adaptation of larvae to waters rather charged with phytoplankton or even to turbid waters, at least during their first days 25 (5) 25 (0) 63 (4) 0 (0) 20 (7) 26 (22) of life. Cataudella et al. (1988a) reported a much faster growth of larvae raised in natural pond compared with those raised in an intensive type of hatchery. For the flathead grey mullet, M. cephalus, larval rearing was tested only using the green water technique that was considered the most suitable for mass production. The larvae at hatching sized lower than those of thicklip grey mullet and their growth rate was slower, but it reversed to high growth during the growing phase (Ben Khemis et al., 2004; Besbes et al., 2010). However, survival rate was low, (i.e. 10 %) 46.199 (9.755) 136.004 (9.692) 0 (0) 2.917 (1.554) resulting to low production of fingerlings. Although weak, these values are encouraging demonstrating the possibility of producing flathead grey mullet larvae in captivity. Also, they are perfectible depending on the capacity of accumulation of the staff’s know-how. All the techniques tested prove to be adapted to grey mullets. As far as the massive production of grey mullets fry is concerned, (i.e. at the order of 5 millions), the extensive or semi-extensive mesocosm systems, which are in general of low productivity and also of random and un- predictable production (because of their dependence on hydroclimatic 52.500 (10.607) 162.500 (17.678) 675.000 (106.066) 418.945 (96.263) 0 (0) 3.500 (2.121) 178.700 (271.199) 120.813 (168.592) parameters, which are often uncontrollable), fall behind hatchery-in- tensive production systems (green or clear water). For the larval growth differences observed between these techniques, they were recoverable during the extensive growth phase in dam reservoirs, which are natural environments favourable to the development of grey mullets (Raïs and Turki., 1989).

5. General conclusion

The control of reproduction of grey mullets was successfully put in practice and fry production at large scale was achieved. The know-how technology of the INSTM)-Aquaculture Laboratory team has been al- ready transferred (since 2012) to a Tunisian farm company (GIPP/ Female bw Latency Period (hrs) Nb of eggs per female Nb of eggs per kg female Fertilization rate (%) Hatching rate (%) 1.370 (354) 1.173 (103) 16 (0) 1.302 (241) 17 (5) COSPE, Tabarka) specialized to grey mullet fry production and grow- out. That collaboration allowed for comparative experimental seeding of potentially exploitable environments i.e the freshwater hill lakes of Bni Atta and Fartout (North of country), the Ickeul lake, the Monastir lagoon and the geothermal waters of the artesian wells of southern Tunisia (South of Country). Results obtained were very encouraging clearly demonstrating that stocking grey mullets populations into con- tinental facilities (lakes/reservoirs for irrigation) could boost activity, into the frame of rationalization of the natural resources 5.000 hCG +200 LHRHa 1.138 (11) 24 (0) creating sources of income for inhabitants of those regions. However, bw female) future projects are needed to further improve fry production, to verify −1 the zootechnical performance of inland aquaculture and finally to as- sess its environmental and socio-economic impact.

CRediT authorship contribution statement Priming injection Resolving injection 5.000 IU hCG 10.000 IU hCG 10.000 hCG +100 LHRHa 1.203 (216) 15 (1) 10.000 IU hCG 10.000 hCG +200 LHRHa 1.625 (120) 12 (3) Hormone dose (kg 10.000 IU hCG 10.000 IU hCG 200 μg LHRHa 200 μg LHRHa Raouf Besbes: Project administration, Supervision, Investigation, Conceptualization, Methodology, Software, Formal analysis, Writing - original draft, Validation. Amina Besbes Benseddik: Data curation, Formal analysis, Writing - original draft, Writing - review & editing. Lambros Kokokiris: Writing - review & editing, Validation. Thomas Changeux: Visualization, Writing - review & editing, Validation. 1 2 3 Mugil cephalus 4 5 Total

Table 4 Egg production data after various experimental trials to induce spawning of flathead grey mullet ( Mugil cephalus ) brooders in INSTM (Centre of Monastir, Tunisia). Ahlem Hamza: Resources, Software, Formal analysis. Fathi

8 R. Besbes, et al. Aquaculture Reports 17 (2020) 100380

Fig. 8. Mugil cephalus. Egg development stages. a: fertilized egg, b: blastula stage, c,d: morula stage, e: gastrula stage, f-i: developing embryo. Ø 950 μm.

Fig. 9. Mugil cephalus. Larvae development. a: Newly hatched larva, b: 1 day old larva, c: 2 day old larva: mouth opening, d: 5 day old larva, e: 9 day old larva and f: 12 day old. Bar scale: 500 μm.

9 R. Besbes, et al. Aquaculture Reports 17 (2020) 100380

Fig. 10. Length growth of flathead grey mullet (Mugil cephalus) larvae in "green water”.

Fig. 11. Weight growth of flathead grey mullet (Mugil cephalus) larvae in "green water”.

Kammoun: Resources, Software, Formal analysis. Hechmi Missaoui: Besbes, R., Geurbej, H., 1996. Procédé de fabrication d’aliments semi-humides pour les Funding acquisition, Validation. élevages du loup Dicentrarchus labrax et de la daurade Sparus aurata. Bull Inst Nat Scien Techn Mer. N° 3 (1996). pp. 46–61. Besbes, R., Guerbej, H., El Ouaer, A., El Abed, A., 1999. Choix de nouvelles espèces de Declaration of Competing Interest poissons marins pour l’aquaculture en Tunisie (Synthèse bibliographique). Institut National des Sciences et Techniques de la Mer. Rapport interne 18 pp. Besbes, R., Ben Jemaa-Najjar, S., Besbes Benseddik, A., Fauvel, C., El Abed, A., Ben The authors declare that they have no known competing financial Hassine, O.K., 2001. Parasitose causée par Caligus pageti (Russel; 1925) chez les interests or personal relationships that could have appeared to influ- géniteurs de Mugil cephalus et Chelon labrosus en stabulation. Description et traite- ence the work reported in this paper. ments de la pathologie. Bull Inst Nat Scie Tech Mer. N° Spécial 6. pp. 39–42. Besbes, R., Besbes Benseddik, A., Ben Khemis, I., Zouiten, D., Zaafrane, S., Maatouk, K., El Abed, A., M’RABET, R., 2010. Développement et croissance compares des larves du References mulet lippu Chelon labrosus (Mugilidae) élevées en conditions intensives: eau verte et eau claire. Cybium 34 (2), 145–150. Besbes, R., Besbes Benseddik, A., 2012. Expertise et assistance technique au démarrage de Aizen, J., Meiria, I., Tzchoria, I., Levavi-Sivanb, B., Hanna Rosenfelda, H., 2005. l’Ecloserie pilote de mulets et d’anguilles de Tabarka. Rapport final de mission. Enhancing spawning in the grey mullet (Mugil cephalus) by removal of dopaminergic COSPE (Italie)/GIPP (Tunisie). Projet “AID 8049/COSPE/TUN”: 37 pp.. . inhibition. Gen. Comp. Endocrinol. 142 (2005), 212–221. Boglione, C., Bertolini, B., Russiello, M., Cataudella, S., 1992. Embryonic and larval de- Arias, A.M., Dreke, P., Rodriguez, R.B., 1984. Los esteros de las salinas se San Fernando velopment of the thick-lipped mullet (Chelon labrosus) under controlled reproduc- (Cadiz, Espana) y el cultivo extensivo de peces marinos. In: Barnabé, R. (Ed.), tion conditions. Aquaculture 101, 349–359. Colloque sur l’aquaculture du bar (loup) et des sparidés. Sète (France), 15 mars 1983, Cataudella, S., Massa, F., Rampacci, M., Crosetti, D., 1988a. Artificial reproduction and INRA, Paris. pp. 447–463. larval rearing of the thick lipped mullet (Chelon labrosus). J. Appl. Ichthyol. 4, Bar-llan, M., 1975. Stocking of Mugil capito and Mugil cephalus at their commercial catch 130–139. in lake Kinneret. Aquaculture 5, 85–89. Cataudella, S., Crosetti, D., Massa, F., Rampacci, M., 1988b. The propagation of juvenile Ben Hassine, O.K., 1983. Les copépods parasites de poisons Mugilidae en Méditerranée mullet (Chelon labrosus) in earthen ponds. Aquaculture 68, 321–323. occidentale (Côtes françaises et tunisiennes). Morphlogie, bio-écologie, cycles Chatterji, A., Ingole, B.S., Parulekar, A.H., 1982. Effectiveness of formaldehyde in Caligus évolutifs. Thèse Doctorat d’Etat, U.S.T.L. Montpellier. 452 p. . infection of laboratory reared grey mullet, Mugil cephalus (L.). Indian J. Mar. Sci. 11 Ben Jemaa-Najar, S., 1995. Dynamique évolutive des ectoparasites bioindicateurs dans le (4), 344–346. complexe lagunaire Ichkeul-Bizerte. D.E.A. Fac. Sciences Tunis. 211 p. . Crosetti, D., 2015. Current state of grey mullet fisheries and culture. Biol. Ecol. Cult. Grey Ben Khemis, I., Kamoun, F., Zouiten, D., Besbes, R., 2004. Croissance comparée des larves Mullets 398–450. du mulet lippu (Chelon labrosus) élevées en conditions intensives et semi extensives Crosetti, D., Cataudella, S., 1994. The mullets. In: Nash, C.E. (Ed.), Production of Aquatic en mésocosmes. Bull Inst Nat Scie Tech Mer. N° spécial 9. pp. 163–166. – . Elsevier, Amsterdam, pp. 253–268. Ben Khemis, I., Zouiten, D., Besbes, R., Kamoun, F., 2006. Larval rearing and weaning of Crosetti, D., Cordisco, C.A., 2004. Induced spawning of the thick-lipped mullet (Chelon thick lipped grey mullet (Chelon labrosus) in mesocosm with semi-extensive tech- labrosus, Mugilidae, ). Mar. Life 14 (1–2), 37–43. nology. Aquaculture 259 (1), 190.

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Corrigendum Corrigendum to “Thicklip (Chelon labrosus) and flathead (Mugil cephalus) grey mullets fry production in Tunisian aquaculture” [Aquacult. Rep. 17 (2020) 100380]

Raouf Besbes a,*, Amina Besbes Benseddik a, Lambros Kokokiris b, Thomas Changeux c, Ahlem Hamza a, Fathi Kammoun a, Hechmi Missaoui a a Institut National des Sciences et Technologie de la Mer (INSTM), Centre of Monastir, Monastir, Tunisia b International Hellenic University, Department of Nutritional Sciences and Dietetics, Sindos University Campus, Thessaloniki, Greece c Institut M´editerran´een d’Oc´eanologie (MIO), Marseille, France

refers to: were initially incorrectly listed as co-authors of this article, as they Raouf Besbes, Amina Besbes Benseddik, Lambros Kokokiris, Thomas consider their involvement insufficient to justify co-authorship. The Changeux, Ahlem Hamza, Fathi Kammoun, Hechmi Missaoui contents of the article remain unchanged. Thicklip (Chelon labrosus) and flathead (Mugil cephalus) grey mullets The correct author list is: Raouf Besbes, Amina Besbes Benseddik, fry production in Tunisian aquaculture Lambros Kokokiris, Ahlem Hamza, Fathi Kammoun. Aquaculture Reports, Volume 17, July 2020, 100380 The authors would like to apologise for any inconvenience caused. The authors regret that Thomas Changeux and Hechmi Missaoui

DOI of original article: https://doi.org/10.1016/j.aqrep.2020.100380. * Corresponding author. E-mail address: [email protected] (R. Besbes). https://doi.org/10.1016/j.aqrep.2020.100536

Available online 24 November 2020 2352-5134/© 2020 Published by Elsevier B.V.