C. Pousis

Comparative study of liver vitellogenin gene expression and oocyte yolk accumulation in wild and captive Atlantic bluefin tuna (Thunnus thynnus L.).

The sequence of vitellogenin A (VgA) and vitellogenin B (VgB) cDNAs in Atlantic bluefin tuna (Thunnus thynnus L.) were determined, and vitellogenin expression levels in the liver and oocyte yolk accumulation were compared in wild and captive-reared individuals. Liver and ovary samples were taken from 31 individuals reared experimentally in three commercial Atlantic bluefin tuna fattening sites in the Mediterranean Sea and from 33 wild individuals caught by commercial traps during the fishs migration towards their Mediterranean spawning grounds. The total length of VgA cDNA was 5585 nucleotides and that of VgB was 5267 nucleotides. The identity and similarity between deduced amino acid sequences of VgA and VgB were 60% and 78%, respectively. The Atlantic bluefin tuna VgA and VgB amino acid sequences have high similarities with those of other teleost fishes. Relative levels of VgA and VgB mRNAs were low in April, increased significantly during the reproductive period in May and June, and declined in July. There was a trend towards higher relative levels of VgA and VgB mRNAs in captive fish compared to wild individuals during the reproductive period. The surface occupied by eosinophilic yolk granules in fully vitellogenic oocytes, as well as the frequency of oocytes in late vitellogenesis, was significantly higher in captive compared to wild individuals. The study suggests that the experimental conditions under which Atlantic bluefin tuna individuals were reared allowed the occurrence of normal vitellogenesis, based on gene expression of VgA and VgB in the liver and yolk accumulation in the oocytes. The higher yolk accumulation and frequency of vitellogenic oocytes observed in the ovaries of captive fish suggest that improvements in feeding practices may result in an improved vitellogenic process.

Expression of vitellogenin receptor gene in the ovary of wild and captive Atlantic bluefin tuna (Thunnus thynnus)

The cDNA sequences of vitellogenin receptor proteins (VgR(+) and VgR(-)), containing or lacking the O-linked sugar domain, were determined in Atlantic bluefin tuna (Thunnus thynnus L.). VgR(-) gene expression in the ovary was compared in captive-reared and wild Atlantic bluefin tuna during the reproductive cycle. Gonad samples from adult fish were sampled from 2008 to 2010 from stocks reared in captivity at different commercial fattening operations in the Mediterranean Sea and from wild individuals caught either by traditional tuna traps during their migration towards the spawning grounds in the Mediterranean Sea or by the long-line artisanal fishery. In addition, juvenile male and female Atlantic bluefin tuna were sampled from a farming facility, to obtain baseline information and pre-adulthood amounts of VgR(-). The total length of VgR(+) cDNA was 4006 nucleotides (nt) and that of VgR(-) was 3946 nt. Relative amounts of VgR(-) were greater in juvenile females and in those adults having only previtellogenic oocytes (119 ± 55 and 146 ± 26 folds more than juvenile males, respectively). Amounts of VgR(-) were less in individuals with yolked oocytes (ripening stage, May-June) and increased after spawning in July (92 ± 20 and 113 ± 13 folds more than juvenile males in ripening and post-spawning fish, respectively). These data suggest that regulation of VgR(-) is not under oestrogen control. During the ripening period, greater VgR(-) gene expression was observed in wild fish than in fish reared in captivity, possibly because of (a) differences in water temperature exposure and/or energy storage, and/or (b) an inadequate diet in reared Atlantic bluefin tuna.

Evidence that severe acute stress and starvation induce rapid atresia of ovarian vitellogenic follicles in Atlantic bluefin tuna, Thunnus thynnus (L.) (Osteichthyes: Scombridae)

The effects of different stressors on the atretic degeneration of ovarian vitellogenic follicles, as well as on the ovarian mass, were examined in female Atlantic bluefin tuna, Thunnus thynnus (L.), from the Mediterranean Sea. The stressors taken into consideration were short-term starvation (up to 14 days), long-term cage rearing (1 year) and crowding-induced severe panic frenzy. Wild-caught individuals were used as a control group. Fish subjected to either severe panic frenzy or starvation exhibited a decrease in gonad mass and had significantly higher intensity of α atresia in the vitellogenic follicles (means: 78% and 58%, respectively; range: 36-100%) than either wild or long-term caged individuals (means: 32% and 30%, respectively; range: 19-44%). The extensive atresia in fish stressed by severe panic frenzy was observed as early as 24 h after the stressing event. The present study represents the first evidence of the extreme susceptibility of Atlantic bluefin tuna to severe acute stress during vitellogenesis; it also shows that starvation is associated with progressive reabsorption of vitellogenic oocytes.

Comparative Study of Reproductive Development in Wild and Captive-Reared Greater Amberjack Seriola dumerili (Risso, 1810).

The greater amberjack Seriola dumerili is a large teleost fish with rapid growth and excellent flesh quality, whose domestication represents an ambitious challenge for aquaculture. The occurrence of reproductive dysfunctions in greater amberjack reared in captivity was investigated by comparing reproductive development of wild and captive-reared individuals. Wild and captive-reared breeders were sampled in the Mediterranean Sea during three different phases of the reproductive cycle: early gametogenesis (EARLY, late April-early May), advanced gametogenesis (ADVANCED, late May-early June) and spawning (SPAWNING, late June-July). Fish reproductive state was evaluated using the gonado-somatic index (GSI), histological analysis of the gonads and determination of sex steroid levels in the plasma, and correlated with leptin expression in the liver and gonad biochemical composition. The GSI and sex steroid levels were lower in captive-reared than in wild fish. During the ADVANCED period, when the wild greater amberjack breeders were already in spawning condition, ovaries of captive-reared breeders showed extensive atresia of late vitellogenic oocytes and spermatogenic activity ceased in the testes of half of the examined males. During the SPAWNING period, all captive-reared fish had regressed gonads, while wild breeders still displayed reproductive activity. Liver leptin expression and gonad proximate composition of wild and captive greater amberjack were similar. However, the gonads of captive-reared fish showed different total polar lipid contents, as well as specific lipid classes and fatty acid profiles with respect to wild individuals. This study underlines the need for an improvement in rearing technology for this species, which should include minimum handling during the reproductive season and the formulation of a specific diet to overcome the observed gonadal decrements of phospholipids, DHA (22:6n-3) and ARA (20:4n-6), compared to wild breeders.

Fin Spine Bone Resorption in Atlantic Bluefin Tuna, Thunnus thynnus, and Comparison between Wild and Captive-Reared Specimens

Bone resorption in the first spine of the first dorsal fin of Atlantic bluefin tuna (ABFT) has long been considered for age estimation studies. In the present paper spine bone resorption was assessed in wild (aged 1 to 13 years) and captive-reared (aged 2 to 11 years) ABFT sampled from the Mediterranean Sea. Total surface (TS), solid surface (SS) and reabsorbed surface (RS) were measured in spine transverse sections in order to obtain proportions of SS and RS. The spine section surface was found to be isometrically correlated to the fish fork length by a power equation. The fraction of solid spine bone progressively decreased according to a logarithmic equation correlating SS/TS to both fish size and age. The values ranged from 57% in the smallest examined individuals to 37% in the largest specimens. This phenomenon was further enhanced in captive-reared ABFT where SS/TS was 22% in the largest measured specimen. The difference between the fraction of SS of wild and captive-reared ABFT was highly significant. In each year class from 1- to 7-year-old wild specimens, the fraction of spine reabsorbed surface was significantly higher in specimens collected from March to May than in those sampled during the rest of the year. In 4-year-old fish the normal SS increase during the summer did not occur, possibly coinciding with their first sexual maturity. According to the correlations between SS/TS and age, the rate of spine bone resorption was significantly higher, even almost double, in captive-reared specimens. This could be attributed to the wider context of systemic dysfunctions occurring in reared ABFT, and may be related to a number of factors, including nutritional deficiencies, alteration of endocrine profile, cortisol-induced stress, and loss of spine functions during locomotion in rearing conditions.

Increased liver apoptosis and tumor necrosis factor expression in Atlantic bluefin tuna (Thunnus thynnus) reared in the northern Adriatic Sea

The Atlantic bluefin tuna Thunnus thynnus (ABFT) is intensely fished in the Mediterranean Sea to supply a prosperous capture-based mariculture industry. Liver apoptotic structures and tumor necrosis factor (TNF) gene expression were determined in: wild ABFT caught in the eastern Atlantic; juvenile ABFT reared in the central Adriatic Sea; juvenile ABFT reared in the northern Adriatic Sea; adult ABFT reared in the western Mediterranean. The highest density of liver apoptotic structures was found in the juveniles from the northern Adriatic. Two partial TNF cDNAs (TNF1 and TNF2) were cloned and sequenced. TNF1 gene expression was higher in juveniles than in adults. The highest expression of TNF2 was found in the juveniles from the northern Adriatic. These findings might be related to the juvenile exposure to environmental pollutants.

Rearing in captivity affects spermatogenesis and sperm quality in greater amberjack, (Risso, 1810)

The greater amberjack, Seriola dumerili (Risso, 1810), is a promising candidate for the diversification of European aquaculture production, but inconsistent reproduction in captivity prevents commercial production. Recent studies showed that greater amberjack confined in sea cages exhibited scarce gonad development and early interruption of gametogenic activity during the reproductive season. The aim of the present study was to improve our understanding of the observed impairment of spermatogenesis. Adult wild and captive-reared males were sampled during 3 different phases of the reproductive cycle: early gametogenesis (EARLY; late April to early May), advanced gametogenesis (ADVANCED; late May to early June), and spawning (SPAWNING; late June to July). Spermatogonial stem cells and proliferating germ cells were identified through the immunohistochemical localization of Pou5f1 and proliferating cell nuclear antigen, respectively. Apoptotic germ cells were identified throughout the terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nick end labeling method. Sperm quality of captive-reared fish was evaluated using computer-assisted sperm analysis. Captive-reared males exhibited seminiferous lobules of a smaller diameter, a precocious and progressive decrease of spermatogonial mitosis, and a high level of apoptosis at the beginning of the reproductive season, concomitant with a many-fold higher 17β-estradiol plasma concentration. The motile spermatozoa percentage of captive greater amberjack was lower than in other teleosts, and a drastic decrease of spermatozoa motility duration, velocity, and ATP content occurred along the reproductive season. An abnormal increase of sperm concentration as well as an increase of dead spermatozoa occurred during the SPAWNING phase, probably because of lack of sperm hydration and ejaculation and consequent sperm ageing...

Micro-anatomical structure of the first spine of the dorsal fin of Atlantic bluefin tuna, Thunnus thynnus (Osteichthyes: Scombridae)

The first spine of the first dorsal fin (FS) of the Atlantic bluefin tuna (ABFT), Thunnus thynnus, is customarily used in age determination research because its transverse sections display well-defined growth marks. In this paper the FS structure was studied to explain its known dramatic age- and season-related morphological modifications, which are evidently caused by bone remodeling. Cross sections of samples from six adult ABFT were in part decalcified to be stained with histological, histochemical and immunohistochemical methods, and in part embedded in methyl-methacrylate to be either observed under a linear polarized light or microradiographed. FS showed an external compact bone zone and an inner trabecular bone zone. The compact bone zone consisted of an outer non-osteonic primary bone layer (C1) and an inner osteonic bone layer (C2). C1 was in turn characterized by alternate translucent and opaque bands. Evidence of spine bone remodeling was shown by the presence of osteoclasts and osteoblasts as well as by tartrate-resistant acid phosphatase (TRAP) positive bands at the boundary between old and newly formed bone. The examination of plain, i.e. not-fixed and not-decalcified, FS from 28 ABFT showed that the average thickness of C1 remained fairly constant during fish growth, whereas C2 increased significantly, indicating that the periosteal primary bone apposition is counterbalanced by the parallel bone remodeling occurring inside the compact bone zone. The present study revealed the structure of the ABFT FS and the pattern of its bone remodeling. Both of them underlay phenomena, never examined in detail before, such as the appearance followed by the progressive disappearance of growth bands.