Larval and juvenile development of dusky grouper Epinephelus marginatus reared in mesocosms

The larval development of the dusky grouper Epinephelus marginatus up to the benthic juvenile stage is described in detail to establish a reference for their larval identification. Development is described in terms of ontogenetic changes in morphology, growth, pigmentation, fin structure and skeletal structure. Larvae were reared in mesocosms at a mean temperature of 24·3° C, salinity of 36·5, dissolved oxygen of 6·4 mg l^?1 and pH of 8·2. Newly hatched larvae had an estimated total length (L_T) of 2·3 mm. On the second day post hatching the yolk was almost fully absorbed with traces of the oil globule still present, the eyes were already pigmented and mouth and gut functional. At this stage the cranial skeletal elements for feeding and breathing (mouth and gills) and the pectoral‐fin support were already present. About 50% of the observed larvae had food in their guts. Pigmentation was very characteristic, consisting of two large chromatophores visible on the edge of the primordial fin, close to the midpoint of the post‐anal region of the body and over the midgut and hindgut and post‐anal portion of the body. At 2·9 mm L_T the emergence of the second dorsal‐fin spine, characteristic of the Epinephilinae, was clearly visible. The pre‐flexion stage started in larva of 3·2 mm L_T. At 5·5 mm L_T the larvae possessed posterior preopercular angle spines, and the dorsal and pelvic spines presented serrated edges and were pigmented. The water surface‐tension‐related death of the yolk sac and pre‐flexion larvae described in the rearing of several other grouper species did not occur during E. marginatus culture. Notochord flexion, with initial ossification of the caudal‐fin supporting elements, started at 6·6 mm L_T. At this stage the major melanophores, preopercular, dorsal and pelvic spines and mandibular teeth were already present. Transformation of larvae into juveniles occurred when larvae averaged 13·8 mm L_T. Juveniles with a mean L_T of 20·1 mm started to settle and most of them were benthic with a mean L_T of 26·8 mm.     

Age and growth of the dusky grouper Epinephelus marginatus (Lowe 1834) in an exploited population of the western Mediterranean Sea

The age and growth of the dusky grouper, Epinephelus marginatus , in the Balearic Islands (western Mediterranean) were studied by otolith analysis from a sample of 358 specimens ranging in total length ( L _T) from 6·6 to 105·6 cm. The specimens came from commercial artisanal and recreational spear fisheries between 1999 and 2003. Otoliths grew asymmetrically throughout the range of L _T studied, showing a clear pattern of alternating translucent and opaque bands. Marginal increment analysis of specimens up to 8 years-old indicated that a single opaque band was formed each year during spring and summer. Whole otoliths allowed ageing specimens up to 10 years old, but above that age whole otoliths yielded lower age estimates than sectioned otoliths. The maximum estimated age was 61 years, which significantly extends the estimated life span of the species from a maximum of 36 years in a previous study. The von Bertalanffy growth parameters were estimated as L _∞= 95·5 cm L _T, K = 0·087 and T _O=−1·12. The study revealed differences in mean L _T at age and age structure between the shallow- and deep-water samples which may be attributed to different fishing pressure and environmental conditions.     

Allozyme and microsatellite loci provide discordant estimates of population differentiation in the endangered dusky grouper (Epinephelus marginatus) within the Mediterranean Sea

The dusky grouper, Epinephelus marginatus, inhabits coastal reefs in the Mediterranean Sea and Atlantic Ocean. A decline in the abundance of this long-lived protogynous hermaphrodite has led to its listing as an endangered species in the Mediterranean, and heightened management concerns regarding its genetic variability and population substructure. To address these concerns, we analysed genetic variation at seven microsatellite and 28 allozyme loci in dusky groupers sampled from seven areas (for microsatellites) and three areas (for allozymes) in the west-central Mediterranean. Levels of genetic variability were higher for microsatellites than for allozymes (mean H(E) = 0.78 and 0.07, respectively), but similar to those observed in other marine fishes with comparable markers. Both microsatellites and allozymes revealed significant genetic differentiation among all areas analysed with each class of marker, but the magnitude of differentiation revealed by allozymes over three locales (F(ST) = 0.214) was greater than that detected with microsatellites over seven areas, or over the three areas shared with the allozyme analysis (F(ST) = 0.018 and approximately 0, respectively). A large proportion of the allozyme differentiation was due to a single locus (ADA*) possibly influenced by selection, but allozyme differentiation over the three areas was still highly significant (F(ST) = 0.06, P < 0.0001), and the 95% confidence intervals for allozyme and microsatellite F(ST) did not overlap when this locus was excluded. There was no evidence of isolation by distance with either class of markers. Our results lead us to conclude that dusky groupers are not panmictic in the Mediterranean Sea and suggest that they should be managed on a local basis. However, more work is needed to elucidate genetic relationships among populations.     

Occurrence of dusky grouper Epinephelus marginatus (Lowe, 1834) in gulfs of northern Patagonia, Argentina

The occurrence of the dusky grouper Epinephelus marginatus in northern Patagonian Gulfs San Matías and Nuevo, Argentina, is recorded. This finding represents the southernmost record of the species for the southern hemisphere.     

Shape and size variation: Growth and development of the dusky grouper (Epinephelus marginatus Lowe, 1834)

Demersal fishes have complex life cycles that involve an ontogenetic change in morphology, physiology, and behavior, as their pelagic larval stages colonize benthic habitats. The developmental transition between larvae and juveniles leads to very complex processes of morphogenesis and differentiation. These processes primarily determine changes in external morphology, which is shaped by selective pressures to optimize performance for basic activities such as swimming, escape from predators, and feeding. Fishes have provided fertile grounds for ecomorphological investigations throughout ontogeny, as the role of changing morphology in inducing ontogenetic niche shifts is not always clear. In this framework, some studies have demonstrated that certain species undergo gradual changes, whereas other species experience threshold effects in their ecomorphological relationships during ontogeny. In this study, the intraspecific allometry of the dusky grouper was examined. Geometric morphometric tools were used to quantify shape changes through the development, and a modular approach was also applied to analyze the pattern of covariation between three distinct blocks (head, trunk, and tail). For this purpose, a two‐block Partial Least Square was computed. This method reveals that the pattern of changes in the overall body shape is the result of the modularized changes of these blocks. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Histological and histochemical study of female germ cell development in the dusky grouper Epinephelus marginatus (Lowe, 1834)

The developmental stages of female germ cells were analysed in a wild population of the protogynous teleost Epinephelus marginatus (Lowe, 1834). 321 wild dusky grouper females were collected in the South Mediterranean Sea during the spawning season and their ovaries analysed using histological and histochemical techniques. Oocyte morphology, nucleus-cytoplasm ratio (N/C) range, location and movements of cytoplasmic inclusions during primary growth, vitellogenesis and final oocyte maturation were described. The distribution of proteins, lipids and carbohydrates through oocyte development was also investigated in 50 females. Lipid vesicles appeared firstly in the mid ooplasm of oocytes larger than 130 microm, at the beginning of the secondary growth phase. Immediately afterwards, small carbohydrate granules (PAS and Alcian blue positive) appeared before the occurrence of the first yolk granules. Tyrosine-enriched proteins were especially evidenced in the zona radiata interna of late vitellogenic oocytes. Specific lectin binding patterns reflected characteristic differences in the content and distribution of specific sugar moieties expressed in the oocytes during vitellogenesis and final maturation. At the end of vitellogenesis and during final maturation, follicular cells, zona radiata, and cortical alveoli were characterised by a strong increase of specific binding for WGA.     

Reproduction in the dusky grouper from the southern Mediterranean

Demographic data and gonad histology confirmed that the dusky grouper Epinephelus marginatus is a protogynous hermaphrodite that follows a monandric pathway to sexual development. Females reached first sexual maturity at 36·7 cm L_s and estimated mean length at first maturity (L₅₀) was 43·8 cm L_s for females and 81·3 cm L_s for males. Adult sex ratios during the reproductive period were c. 3·5: 1 females to males. Females exhibited group-synchronous ovarian development and multiple ovulation occurred over the spawning period. Gonads were ripe from early May and spawning occurred from June until early September. The size of ripe testes (0·6% W )indicated strong oligospermy and suggested a mating system with no sperm competition. Sexual transition was protogynous involving regression of ovarian tissue and proliferation of testicular tissue in the gonads. Transitional individuals occurred from May through November and accounted for 9% of sampled adult population. Sex change occurred in fish 69–93 cm (L_s) long and the size distributions of males and females overlapped over 27% of the L_s range. Special zones were recognized as gathering areas for sexually mature dusky groupers during the reproductive period.     

Size related dietary shifts of Epinephelus marginatus in a western Mediterranean littoral ecosystem: an isotope and stomach content analysis

The diet of the dusky grouper Epinephelus marginatus , from the Balearic Islands, western Mediterranean, consisted primarily of crustaceans, molluscs and fishes, but diet composition varied with body size. The smaller dusky groupers (<300 mm L _T) fed primarily on crustaceans, and particularly on brachyurans, which accounted for 46% of the prey identified. As dusky groupers grew, cephalopods became increasingly important and constituted 10 to 40% of the prey identified in subadult and adult specimens. The largest dusky groupers fed primarily on fishes that represented 40·9% of prey identified. These shifts in diet were accompanied by a positive selection of increasingly large prey and by an expansion of trophic niche. δ ¹⁵N values of dusky grouper white muscle ranged from 8·8 to 13·1% and 71% of the variation in δ ¹⁵N was explained by differences in dusky grouper size. δ ¹³C values ranged from – 17·9 to – 15·9%, and no significant body size effect on δ ¹³C was detected. Stomach content and ¹³C values indicated that from 1 year old, the diet of E. marginatus was based on the benthic food web. The enrichment in δ ¹⁵N registered from juvenile to large males was c . 3·8%. Overall, there was good agreement between gut content and stable isotope data indicating that the latter may be useful as a tool in trophic studies in marine protected areas where sampling for fishes is not allowed.     

A wireless communication system for determining site fidelity of juvenile dusky groupers Epinephelus marginatus (Lowe, 1834) using coded acoustic transmitters

The value of marine reserves to protect threatened and vulnerable species, such as the dusky grouper (Epinephelus marginatus), is often uncertain because of a lack of knowledge about their ecology and habitat utilisation. To address this issue, a Wireless Communication System for location-based positioning of fish tagged with coded acoustic transmitters was installed to determine site fidelity of juvenile dusky groupers (mean total length = 30.4 cm, range: 20.5–40.0 cm; mean total weight = 470 g, range: 113–950 g) within a marine reserve (Island of Ustica, Sicily, Italy). The system was based on five wireless hydrophones distributed approximately 200 m apart along the coastline of the reserve, each monitoring acoustic signals from transmitters within a reception cell. The hydrophones retransmitted detected signals as a radio signal to a land-based receiver and data logger. Unique identifiers for each hydrophone permitted the identification of the detection cell containing the tagged fish. The wireless hydrophones had partly overlapping detection ranges, dividing the marine reserve into more than five different detection cells. The average detection range was 232 m, but coverage varied from site to site and with time, depending on many factors, such as ambient noise, wave action and signal strength of the transmitters. Despite some limitations, the Wireless Hydrophone System (WHS) combined with manual tracking provided a valuable tool for investigating site fidelity in the dusky grouper. Preliminary home ranges for the fish were estimated by the kernel method (probability level of 95%), based on a limited set of manual tracking data, to be a median 5312 m² (range 1673–18626 m²).     

Growth and reproduction of the highfin grouper Epinephelus maculatus

Highfin grouper Epinephelus maculatus sampled in Chuuk, Micronesia, exhibited a moderate growth rate and a relatively short life span compared to other epinephelids of a similar size. Combined gonad and otolith analysis provide preliminary evidence that the species conforms to a protogynous sexual pattern. Mean total length at maturity for females was 308 mm with first age at maturity 2·8 years for females and 4 years for males, which differs from other regional studies. Based on the gonado‐somatic index and microscopic analysis of gonads, E. maculatus in Chuuk have a 4 month spawning season (January to April) that corresponds with seasonal lows in sea surface water temperature and overlaps with that of other aggregating epinephelids. The estimated von Bertalanffy growth factor (K) was 0·51 year^?1, while total mortality was 0·34 year^?1. Current management for E. maculatus in Chuuk includes a January to May catch, sale and export ban, which overlaps with its reproductive season. The effectiveness of these arrangements will require on‐going monitoring to determine whether alternative management strategies are required to ensure population persistence.     

Molecular cloning and expression analysis of interferon-gamma-inducible-lysosomal thiol reductase gene in orange-spotted grouper, Epinephelus coioides

In mammals, interferon-gamma-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. In this study a cDNA containing the orange-spotted grouper GILT (OsgGILT) coding sequence has been cloned and its complete sequence determined. The full-length cDNA of OsgGILT gene is 1066 bp nucleotides (nt) encoding a protein of 260 amino acids (aa), with a putative molecular weight of 28.7 kDa. The deduced OsgGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 10 conserved cysteines. The result of real-time PCR showed that OsgGILT mRNA was expressed in heart, liver, brain, gill, kidney and muscle and more highly expressed in spleen. The OsgGILT expression is obviously up-regulated in spleen and kidney after induction with LPS, these results suggest that OsgGILT may be involved in the immune response to LPS challenge in orange-spotted grouper.     

Molecular cloning of GnRH1 gene and GTH cDNAs of the protogynous longtooth grouper, Epinephelus bruneus

In teleosts, gonadotropin-releasing hormone (GnRH) and gonadotropin hormone (GTH) play important roles in regulating gonadal development and maturation. In Southeast Asia, the longtooth grouper, Epinephelus bruneus, is a commercially important aquaculture fish. In this study, we cloned and characterized the longtooth grouper GnRH1 gene and cDNAs of three gonadotropin subunits (GTHα, FSHβ, LHβ). The GnRH1 gene of longtooth grouper was 4, 032 bp long, and contained four exons, 61, 151, 99, and 423 bp long. GTHα, FSHβ, and LHβ cDNAs were 509, 576, and 579 bp, respectively. Phylogenetic and Southern hybridization analyses revealed that the longtooth grouper GTH subunits were evolutionarily close to those of groupers and are one-copy genes. RT-PCR analyses showed that GTH subunit mRNAs were expressed at a higher level in the pituitary glands of immature fish than in those of mature fish, suggesting a role in gonadal maturation.