Detection of species-specific long VNTRs in mitochondrial control region and their application to identifying sympatric Hong Kong grouper (Epinephelus akaara) and yellow grouper (Epinephelus awoara)

Length variation and heteroplasmy were observed in PCR products of the first half of mtDNA control region of both Hong Kong grouper (Epinephelus akaara) and yellow grouper (Epinephelus awoara). DNA sequencing unveiled the phenomena were caused by the presence of species-specific long variable number tandem repeats (VNTRs). This is the first report on the mtDNA VNTRs and their heteroplasmy in groupers. Moreover, these VNTRs are also the longest such structure found in teleost fish. Thereafter, we designed two species-specific PCR reverse primers according to the 3' end sequences of the VNTRs and successfully established assays for the identification of these two sympatric grouper species.     

Sexual development and sexuality in the Nassau grouper

Hermaphroditic sexual patterns are widespread among teleosts and are especially characteristic of certain perciform families. Among the most diverse groups of hermaphroditic species are the groupers, hamlets and sea basses of the family Serranidae. Like other groupers, the epinepheline serranid, Epinephelus striatus , the Nassau grouper, has long been assumed to exhibit monandric protogyny (a form of hermaphroditism in which all males derive from adult females by sex change). Histological and demographic data, however, indicate that the sexual pattern of this species is not monandric, and that, unlike other group species, the juveniles pass through a bisexual stage of gonadal development. Sexuality in the Nassau grouper is essentially gonochoristic, with potential for sex change. The combination of bisexuality and gonochorism is previously undescribed in the serranids.     

Molecular phylogeography of western Mediterranean dusky grouper Epinephelus marginatus

Intraspecific sequence variation in a portion of the gene coding for cytochrome b in the dusky grouper (Epinephelus marginatus Lowe 1834), an endangered fish species in various regions of the Mediterranean sea, was examined in 29 individuals from the western Mediterranean sea. Sixty-four phylogenetically informative nucleotide positions were present in a 353-base pair cytochrome b sequence, amplified using the polymerase chain reaction. Statistical analysis of the sequence data using a variety of tree-building algorithms separated the taxa into one group of dusky groupers corresponding to some of the Algerian individuals and another regrouped set of fishes originating in France, Tunisia and the remaining Algerian specimens. Although, on the basis of their morphology, E. marginatus are now considered as a single species, our results suggest that a subgroup of the Algerian dusky grouper constitutes a cryptic (undescribed) species. These results suggest that morphological and genetic evolution may be uncoupled in dusky grouper, resulting in morphological similarity between species despite extensive genetic divergence. In addition, we cannot rule out the possibility of gene introgression with other species of grouper. A more in depth phylogenetic analysis (i.e. between and within the different Epinephelus species) would likely affect many conservation management decisions about this assemblage of groupers.     

Evaluation of dorsal aorta cannulation for immunological studies of grouper (Epinephelus malabaricus)

Blood is often withdrawn to study the immune responses of fish. However, netting, handling and anaesthetising the experimental fish, and drawing blood samples cause severe stress that may alter the effects of immune study protocols and treatments. We evaluated the effect of aorta cannulation, for use in immune studies, on grouper (Epinephelus malabaricus) plasma cortisol, total red and white blood cell counts and phagocytosis.Plasma cortisol increased from 30 to 88 ng/ml 1 day after insertion of the cannula, to a maximum of 951 ng/ml 3 to 5 days after surgery, indicating the groupers were stressed by cannulation and post-cannulation inflammation. Total RBC count decreased, and total WBC count increased after surgery. Following cannulation, the phagocytic index of peripheral blood leukocytes decreased from 100% to 46%. The adverse effects of cannulation were mitigated by continuously immersing groupers in oxytetracycline (OTC), which decreased the recovery period for treated fish. In contrast, OTC-treatment did not markedly improve the recovery of groupers subjected to caudal vessel puncture. Cortisol levels in OTC-treated grouper with caudal vessel puncture were significantly higher than in OTC-treated, cannulated grouper, and remained at a high level until day 13 of the experiment. From day 7 to 13, total RBC and WBC counts in OTC-treated, cannulated groupers were significantly different from those in OTC-treated groupers with caudal vessel puncture. OTC treatment improved the phagocytic index of groupers subjected to caudal vessel puncture, but the phagocytic index was lower than that of groupers subjected to cannulation. Cannulation minimises visual and handling disturbances, and facilitates standardisation of experimental conditions and quick and easy sampling via the dorsal aorta cannula. Therefore, dorsal aorta cannulation minimises the stress of blood sampling and should prove useful for immune studies in fish.     

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.     

Genetic characteristics and growth patterns of the hybrid grouper derived from the hybridization of Epinephelus fuscoguttatus (female) × Epinephelus polyphekadion (male)

Hybridization is one of the primary methods used to cultivate farmed grouper species. The hybrid grouper derived from crossing Epinephelus fuscoguttatus (♀) and E. polyphekadion (♂) exhibits growth superiority over its parents. The genetic characteristics and growth patterns of the hybrid grouper have not yet been defined. This study confirms the ploidy level of the hybrid grouper (2n = 48) using chromosome count analysis and flow cytometry. The 5S rDNA family was used to evaluate genetic diversity. Only one 5S class (~400 bp) was detected in the hybrid grouper, which could be used to distinguish between two different types based on nucleotide sequences, likely representing homologous unit classes from the female and male parental species. Growth patterns of 5–8-month-old hybrid groupers were also monitored. In this phase, a positive allometric growth pattern in body mass with total length was found. Body height and body mass were significantly correlated based on correlation and path coefficient, suggesting that body height could serve as an excellent index to increase body mass. These results aid our understanding of the genetic evolution of the hybrid grouper and inform the development of improved rearing techniques.     

Characterization of eight polymorphic microsatellite loci for the giant grouper (Epinephelus lanceolatus Bloch)

Eight polymorphic microsatellite loci were isolated and characterized using a small insert genomic DNA library for the giant grouper (Epinephelus lanceolatus Bloch, 1790), a commercially valuable marine fish in tropical waters. They showed polymorphism information content ranging from 0.177 to 0.775, allele numbers ranging from two to 10, effective allele numbers ranging from 1.227 to 5.012, and observed and expected heterozygosities from 0.2 to 0.733 and from 0.185 to 0.801, respectively, which we anticipate will be useful for population genetic studies of the giant grouper.     

Acoustic telemetry survey of the dusky grouper (Epinephelus marginatus) in the Marine Reserve of Cerbère-Banyuls: informations on the territoriality of this emblematic species

The Marine Reserve of Cerbère-Banyuls represents a refuge for dusky grouper populations (Epinephelus marginatus). Density survey and observation of the reproductive behavior of this species have been conducted there for the last 30 years. However, information on the behavior or habitat utilization for this species during winter time is limited. We conducted acoustic telemetry surveys from September 2005 to June 2006 in the Marine Reserve of Cerbère-Banyuls for a dusky grouper population. Six groupers were tagged and followed within the integral reserve and in the adjacent partial reserve. The results from the tracking of these individuals indicated that the largest dusky groupers are sedentary fishes with a high degree of site fidelity and a high sensitivity to variations of environmental conditions such as storms. This together with the life history characteristics of this species encourages the conservation and the protection of essential habitats of this species. Acoustic tracking coupled to the use of a fixed Kernel can be proposed to follow fish populations. To cite this article: J. Pastor et al., C. R. Biologies 332 (2009).     

Influence of habitat on grouper abundance in the Florida Keys, U.S.A.

There was a significant inshore to offshore pattern in grouper species composition and density on Florida Keys coral reefs. Red grouper Epinephelus morio and black grouper Mycteroperca bonaci were most abundant on inshore patch reefs. Graysby Cephalopholis cruentata was most abundant on offshore reef types. Density among sites ranged from 0 to 0·43, 0 to 0·26 and 0 to 1·23 100 m⁻² for red grouper, black grouper, and graysby, respectively. Grouper density exhibited no consistency among sites within similar reef types. The densities of red and black grouper were correlated significantly with either physical or biotic habitat parameters. Graysby were observed in microhabitats with whose benthic features differed significantly from the surrounding habitat. This species was observed in high-relief areas with significantly less octocoral species and colony densities. Larger graysby were observed in areas of higher relief and less octocorals than smaller graysby. Habitat was one of several important factors influencing the distribution of groupers among coral reefs types in the Florida Keys.     

Development and characterization of 32 microsatellite loci in the giant grouper Epinephelus lanceolatus (Serranidae)

An economically important marine fish species, the giant grouper Epinephelus lanceolatus (Serranidae) is widely cultured in Taiwan and costal areas of China. We isolated and characterized 32 polymorphic microsatellite loci from a CA-enriched genomic library of giant grouper. The number of alleles per locus ranged from 3 to 7, with a mean of 4.69. Observed and expected heterozygosities per locus varied from 0.387 to 1.000 and from 0.377 to 0.843, respectively. Six loci significantly deviated from Hardy-Weinberg equilibrium. After sequential Bonferroni's correction, only two loci showed deviation from Hardy-Weinberg equilibrium, and no linkage disequilibrium was found between any pair of loci. These microsatellites can be useful tools for the study of population genetics in the giant grouper.     

A genome-wide association study on growth traits in orangespotted grouper(Epinephelus coioides) with RAD-seq genotyping

The orange-spotted grouper, Epinephelus coioides, is one of the most popular fish in China and Southeast Asian countries because of its important economic value. However, molecular mechanism underlying the growth of orange-spotted grouper has never been fully understood. Herein, we performed a genome-wide association study(GWAS) on a natural population of 198 individuals aiming to screen the whole genome of orange-spotted grouper for identification of growth-related loci by restrictionsite associated DNA sequencing. In this research, 261,366 single nucleotide polymorphisms(SNPs) were developed, in which110 SNPs were identified to be correlated with growth and 20 SNPs were further confirmed to be associated with both body weight and total length. From these identified SNPs, we annotated a total of 34 genes, including adgrb2, csnkza1, cers5, col22 a1,creb5, dnd1, dzank1, dnai1, npy2 r, fat3, lrrk2, lrp5, map3 k9, and so on. Among these candidate genes, npy2 r(neuropeptide Y receptor Y2) was reported to play a critical role in growth of the orange-spotted grouper. In addition, population structure,principal component analysis, kinship matrix and linkage disequilibrium were examined to verify the accuracy and reliability of our GWAS results. Our data will also provide a valuable genetic resource for further marker-assisted selection program to improve growth quality in groupers.     

Twelve novel polymorphic microsatellite loci for the Yellow grouper (Epinephelus awoara) and cross-species amplifications

Yellow grouper (Epinephelus awoara) is a commercially important marine fish species. A dinucleotide-enriched genomic library of E. awoara was constructed using the method of FIASCO. Twelve loci were polymorphic in a test population with alleles per locus ranging from two to eight, and observed and expected heterozygosities per locus from 0.13 to 1.00 and from 0.20 to 0.86, respectively. Five loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic diversity of E. awoara and related species. L. Zhao and C. Shao have contributed equally to this work.     

Characterization of polymorphic microsatellite loci from black grouper,Mycteroperca bonaci (Teleostei: Serranidae)

Many groupers in the western Atlantic are vulnerable to overfishing, and appropriate management requires knowledge of the population structure of these fish. To this end microsatellite markers were developed from black grouper, Mycteroperca bonaci, using an enrichment procedure. Five loci with levels of polymorphism ranging from 0.5 to 0.9 were selected. These markers also proved to be useful in scamp, M. phenax; red grouper, Epinephelus morio; and goliath grouper, E. itajara. Conformation to Hardy–Weinberg equilibrium was observed except for heterozygote deficiencies noted for E. morio at two of the loci used for this species.     

Monitoring feed amounts in goliath groupers (Epinephelus itajara) using behavioral conditioning in a large mixed species exhibit

Goliath groupers (Epinephelus itajara) are large charismatic species, which are often residents in public aquaria. This study reports a novel approach to feeding techniques for three resident goliath groupers. Because of the size and depth of their exhibit, the groupers were conditioned to hand feed from aquarists. Daily food logs were recorded including the type and number of species, how often the groupers were fed, and how often they accepted the offered food. Mackerel, herring, and sardine represented the highest percentage of the diet, whereas capelin, squid, and shrimp were the lowest percentage. Over a one‐year period, records showed that grouper 1 and grouper 3 ate fairly consistently throughout the year. Grouper 2 had a higher degree of variation in his monthly feeding average with a decrease from May to September and an increase through April. Grouper 1 and grouper 3 took food from the aquarists most consistently with a monthly average of 88.8±10.8 and 89.7±6.15% of the time, respectively. Grouper 2 was not as consistent, hand feeding at only 74.5±16.2% of the time offered. Diet management and behavioral conditioning with the goliath groupers have established consistent husbandry records and therefore better monitoring of the individual fish's long‐term health status. Zoo Biol 27:414–419, 2008. © 2008 Wiley‐Liss, Inc.     

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.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of seven-band grouper (Epinephelus septemfasciatus) and cross-species amplification

Seven-band grouper (Epinephelus septemfasciatus) is a commercially important fishery species. Sixty-six microsatellite loci were isolated from a dinucleotide-enriched genomic library of E. septemfasciatus. Twelve of these loci were polymorphic in a test population with alleles per locus ranging from two to five, and observed and expected heterozygosities per locus from 0.04 to 1.00 and from 0.28 to 0.76, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci after Bonferroni correction. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of E. septemfasciatus and other related species. Lili Zhao and Changwei Shao have contributed equally.     

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.     

9种石斑鱼遗传多样性和系统发生关系的微卫星分析

利用实验室克隆的13个青石斑鱼微卫星分子标记, 对中国南海海域9种石斑鱼(青石斑鱼、蜂巢石斑鱼、鲑点石斑鱼、黑边石斑鱼、鞍带石斑鱼、赤点石斑鱼、七带石斑鱼、斜带石斑鱼和棕点石斑鱼)进行了遗传多样性和系统发生关系的分析。研究结果显示, 13个微卫星标记共检测到了84个等位基因, 9种石斑鱼中的平均等位基因数、平均多态信息含量(PIC)、平均观测杂合度(Ho)、平均期望杂合度(He)和平均Hardy-Weinberg遗传偏离指数(D)分别在2.69~5.38、0.1976~0.4267、0.4615~0.6239、0.3510~0.4754和0.1097~0.2836之间变动, 说明9种石斑鱼的遗传多样性都处于中等水平。用NJ法进行聚类分析的结果将9种石斑鱼分为3个支系：斜带石斑鱼、棕点石斑鱼和鞍带石斑鱼为第1支; 青石斑鱼、赤点石斑鱼和七带石斑鱼为第2支系; 蜂巢石斑鱼、黑边石斑鱼和鲑点石斑鱼为第3支系, 该支系与第2支系的关系较近。本研究支持将宽额鲈(鞍带石斑鱼)归入石斑鱼属。     

Sex inversion of sexually immature honeycomb grouper (Epinephelus merra) by aromatase inhibitor

Previous studies have shown that estrogen plays an important role in sex change of protogynous honeycomb grouper, and that the treatments with aromatase inhibitor (AI) cause estrogen depletion and complete sex inversion of pre-spawning females into functional males. In the present study, we examined whether AI causes sex inversion of sexually immature females. Female honeycomb groupers were implanted with various doses of Fadrozole (0, 100, 500 and 1000 microg/fish) in the non-breeding season, and resultant changes in the gonadal structures and the plasma levels of sex steroid hormones (estradiol-17 beta, E2; testosterone, T; 11-ketotestosterone, 11-KT) were examined three months after implantation. Vehicle-implanted groups did not change sex, while 100 and 500 microg AI-implanted groups had turned into transitionals with intersex gonad. In contrast, the highest dose receiving group exhibited both transitional and male phases. Transitional phase gonad had atretic oocytes and spermatogenic germ cells at the late stages of spermatogenesis, while male phase testis contained spermatozoa accumulated in the seminiferous tubules. All males released sperm upon slight pressure on the abdomen. In the AI-implanted fish, plasma levels of E2 decreased in a dose-dependent manner, while the levels of 11-KT were high in the highest dose receiving group. Present results suggest that estrogen plays an important role in sex change of protogynous honeycomb grouper, and that treatments with AI potentially inhibits endogenous E2 production in vivo, causing oocyte degeneration and subsequently the sex inversion from female to male. The Fadrozole could be an important tool for manipulating the sex of hermaphrodite fishes.